bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of an automated ultraviolet radiation device for decontamination of Clostridium difficile and other healthcare-associated pathogens in hospital rooms
Authors: Nerandzic Michelle M , Cadnum Jennifer L , Pultz Michael J , Donskey Curtis J
Published: July 2010

Background

Environmental surfaces play an important role in transmission of healthcare-associated pathogens. There is a need for new disinfection methods that are effective against Clostridium difficile spores, but also safe, rapid, and automated.

Methods

The Tru-D™ Rapid Room Disinfection device is a mobile, fully-automated room decontamination technology that utilizes ultraviolet-C irradiation to kill pathogens. We examined the efficacy of environmental disinfection using the Tru-D device in the laboratory and in rooms of hospitalized patients. Cultures for C. difficile, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE) were collected from commonly touched surfaces before and after use of Tru-D.

Results

On inoculated surfaces, application of Tru-D at a reflected dose of 22,000 μWs/cm2 for ~45 minutes consistently reduced recovery of C. difficile spores and MRSA by >2-3 log10 colony forming units (CFU)/cm2 and of VRE by >3-4 log10 CFU/cm2. Similar killing of MRSA and VRE was achieved in ~20 minutes at a reflected dose of 12,000 μWs/cm2, but killing of C. difficile spores was reduced. Disinfection of hospital rooms with Tru-D reduced the frequency of positive MRSA and VRE cultures by 93% and of C. difficile cultures by 80%. After routine hospital cleaning of the rooms of MRSA carriers, 18% of sites under the edges of bedside tables (i.e., a frequently touched site not easily amenable to manual application of disinfectant) were contaminated with MRSA, versus 0% after Tru-D (P < 0.001). The system required <5 minutes to set up and did not require continuous monitoring.

Conclusions

The Tru-D Rapid Room Disinfection device is a novel, automated, and efficient environmental disinfection technology that significantly reduces C. difficile, VRE and MRSA contamination on commonly touched hospital surfaces.
A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts
Authors: Burger Katharina , Matt Katja , Kieser Nicole , Gebhard Daniel , Bergemann Jörg
Published: July 2010

Background

The Host Cell Reactivation Assay (HCRA) is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances.

A sensitive and repeatable method to detect the repair capacity of skin cells would be useful in two different aspects: On the one hand, to identify substances influencing the repair capacity in a positive manner (these substances could be promising ingredients for cosmetic products) and on the other hand, to exclude the negative effects of substances on the repair capacity (this could serve as one step further towards replacing or at least reducing animal testing).

Results

In this paper, we present a rapid and sensitive assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts based on two wave-length Green Fluorescent Protein (GFP) and DsRed reporter technology in order to test different substances and their potential to influence the DNA repair capacity. For the detection of plasmid restoration, we used FACS technology, which, in comparison to luminometer technology, is highly sensitive and allows single cell based analysis.

The usefulness of this assay and studying the repair capacity is demonstrated by the evidence that DNA repair is repressed by Cyclosporin A in fibroblasts.

Conclusions

The methodology described in this paper determines the DNA repair capacity in different types of human skin cells. The described transfection protocol is suitable for the transfection of melanocytes, keratinocytes and fibroblasts, reaching efficacies suitable for the detection of the restored plasmids by FACS technology. Therefore the repair capacity of different cell types can be compared with each other. The described assay is also highly flexible, and the activity of other repair mechanisms can be determined using modifications of this method.
Addressing conflicts of interest in Public Private Partnerships
Authors: Omobowale Emmanuel B , Kuziw Michael , Naylor Melinda Treurnicht , Daar Abdallah S , Singer Peter A
Published: July 2010

Background

Many articles have been written on conflicts of interests (COIs) in fields such as medicine, business, politics, public service and education. With the growing abundance of Public Private Partnerships (PPPs), often involving complex relationships among the partners, it is important to understand how COIs can be mitigated and managed in PPPs.

Discussion

We wanted to study PPPs, particularly in the areas of global health and agriculture, but discovered no single source of information available to identify and compare various approaches for avoiding and managing COIs in PPPs. This is a significant gap, especially for those wishing to study, compare and strengthen existing COI policies related to PPPs. In order to bridge this gap, we reviewed how PPPs currently address COIs and highlight what might be considered good practice in developing COI policies. We reviewed the online COI policies of 10 PPPs in global health and agriculture, and interviewed two global health PPP chief executives.

Based on our review of policies and interviews, we conclude that there exists a range of good practices including attention to accountability and governance, acknowledgement and disclosure, abstention and withdrawal, reporting and transparency, and independent monitoring. There appears to be a need for PPPs to interact closely and learn from each other on these parameters and to also place more emphasis on independent external monitoring of COIs as a means of strengthening their major social objectives on which their activities are largely predicated. We also recommend the establishment of a web based database, which would serve as a forum to discuss COI issues and how they can be resolved.
An improved method for the in vitro differentiation of Plasmodium falciparum gametocytes into ookinetes
Authors: Ghosh Anil K , Dinglasan Rhoel R , Ikadai Hiromi , Jacobs-Lorena Marcelo
Published: July 2010

Background

Ookinete is the form of the malaria parasite that invades the mosquito midgut epithelium to initiate sporogony. Differentiation of ingested gametocytes into ookinetes in the mosquito midgut lumen and subsequent interaction with the lumenal surface of the midgut epithelium in preparation for invasion is a complex and multi-stepped process. To facilitate the study of these events in detail it is necessary to produce sufficient numbers of pure, fully mature and functional ookinetes. However, production of even a small number of Plasmodium
falciparum ookinetes in vitro has proven to be a daunting task. Consequently, over the past four decades our collective understanding of the biology of this parasite form remains sorely deficient. This article reports on investigations of five different ookinete media, in an effort to improve the in vitro transformation efficiency of P. falciparum gametocytes into mature ookinetes and their infectivity of the mosquito midgut.

