bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and function of soluble guanylate cyclase in pulmonary arterial hypertension
Authors: Schermuly R. T. , Stasch J-P. , Pullamsetti S. S. , Middendorff R. , Müller D. , Schlüter K-D. , Dingendorf A. , Hackemack S. , Kolosionek E. , Kaulen C. , Dumitrascu R. , Weissmann N. , Mittendorf J. , Klepetko W. , Seeger W. , Ghofrani H. A. , Grimminger F.
Published: June 2008

Alterations of the nitric oxide receptor, soluble guanylate cyclase (sGC) may contribute to the pathophysiology of pulmonary arterial hypertension (PAH). In the present study, the expression of sGC in explanted lung tissue of PAH patients was studied and the effects of the sGC stimulator BAY 63-2521 on enzyme activity, and haemodynamics and vascular remodelling were investigated in two independent animal models of PAH.

Strong upregulation of sGC in pulmonary arterial vessels in the idiopathic PAH lungs compared with healthy donor lungs was demonstrated by immunohistochemistry. Upregulation of sGC was detected, similarly to humans, in the structurally remodelled smooth muscle layer in chronic hypoxic mouse lungs and lungs from monocrotaline (MCT)-injected rats. BAY 63-2521 is a novel, orally available compound that directly stimulates sGC and sensitises it to its physiological stimulator, nitric oxide. Chronic treatment of hypoxic mice and MCT-injected rats, with fully established PAH, with BAY 63-2521 (10 mg·kg−1·day−1) partially reversed the PAH, the right heart hypertrophy and the structural remodelling of the lung vasculature.

Upregulation of soluble guanylate cyclase in pulmonary arterial smooth muscle cells was noted in human idiopathic pulmonary arterial hypertension lungs and lungs from animal models of pulmonary arterial hypertension. Stimulation of soluble guanylate cyclase reversed right heart hypertrophy and structural lung vascular remodelling. Soluble guanylate cyclase may thus offer a new target for therapeutic intervention in pulmonary arterial hypertension.
Collapsibility of the relaxed pharynx and risk of sleep apnoea
Authors: Oliven A. , Aspandiarov E. , Gankin I. , Gaitini L. , Tov N.
Published: June 2008

The present study measured hypotonic pharyngeal collapsibility in subjects not known to have obstructive sleep apnoea (OSA), and assessed the variables that affect collapsibility and the relationship with OSA.

The critical value of positive end-expiratory pressure (Pcrit) was measured under the hypotonic condition of anaesthesia in 227 subjects who underwent elective surgery. The risk of OSA in this population was estimated using the Berlin questionnaire.

The meanPcritfor all subjects was positive (above atmospheric), ranging from 0.69 (95% confidence interval (CI) -7.39–8.77) to 4.0 (CI -4.82–12.82) cmH2O for subjects with low and high prevalence of OSA, respectively.Pcrit≤-5 cmH2O was only found in 3.1% of the study subjects. In the general population,Pcritwas similar in males and females and correlated positively with increasing age, while a correlation with neck circumference was found only in males.Pcritaccounted for only 12.25% of the variability in OSA risk score.

In conclusion, subjects with high critical value of positive end-expiratory pressure are at an increased risk for developing obstructive sleep apnoea. However, the human pharynx is prone to collapse and occludes in most people in the absence of neuromuscular support. Therefore, in most subjects, the level of neuromuscular activity may ultimately determine the occurrence of sleep apnoea.
Effects of hyperinflation on the oxygen pulse as a marker of cardiac performance in COPD
Authors: Vassaux C. , Torre-Bouscoulet L. , Zeineldine S. , Cortopassi F. , Paz-Díaz H. , Celli B. R. , Pinto-Plata V. M.
Published: June 2008

A decreased inspiratory capacity (IC)/total lung capacity (TLC) ratio is associated with dynamic hyperinflation and decreased exercise capacity. The present authors hypothesised that static (low IC/TLC) and dynamic hyperinflation impair cardiac function as assessed by oxygen pulse at rest and during cardiopulmonary exercise testing (CPET).

Lung function, body mass index, hand grip strength and CPET parameters were measured (oxygen uptake (mL·kg−1·min−1) and oxygen pulse (mL·beat−1)) in 87 chronic obstructive pulmonary disease (COPD) patients (American Thoracic Society/European Respiratory Society/Global Initiative for Chronic Obstructive Lung Disease stage 3–4) and 46 controls. The patients were divided into those with IC/TLC >25% or ≤25%.

The IC/TLC ratio at rest and at peak exercise was associated significantly with oxygen pulse. Patients with IC/TLC ≤25% (n = 45) had significantly lower exercise capacity, peak oxygen pulse, peak minus baseline oxygen pulse, peak IC, peak IC/TLC ratio and % change from baseline to peak IC/TLC ratio compared with patients with IC/TLC >25% and controls. During CPET, the oxygen pulse was lower at iso-work in patients with IC/TLC ≤25% than in those with IC/TLC >25%.