Methods

Five different ookinete media were evaluated for their ability to support the differentiation of gametocytes into gametes and further into mature stage V ookinetes. Moreover, infectivity of the in vitro-transformed ookinetes was evaluated by feeding them to vector mosquitoes and measuring their ability to traverse the midgut and form oocysts.

Results

One of the five media (medium E) was clearly superior in that the cultured ookinetes produced the largest number of oocysts when fed to mosquitoes. Key components were additions of human serum, human red blood cell lysate and mosquito pupal extract, resulting in the production of larger numbers of ookinetes able to develop into oocysts when fed to mosquitoes.

Conclusion

This simple and practical improvement over the prevailing methodology will facilitate the investigation of how this important human malaria parasite initiates its development in the mosquito and will contribute to the understanding of its transmission biology.
Deletion analysis of BMI1 oncoprotein identifies its negative regulatory domain
Authors: Yadav Ajay K , Sahasrabuddhe Anagh A , Dimri Manjari , Bommi Prashant V , Sainger Rachana , Dimri Goberdhan P
Published: July 2010

Background

The polycomb group (PcG) protein BMI1 is an important regulator of development. Additionally, aberrant expression of BMI1 has been linked to cancer stem cell phenotype and oncogenesis. In particular, its overexpression has been found in several human malignancies including breast cancer. Despite its established role in stem cell maintenance, cancer and development, at present not much is known about the functional domains of BMI1 oncoprotein. In the present study, we carried out a deletion analysis of BMI1 to identify its negative regulatory domain.

Results

We report that deletion of the C-terminal domain of BMI1, which is rich in proline-serine (PS) residues and previously described as PEST-like domain, increased the stability of BMI1, and promoted its pro-oncogenic activities in human mammary epithelial cells (HMECs). Specifically, overexpression of a PS region deleted mutant of BMI1 increased proliferation of HMECs and promoted an epithelial-mesenchymal transition (EMT) phenotype in the HMECs. Furthermore, when compared to the wild type BMI1, exogenous expression of the mutant BMI1 led to a significant downregulation of p16INK4a and an efficient bypass of cellular senescence in human diploid fibroblasts.

Conclusions

In summary, our data suggest that the PS domain of BMI1 is involved in its stability and that it negatively regulates function of BMI1 oncoprotein. Our results also suggest that the PS domain of BMI1 could be targeted for the treatment of proliferative disorders such as cancer and aging.
AIDS‐associated Kaposi's sarcoma is linked to advanced disease and high mortality in a primary care HIV programme in South Africa
Authors: Chu Kathryn M , Mahlangeni Gcina , Swannet Sarah , Ford Nathan P , Boulle Andrew , Van Cutsem Gilles
Published: July 2010

Background

AIDS‐associated Kaposi's sarcoma is an important, life‐threatening opportunistic infection among people living with HIV/AIDS in resource‐limited settings. In western countries, the introduction of combination antiretroviral therapy (cART) and new chemotherapeutic agents has resulted in decreased incidence and improved prognosis of AIDS‐associated Kaposi's sarcoma. In African cohorts, however, mortality remains high. In this study, we describe disease characteristics and risk factors for mortality in a public sector HIV programme in South Africa.

Methods

We analysed data from an observational cohort study of HIV‐infected adults with AIDS‐associated Kaposi's sarcoma, enrolled between May 2001 and January 2007 in three primary care clinics. Paper records from primary care and tertiary hospital oncology clinics were reviewed to determine the site of Kaposi's sarcoma lesions, immune reconstitution inflammatory syndrome stage, and treatment. Baseline characteristics, cART use and survival outcomes were extracted from an electronic database maintained for routine monitoring and evaluation. Cox regression was used to model associations with mortality.

Results

Of 6292 patients, 215 (3.4%) had AIDS‐associated Kaposi's sarcoma. Lesions were most commonly oral (65%) and on the lower extremities (56%). One quarter of patients did not receive cART. The mortality and lost‐to‐follow‐up rates were, respectively, 25 (95% CI 19‐32) and eight (95% CI 5‐13) per 100 person years for patients who received cART, and 70 (95% CI 42‐117) and 119 (80‐176) per 100 person years for patients who did not receive cART. Advanced T stage (adjusted HR, AHR = 5.3, p < 0.001), advanced S stage (AHR = 5.1, p = 0.008), and absence of chemotherapy (AHR = 2.4, p = 0.012) were associated with mortality.

Patients with AIDS‐associated Kaposi's sarcoma presented with advanced disease and high rates of mortality and loss to follow up. Risk factors for mortality included advanced Kaposi's sarcoma disease and lack of chemotherapy use. Contributing factors to the high mortality for patients with AIDS‐associated Kaposi's sarcoma likely included late diagnosis of HIV disease, late accessibility to cART, and sub‐optimal treatment of advanced Kaposi's sarcoma.

Conclusions

These findings confirm the importance of early access to both cART and chemotherapy for patients with AIDS‐associated Kaposi's sarcoma. Early diagnosis and improved treatment protocols in resource‐poor settings are essential.
Innate immunity triggers IL-32 expression by fibroblast-like synoviocytes in rheumatoid arthritis
Authors: Alsaleh Ghada , Sparsa Laetitia , Chatelus Emmanuel , Ehlinger Mathieu , Gottenberg Jacques-Eric , Wachsmann Dominique , Sibilia Jean
Published: July 2010

Introduction

Interleukin-32 (IL-32) is a recently described cytokine that is a strong inducer of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, IL-1β, IL-6, and IL-8. The expression of this cytokine is highly increased in the rheumatoid synovium and correlated with the severity of joint inflammation. Little is known regarding the innate immune-related regulation of IL-32 by fibroblast-like synoviocytes (FLSs). We therefore investigated the effect of innate immune stimulation by ligands of Toll-like receptor (TLR)2, TLR3, and TLR4, and cytokines such as TNF-α and interferon (IFN)-γ, on IL-32 expression by FLSs.

Methods

FLSs were isolated from patients with rheumatoid arthritis (RA) according to the ACR criteria. Quantitative RT-PCR, confocal analysis, and ELISA were performed to evaluate IL-32 mRNA induction and IL-32 release by FLSs stimulated with TLR2 (BLP), TLR3 (poly I:C), and TLR4 (lipopolysaccharide) ligands, TNF-α and IFN-γ.

Results

TLR2, -3, and -4 ligands as well as IFN-γ and TNF-α induced IL-32 β, γ and δ mRNA expression by RA FLSs. Mature IL-32 was expressed intracellularly and released by cells stimulated with the various activators. The IL-32α isoform was expressed intracellularly in response to TNF-α and poly I:C and not released in culture supernatants. Stimulation of FLS with TNF-α, BLP, lipopolysaccharide, or poly I:C concomitant with IFN-γ increased IL-32 expression compared with stimulation with IFN-γ alone.

Conclusions

IL-32 synthesis by FLSs is tightly regulated by innate immunity in rheumatoid arthritis. Thus TNF-α, IFN-γ, double-strand RNA, hyaluronic acid, or other damage-associated molecular patterns (DAMPs), highly secreted in synovial tissues of RA patients, might trigger IL-32 secretion by FLSs. IL-32 might therefore represent a relevant therapeutic target in RA.
Mammostrat®as a tool to stratify breast cancer patients at risk of recurrence during endocrine therapy
Authors: Bartlett John MS , Thomas Jeremy , Ross Douglas T , Seitz Robert S , Ring Brian Z , Beck Rodney A , Pedersen Hans Christian , Munro Alison , Kunkler Ian H , Campbell Fiona M , Jack Wilma , Kerr Gillian R , Johnstone Laura , Cameron David A , Chetty Udi
Published: July 2010

Introduction

Patients with early-stage breast cancer, treated with endocrine therapy, have approximately 90% 5-year disease-free survival. However, for patients at higher risk of relapse despite endocrine therapy, additional adjuvant therapy, such as chemotherapy, may be indicated. The challenge is to prospectively identify such patients. The Mammostrat® test uses five immunohistochemical markers to stratify patients on tamoxifen therapy into risk groups to inform treatment decisions. We tested the efficacy of this panel in a mixed population of cases treated in a single center with breast-conserving surgery and long-term follow-up.

Methods

Tissue microarrays from a consecutive series (1981 to 1998) of 1,812 women managed by wide local excision and postoperative radiotherapy were collected following appropriate ethical review. Of 1,390 cases stained, 197 received no adjuvant hormonal or chemotherapy, 1,044 received tamoxifen only, and 149 received a combination of hormonal therapy and chemotherapy. Median age at diagnosis was 57, 71% were postmenopausal, 23.9% were node-positive and median tumor size was 1.5 cm. Samples were stained using triplicate 0.6 mm2 tissue microarray cores, and positivity for p53, HTF9C, CEACAM5, NDRG1 and SLC7A5 was assessed. Each case was assigned a Mammostrat® risk score, and distant recurrence-free survival (DRFS), relapse-free survival (RFS) and overall survival (OS) were analyzed by marker positivity and risk score.

Results

Increased Mammostrat® scores were significantly associated with reduced DRFS, RFS and OS in estrogen receptor (ER)-positive breast cancer (P < 0.00001). In multivariate analyses the risk score was independent of conventional risk factors for DRFS, RFS and OS (P < 0.05). In node-negative, tamoxifen-treated patients, 10-year recurrence rates were 7.6 ± 1.5% in the low-risk group versus 20.0 ± 4.4% in the high-risk group. Further, exploratory analyses revealed associations with outcome in both ER-negative and untreated patients.

Conclusions

This is the fifth independent study providing evidence that Mammostrat® can act as an independent prognostic tool for ER-positive, tamoxifen-treated breast cancer. In addition, this study revealed for the first time a possible association with outcome regardless of node status and ER-negative tumors. When viewed in the context of previous results, these data provide further support for this antibody panel as an aid to patient management in early-stage breast cancer.
Stimulation of human butyrophilin 3 molecules results in negative regulation of cellular immunity
Authors: Yamashiro Hiromichi , Yoshizaki Shinji , Tadaki Toshimasa , Egawa Kohji , Seo Naohiro
Published: July 2010

Stimulation of butyrophilin 3 molecules by specific agonistic mAbs results in down-regulation of proliferation and cytokine secretion in CD4 and CD8 T lymphocyte subsets.

The BTN molecule consists of three subfamilies, BTN1, BTN2. and BTN3, and possesses interesting properties for biological regulation. Although the biological significance of BTN1 and BTN2 has been progressively clarified, the receptor function of BTN3 remains to be elucidated as a result of the absence of appropriate agonists. To clarify the participation of BTN3 in immune regulation, BTN3-specific mAb, referred to as 34-7 and 232-5, were generated from BTN3 gene-immunized mice. The 232-5 mAb, specific to the extracellular domain of the BTN3 molecule, stained almost all populations of human PBMCs, including T, NK, NKT, and B cells. Notably, treatment with the 232-5 mAb resulted in phosphorylation of BTN3A3 molecules, leading to attenuated proliferation and cytokine secretion by CD4+ and CD8+ T cells in a CD4+ CD25+ Treg cell-independent manner, demonstrating the agonistic property of the 232-5 mAb in BTN3-mediated negative signal transduction. The magnitude of the cell surface expression of BTN3 molecules correlated inversely with lymphocyte activity, suggesting that BTN3 molecules contribute to the maintenance of the immune system. Taken together, our findings provide new insights for the role of BTN3 as an inhibitor of excessive cellular immune responses.
Bone marrow chimeric mice reveal a role for CX3CR1 in maintenance of the monocyte-derived cell population in the olfactory neuroepithelium
Authors: Vukovic Jana , Blomster Linda V , Chinnery Holly R , Weninger Wolfgang , Jung Steffen , McMenamin Paul G , Ruitenberg Marc J
Published: July 2010

Definition of the heterogeneity of monocyte-derived cells in the neurogenic olfactory neuroepithelium, their turnover, and the role of CX3CR1 in this process.

Macrophages in the olfactory neuroepithelium are thought to play major roles in tissue homeostasis and repair. However, little information is available at present about possible heterogeneity of these monocyte-derived cells, their turnover rates, and the role of chemokine receptors in this process. To start addressing these issues, this study used Cx3cr1gfp mice, in which the gene sequence for eGFP was knocked into the CX3CR1 gene locus in the mutant allele. Using neuroepithelial whole-mounts from Cx3cr1gfp/+ mice, we show that eGFP+ cells of monocytic origin are distributed in a loose network throughout this tissue and can be subdivided further into two immunophenotypically distinct subsets based on MHC-II glycoprotein expression. BM chimeric mice were created using Cx3cr1gfp/+ donors to investigate turnover of macrophages (and other monocyte-derived cells) in the olfactory neuroepithelium. Our data indicate that the monocyte-derived cell population in the olfactory neuroepithelium is actively replenished by circulating monocytes and under the experimental conditions, completely turned over within 6 months. Transplantation of Cx3cr1gfp/gfp (i.e., CX3CR1-deficient) BM partially impaired the replenishment process and resulted in an overall decline of the total monocyte-derived cell number in the olfactory epithelium. Interestingly, replenishment of the CD68lowMHC-II+ subset appeared minimally affected by CX3CR1 deficiency. Taken together, the established baseline data about heterogeneity of monocyte-derived cells, their replenishment rates, and the role of CX3CR1 provide a solid basis to further examine the importance of different monocyte subsets for neuroregeneration at this unique frontier with the external environment.
Inflammatory mediators promote production of shed LRP1/CD91, which regulates cell signaling and cytokine expression by macrophages
Authors: Gorovoy Matvey , Gaultier Alban , Campana W Marie , Firestein Gary S , Gonias Steven L
Published: July 2010

The shed form of LRP1/CD91, which is generated at increased levels in inflammation, regulates cell-signaling and cytokine expression by macrophages.

LRP1 is a type-1 transmembrane receptor that mediates the endocytosis of diverse ligands. LRP1 β-chain proteolysis results in release of sLRP1 that is present in human plasma. In this study, we show that LPS and IFN-γ induce shedding of LRP1 from RAW 264.7 cells and BMMs in vitro. ADAM17 was principally responsible for the increase in LRP1 shedding. sLRP1 was also increased in vivo in mouse plasma following injection of LPS and in plasma from human patients with RA or SLE. sLRP1, which was purified from human plasma, and full-length LRP1, purified from mouse liver, activated cell signaling when added to cultures of RAW 264.7 cells and BMMs. Robust activation of p38 MAPK and JNK was observed. The IKK-NF-κB pathway was transiently activated. Proteins that bind to the ligand-binding clusters in LRP1 failed to inhibit sLRP1-initiated cell signaling, however an antibody that targets the sLRP1 N terminus was effective. sLRP1 induced expression of regulatory cytokines by RAW 264.7 cells, including TNF-α, MCP-1/CCL2, and IL-10. These results demonstrate that sLRP1 is generated in inflammation and may regulate inflammation by its effects on macrophage physiology.
More than a sidekick: the IL-6 family cytokine IL-11 links inflammation to cancer
Authors: Putoczki Tracy , Ernst Matthias
Published: July 2010

Review discusses newly emerging role for IL-11 in inflammation-associated cancers of the gastrointestinal tract.

IL-11, a member of the IL-6 family of cytokines, exerts pleiotropic activities by stimulating hemopoiesis and thrombopoiesis, regulating macrophage differentiation, and conferring mucosal protection in the intestine. These effects are mediated by a multimeric complex comprising the ligand-binding IL-11Rα and the ubiquitously expressed gp130R β-subunit, which together, trigger intracellular signaling and engagement of Stat3. In turn, activated Stat3 promotes cell survival and proliferation as well as immune responses associated with inflammatory diseases and tumor progression. IL-6 and IL-11 compete for interaction with gp130, resulting in tissue-specific functions depending on the expression patterns of their respective α-subunit receptors. Although traditionally, IL-6 has been associated with aberrant Stat3 activation and associated pathologies, here, we discuss newly emerging roles for IL-11 in linking inflammation to cancer progression. We propose that in light of the recurrence of persistent STAT3 activation and elevated IL-11 expression in inflammation-associated gastrointestinal cancers in humans, inhibition of Stat3 or pharmacologically, more amenable upstream molecules such as IL-11 may represent novel, therapeutic targets.
Genetic redirection of T cells for cancer therapy
Authors: McCaffrey Ramona L , Schwartz Justin T , Lindemann Stephen R , Moreland Jessica G , Buchan Blake W , Jones Bradley D , Allen Lee-Ann H
Published: July 2010

Francisella tularensis uses both pre- and post-assembly mechanisms to inhibit NADPH oxidase activity at its own phagosome and throughout infected human neutrophils.

Ft is a facultative intracellular pathogen that infects many cell types, including neutrophils. In previous work, we demonstrated that the type B Ft strain LVS disrupts NADPH oxidase activity throughout human neutrophils, but how this is achieved is incompletely defined. Here, we used several type A and type B strains to demonstrate that Ft-mediated NADPH oxidase inhibition is more complex than appreciated previously. We confirm that phagosomes containing Ft opsonized with AS exclude flavocytochrome b558 and extend previous results to show that soluble phox proteins were also affected, as indicated by diminished phosphorylation of p47phox and other PKC substrates. However, a different mechanism accounts for the ability of Ft to inhibit neutrophil activation by formyl peptides, Staphylococcus aureus, OpZ, and phorbol esters. In this case, enzyme targeting and assembly were normal, and impaired superoxide production was characterized by sustained membrane accumulation of dysfunctional NADPH oxidase complexes. A similar post-assembly inhibition mechanism also diminished the ability of anti-Ft IS to confer neutrophil activation and bacterial killing, consistent with the limited role for antibodies in host defense during tularemia. Studies of mutants that we generated in the type A Ft strain Schu S4 demonstrate that the regulatory factor fevR is essential for NADPH oxidase inhibition, whereas iglI and iglJ, candidate secretion system effectors, and the acid phosphatase acpA are not. As Ft uses multiple mechanisms to block neutrophil NADPH oxidase activity, our data strongly suggest that this is a central aspect of virulence.
Systemic Delivery of Bioactive Glucagon-Like Peptide 1 after Adenoviral-Mediated Gene Transfer in the Murine Salivary Gland
Authors: Voutetakis Antonis , Cotrim Ana P. , Rowzee Anne , Zheng Changyu , Rathod Trushar , Yanik Tulin , Loh Y. Peng , Baum Bruce J. , Cawley Niamh X.
Published: July 2010

An adenoviral (Ad) vector that expresses bioactive glucagon-like peptide 1 (GLP-1) was generated, and its effectiveness at modulating glucose homeostasis was evaluated after transduction of murine salivary glands. The construct was engineered with the signal sequence of mouse GH to direct the peptide into the secretory pathway, followed by a furin cleavage site and the GLP-1(7–37) sequence encoding an Ala to Gly substitution at position 8 to achieve resistance to degradation. When expressed in Neuro2A and COS7 cells, an active form of GLP-1 was specifically detected by RIA in the conditioned medium of transduced cells, showed resistance to degradation by dipeptidyl-peptidase IV, and induced the secretion of insulin from NIT1 pancreatic β-cells in vitro. In vivo studies demonstrated that healthy mice transduced with Ad-GLP-1 in both submandibular glands had serum GLP-1 levels approximately 3 times higher than mice transduced with the control Ad-luciferase vector. In fasted animals, serum glucose levels were similar between Ad-GLP-1 and Ad-luciferase transduced mice in keeping with GLP-1’s glucose-dependent action. However, when challenged with glucose, Ad-GLP-1 transduced mice cleared the glucose significantly faster than control mice. In an animal model of diabetes induced by alloxan, progression of hyperglycemia was significantly attenuated in mice given the Ad-GLP-1 vector compared with control mice. These studies demonstrate that the bioactive peptide hormone, GLP-1, normally secreted from endocrine cells in the gut through the regulated secretory pathway, can be engineered for secretion into the circulatory system from exocrine cells of the salivary gland to affect glucose homeostasis.
Essential Role for p38α MAPK But Not p38γ MAPK in Igf2 Expression and Myoblast Differentiation
Authors: Lovett Fiona A. , Cosgrove Ruth A. , Gonzalez Ivelisse , Pell Jennifer M.
Published: July 2010

The muscle satellite cell is established as the major stem cell contributing to fiber growth and repair. p38 MAPK signaling is essential for myoblast differentiation and in particular for up-regulation of promyogenic Igf2 expression. p38 exists as four isoforms (α, β, γ, and δ), of which p38γ is uniquely abundant in muscle. The aim of this study was to characterize p38 isoform expression and importance (using shRNA knockdown; demonstrated via both reduced protein and kinase activities) during myoblast differentiation. p38α and -γ mRNA levels were most abundant in differentiating C2 cells with low/negligible contributions from p38β and -δ, respectively. Increased phosphorylation of p38α and -γ occurred during differentiation but via different mechanisms: p38α protein levels remained constant, whereas total p38γ levels increased. Following shRNA knockdown of p38α, myoblast differentiation was dramatically inhibited [reduced myosin heavy chain (MHC), myogenin, pAkt protein levels]; significantly, Igf2 mRNA levels and promoter-reporter activities decreased. In contrast, knockdown of p38γ induced a transient increase in both myogenin and MHC protein levels with no effect on Igf2 mRNA levels or promoter-reporter activity. Knockdown of p38α/β markedly increased but that of p38γ decreased caspase 3 activity, suggesting opposite actions on apoptosis. p38γ was initially proposed to have a promyogenic function; however, p38γ overexpression could not rescue reduced myoblast differentiation following p38α/β inhibition. Therefore, p38α is essential for myoblast differentiation, and part of its action is to convert signals that indicate cell density into promyogenic gene expression in the form of the key peptide, IGF-II; p38γ has a minor, yet opposing antimyogenic, function.
Serum Cathepsin S Is Associated with Serum C-Reactive Protein and Interleukin-6 Independently of Obesity in Elderly Men
Authors: Jobs E. , Risérus U. , Ingelsson E. , Helmersson J. , Nerpin E. , Jobs M. , Sundström J. , Lind L. , Larsson A. , Basu S. , Ärnlöv J.
Published: July 2010

Objective: Cathepsin S has been suggested provide a mechanistic link between obesity and atherosclerosis, possibly mediated via adipose tissue-derived inflammation. Previous data have shown an association between circulating cathepsin S and inflammatory markers in the obese, but to date, community-based reports are lacking. Accordingly, we aimed to investigate the association between serum levels of cathepsin S and markers of cytokine-mediated inflammation in a community-based sample, with prespecified subgroup analyses in nonobese participants.

Methods: Serum cathepsin S, C-reactive protein (CRP), and IL-6 were measured in a community-based cohort of elderly men (Uppsala Longitudinal Study of Adult Men; mean age 71 years, n = 991). CRP and IL-6 were also measured at a reexamination after 7 yr.

Results: After adjustment for age, body mass index, fasting plasma glucose, diabetes treatment, systolic blood pressure, diastolic blood pressure, hypertension treatment, serum cholesterol, serum high-density lipoprotein cholesterol, prior cardiovascular disease, smoking, and leisure time physical activity, higher cathepsin S was associated with higher CRP (regression coefficient for 1 sd increase, 0.13; 95% confidence interval 0.07–0.19; P < 0.001) and higher serum IL-6 (regression coefficient for 1 sd increase, 0.08; 95% confidence interval 0.01–0.14; P = 0.02). These associations remained similar in normal-weight participants (body mass index <25 kg/m2, n = 375). In longitudinal analyses, higher cathepsin S at baseline was associated with higher serum CRP and IL-6 after 7 yr.

Conclusions: These results provide additional evidence for the interplay between cathepsin S and inflammatory activity and suggest that this association is present also in normal-weight individuals in the community.
The Effect of a High-Fat Meal on Postprandial Arterial Stiffness in Men with Obesity and Type 2 Diabetes
Authors: Phillips L. K. , Peake J. M. , Zhang X. , Hickman I. J. , Kolade O. , Sacre J. W. , Huang B. E. , Simpson P. , Li S. H. , Whitehead J. P. , Sharman J. E. , Martin J. H. , Prins J. B.
Published: July 2010

Context: Postprandial dysmetabolism is emerging as an important cardiovascular risk factor. Augmentation index (AIx) is a measure of systemic arterial stiffness and independently predicts cardiovascular outcome.

Objective: The objective of this study was to assess the effect of a standardized high-fat meal on metabolic parameters and AIx in 1) lean, 2) obese nondiabetic, and 3) subjects with type 2 diabetes mellitus (T2DM).

Design and Setting: Male subjects (lean, n = 8; obese, n = 10; and T2DM, n = 10) were studied for 6 h after a high-fat meal and water control. Glucose, insulin, triglycerides, and AIx (radial applanation tonometry) were measured serially to determine the incremental area under the curve (iAUC).

Results: AIx decreased in all three groups after a high-fat meal. A greater overall postprandial reduction in AIx was seen in lean and T2DM compared with obese subjects (iAUC, 2251 ± 1204, 2764 ± 1102, and 1187 ± 429% · min, respectively; P < 0.05). The time to return to baseline AIx was significantly delayed in subjects with T2DM (297 ± 68 min) compared with lean subjects (161 ± 88 min; P < 0.05). There was a significant correlation between iAUC AIx and iAUC triglycerides (r = 0.50; P < 0.05).

Conclusions: Obesity is associated with an attenuated overall postprandial decrease in AIx. Subjects with T2DM have a preserved, but significantly prolonged, reduction in AIx after a high-fat meal. The correlation between AIx and triglycerides suggests that postprandial dysmetabolism may impact on vascular dynamics. The markedly different response observed in the obese subjects compared with those with T2DM was unexpected and warrants additional evaluation.
β-Catenin (CTNNB1) Promotes Preovulatory Follicular Development but Represses LH-Mediated Ovulation and Luteinization
Authors: Fan Heng-Yu , O'Connor Annalouise , Shitanaka Manami , Shimada Masayuki , Liu Zhilin , Richards JoAnne S.
Published: July 2010

Wingless-type mouse mammary tumor virus integration site family (WNT)/β-catenin (CTNNB1) pathway components are expressed in ovarian granulosa cells, direct female gonad development, and are regulated by the pituitary gonadotropins. However, the in vivo functions of CTNNB1 during preovulatory follicular development, ovulation, and luteinization remain unclear. Using a mouse model Ctnnb1(Ex3)fl/fl;Cyp19-Cre (Ctnnb1(Ex3)gc−/−), expressing dominant stable CTNNB1 in granulosa cells of small antral and preovulatory follicles, we show that CTNNB1 facilitates FSH-induced follicular growth and decreases the follicle atresia (granulosa cell apoptosis). At the molecular level, WNT signaling and FSH synergistically promote the expression of genes required for cell proliferation and estrogen biosynthesis, but decrease FOXO1, which negatively regulates proliferation and steroidogenesis. Conversely, dominant stable CTNNB1 represses LH-induced oocyte maturation, ovulation, luteinization, and progesterone biosynthesis. Specifically, granulosa cells in the Ctnnb1(Ex3)gc−/− mice showed compromised responses to the LH surge and decreased levels of the epidermal growth factor-like factors (Areg and Ereg) that in vivo and in vitro mediate LH action. One underlying mechanism by which CTNNB1 prevents LH responses is by reducing phosphorylation of cAMP-responsive element-binding protein, which is essential for the expression of Areg and Ereg. By contrast, depletion of Ctnnb1 using the Ctnnb1fl/fl;Cyp19-Cre mice did not alter FSH regulation of preovulatory follicular development or female fertility but dramatically enhanced LH induction of genes in granulosa cells in culture. Thus, CTNNB1 can enhance FSH and LH actions in antral follicles but overactivation of CTNNB1 negatively effects LH-induced ovulation and luteinization, highlighting the cell context-dependent and developmental stage-specific interactions of WNT/CTNNB1 pathway and G protein-coupled gonadotropin receptors in female fertility.
Activation of paternally expressed genes and perinatal death caused by deletion of theGtl2gene
Authors: Zhou Yunli , Cheunsuchon Pornsuk , Nakayama Yuki , Lawlor Michael W. , Zhong Ying , Rice Kimberley A. , Zhang Li , Zhang Xun , Gordon Francesca E. , Lidov Hart G. W. , Bronson Roderick T. , Klibanski Anne
Published: July 2010

The Dlk1-Gtl2 imprinting locus is located on mouse distal chromosome 12 and consists of multiple maternally expressed non-coding RNAs and several paternally expressed protein-coding genes. The imprinting of this locus plays a crucial role in embryonic development and postnatal growth. At least one cis-element, the intergenic differentially methylated region (IG-DMR) is required for expression of maternally expressed genes and repression of silenced paternally expressed genes. The mechanism by which the IG-DMR functions is largely unknown. However, it has been suggested that the unmethylated IG-DMR acts as a positive regulator activating expression of non-coding RNAs. Gtl2 is the first non-coding RNA gene downstream of the IG-DMR. Although its in vivo function in the mouse is largely unknown, its human ortholog MEG3 has been linked to tumor suppression in human tumor-derived cell lines. We generated a knockout mouse model, in which the first five exons and adjacent promoter region of the Gtl2 gene were deleted. Maternal deletion of Gtl2 resulted in perinatal death and skeletal muscle defects, indicating that Gtl2 plays an important role in embryonic development. The maternal deletion also completely abolished expression of downstream maternally expressed genes, activated expression of silenced paternally expressed genes and resulted in methylation of the IG-DMR. By contrast, the paternal inherited deletion did not have this effect. These data strongly indicate that activation of Gtl2 and its downstream maternal genes play an essential role in regulating Dlk1-Gtl2 imprinting, possibly by maintaining active status of the IG-DMR.
Conserved and novel roles for the Gsh2 transcription factor in primary neurogenesis
Authors: Winterbottom Emily F. , Illes Jean C. , Faas Laura , Isaacs Harry V.
Published: July 2010

The Gsx genes encode members of the ParaHox family of homeodomain transcription factors, which are expressed in the developing central nervous system in members of all major groups of bilaterians. The Gsx genes in Xenopus show similar patterns of expression to their mammalian homologues during late development. However, they are also expressed from early neurula stages in an intermediate region of the open neural plate where primary interneurons form. The Gsx homologue in the protostome Drosophila is expressed in a corresponding intermediate region of the embryonic neuroectoderm, and is essential for the correct specification of the neuroblasts that arise from it, suggesting that Gsx genes may have played a role in intermediate neural specification in the last common bilaterian ancestor. Here, we show that manipulation of Gsx function disrupts the differentiation of primary interneurons. We demonstrate that, despite their similar expression patterns, the uni-directional system of interactions between homeodomain transcription factors from the Msx, Nkx and Gsx families in the Drosophila neuroectoderm is not conserved between their homologues in the Xenopus open neural plate. Finally, we report the identification of Dbx1 as a direct target of Gsh2-mediated transcriptional repression, and show that a series of cross-repressive interactions, reminiscent of those that exist in the amniote neural tube, act between Gsx, Dbx and Nkx transcription factors to pattern the medial aspect of the central nervous system at open neural plate stages in Xenopus.
Arteries provide essential guidance cues for lymphatic endothelial cells in the zebrafish trunk
Authors: Bussmann Jeroen , Bos Frank L. , Urasaki Akihiro , Kawakami Koichi , Duckers Henricus J. , Schulte-Merker Stefan
Published: July 2010

The endothelial cells of the vertebrate lymphatic system assemble into complex networks, but local cues that guide the migration of this distinct set of cells are currently unknown. As a model for lymphatic patterning, we have studied the simple vascular network of the zebrafish trunk consisting of three types of lymphatic vessels that develop in close connection with the blood vasculature. We have generated transgenic lines that allow us to distinguish between arterial, venous and lymphatic endothelial cells (LECs) within a single zebrafish embryo. We found that LECs migrate exclusively along arteries in a manner that suggests that arterial endothelial cells serve as the LEC migratory substrate. In the absence of intersegmental arteries, LEC migration in the trunk is blocked. Our data therefore demonstrate a crucial role for arteries in LEC guidance.
Sperm entry is sufficient to trigger division of the central cell but the paternal genome is required for endosperm development in Arabidopsis
Authors: Aw Sze Jet , Hamamura Yuki , Chen Zhong , Schnittger Arp , Berger Frédéric
Published: July 2010

Fertilization in flowering plants involves two sperm cells and two female gametes, the egg cell and the central cell, progenitors of the embryo and the endosperm, respectively. The mechanisms triggering zygotic development are unknown and whether both parental genomes are required for zygotic development is unclear. In Arabidopsis, previous studies reported that loss-of-function mutations in CYCLIN DEPENDENT KINASE A1 (CDKA;1) impedes cell cycle progression in the pollen leading to the production of a single sperm cell. Here, we report that a significant proportion of single cdka;1 pollen delivers two sperm cells, leading to a new assessment of the cdka;1 phenotype. We performed fertilization of wild-type ovules with cdka;1 mutant sperm cells and monitored in vivo the fusion of the male and female nuclei using fluorescent markers. When a single cdka;1 sperm was delivered, either female gamete could be fertilized leading to similar proportions of seeds containing either a single endosperm or a single embryo. When two cdka;1 sperm cells were released, they fused to each female gamete. Embryogenesis was initiated but the fusion between the nuclei of the sperm cell and the central cell failed. The failure of karyogamy in the central cell prevented incorporation of the paternal genome, impaired endosperm development and caused seed abortion. Our results thus support that the paternal genome plays an essential role during early seed development. However, sperm entry was sufficient to trigger central cell mitotic division, suggesting the existence of signaling events associated with sperm cell fusion with female gametes.
SoxB1 transcription factors restrict organizer gene expression by repressing multiple events downstream of Wnt signalling
Authors: Shih Yu-Huan , Kuo Cheng-Liang , Hirst Caroline S. , Dee Chris T. , Liu Yu-Ru , Laghari Zulfiqar Ali , Scotting Paul J.
Published: July 2010

Formation of the organizer is one of the most central patterning events in vertebrate development. Organizer-derived signals are responsible for establishing the CNS and patterning the dorsal ventral axis. The mechanisms promoting organizer formation are known to involve cooperation between Nodal and Wnt signalling. However, the organizer forms in a very restricted region, suggesting the presence of mechanisms that repress its formation. Here, we show in zebrafish that the transcription factor Sox3 represses multiple steps in the signalling events that lead to organizer formation. Although β-catenin, Bozozok and Squint are known to play major roles in establishing the dorsal organizer in vertebrate embryos, overexpression of any of these is insufficient to induce robust expression of markers of the organizer in ectopic positions in the animal pole, where Sox3 is strongly expressed. We show that a dominant-negative nuclear localisation mutant of Sox3 can cause ectopic expression of organizer genes via a mechanism that activates all of these earlier factors, resulting in later axis duplication including major bifurcations of the CNS. We also find that the related SoxB1 factor, Sox19b, can act redundantly with Sox3 in these effects. It therefore seems that the broad expression of these SoxB1 genes throughout the early epiblast and their subsequent restriction to the ectoderm is a primary regulator of when and where the organizer forms.
Why Have College Completion Rates Declined? An Analysis of Changing Student Preparation and Collegiate Resources
Authors: Bound John , Lovenheim Michael F , Turner Sarah
Published: July 2010

Rising college enrollment over the last quarter century has not been met with a proportional increase in college completion. Comparing the high school classes of 1972 and 1992, we show declines in college completion rates have been most pronounced for men who first enroll in less selective public universities and community colleges. We decompose the decline into the components due to changes in preparedness of entering students and due to changes in collegiate characteristics, including type of institution and resources per student. While both factors play some role, the supply-side characteristics are most important in explaining changes in college completion. (JEL I23)
US Environmental Regulation and FDI: Evidence from a Panel of US-Based Multinational Firms
Authors: Hanna Rema
Published: July 2010

This paper measures the response of US-based multinationals to the Clean Air Act Amendments (CAAA). Using a panel of firm-level data over the period 1966–1999, I estimate the effect of regulation on a multinational's foreign production decisions. The CAAA induced substantial variation in the degree of regulation faced by firms, allowing for the estimation of econometric models that control for firm-specific characteristics and industrial trends. I find that the CAAA caused regulated multinational firms to increase their foreign assets by 5.3 percent and their foreign output by 9 percent. Heavily regulated firms did not disproportionately increase foreign investment in developing countries. (JEL F23, K32, L51, Q52, Q53, Q58)
Service Delivery and Corruption in Public Services: How Does History Matter?
Authors: Pandey Priyanka
Published: July 2010

This paper provides microlevel evidence of how past institutions impact present economic outcomes. It looks at the impact of colonial land tenure institutions on local governance and education outcomes in northern India. Outcomes are worse in villages that belong to areas with a history of concentration of power with the elites. Such areas continue to retain a greater political presence of socially and economically dominant classes. Future research should examine the success of policies that attempt to break such persistence through empowerment of nonelite groups. (JEL D02, H70, I20, N35, N45, O15, O18)