Resting hyperinflation (inspiratory capacity/total lung capacity) is associated with lower oxygen pulse, peak exercise inspiratory capacity/total lung capacity and exercise capacity in patients with severe chronic obstructive pulmonary disease. The present results support an interaction between hyperinflation and decreased cardiac function that may contribute to exercise limitation in these patients.
Adherence to guidelines’ empirical antibiotic recommendations and community-acquired pneumonia outcome
Authors: Dambrava P. G. , Torres A. , Vallès X. , Mensa J. , Marcos M. A. , Peñarroja G. , Camps M. , Estruch R. , Sánchez M. , Menéndez R. , Niederman M. S.
Published: June 2008

The American Thoracic Society (ATS) published guidelines for the treatment and management of community-acquired pneumonia in 2001, but the impact of adherence on outcomes such as mortality and length of stay is not well defined.

A study of 780 patients with community-acquired pneumonia consecutively admitted to hospital over 1 yr was carried out. Nursing home patients were excluded.

Overall adherence to antibiotics recommended in the ATS guidelines was 84%. The lowest adherence was found in patients admitted to an intensive care unit (52%), especially those at risk of infection withPseudomonas aeruginosa(ATS group IVb). However, very few patients from this group were indeed infected withP. aeruginosa. This could be explained by the exclusion of the nursing home patients. There was a difference in mortality between patients that received adherent and nonadherent regimens (3versus10.6%). There was a difference in length of stay between patients receiving adherent and nonadherent regimens (7.6versus10.4 days). This result was confirmed on multivariate analysis.

Adherence to the 2001 American Thoracic Society guidelines was high except in community-acquired pneumonia patients admitted to an intensive care unit. Length of stay was shorter in patients who received adherent rather than nonadherent antibiotic regimens.
Current clinical guideline definitions of airflow obstruction and COPD overdiagnosis in primary care
Authors: Schermer T. R. J. , Smeele I. J. M. , Thoonen B. P. A. , Lucas A. E. M. , Grootens J. G. , van Boxem T. J. , Heijdra Y. F. , van Weel C.
Published: June 2008

The aim of the present study was to establish the agreement between two recommended definitions of airflow obstruction in symptomatic adults referred for spirometry by their general practitioner, and investigate how rates of airflow obstruction change when pre-bronchodilator instead of post-bronchodilator spirometry is performed.

The diagnostic spirometric results of 14,056 adults with respiratory obstruction were analysed. Differences in interpretation between a fixed 0.70 forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) cut-off point and a sex- and age-specific lower limit of normal cut-off point for this ratio were investigated.

Of the subjects, 53% were female and 69% were current or ex-smokers. The mean post-bronchodilator FEV1/FVC was 0.73 in males and 0.78 in females. The sensitivity of the fixed relative to the lower limit of normal cut-off point definition was 97.9%, with a specificity of 91.2%, positive predictive value of 72.0% and negative predictive value of 99.5%. For the subgroup of current or ex-smokers aged ≥50 yrs, these values were 100, 82.0, 69.2 and 100%, respectively. The proportion of false positive diagnoses using the fixed cut-off point increased with age. The positive predictive value of pre-bronchodilator airflow obstruction was 74.7% among current or ex-smokers aged ≥50 yrs.

The current clinical guideline-recommended fixed 0.70 forced expiratory volume in one second/forced vital capacity cut-off point leads to substantial overdiagnosis of obstruction in middle-aged and elderly patients in primary care. Using pre-bronchodilator spirometry leads to a high rate of false positive interpretations of obstruction in primary care.
Sources of variation in baseline gene expression levels from toxicogenomics study control animals across multiple laboratories
Authors: Boedigheimer Michael J , Wolfinger Russell D , Bass Michael B , Bushel Pierre R , Chou Jeff W , Cooper Matthew , Corton J Christopher , Fostel Jennifer , Hester Susan , Lee Janice S , Liu Fenglong , Liu Jie , Qian Hui-Rong , Quackenbush John , Pettit Syril , Thompson Karol L
Published: June 2008

Background

The use of gene expression profiling in both clinical and laboratory settings would be enhanced by better characterization of variance due to individual, environmental, and technical factors. Meta-analysis of microarray data from untreated or vehicle-treated animals within the control arm of toxicogenomics studies could yield useful information on baseline fluctuations in gene expression, although control animal data has not been available on a scale and in a form best served for data-mining.

Results

A dataset of control animal microarray expression data was assembled by a working group of the Health and Environmental Sciences Institute's Technical Committee on the Application of Genomics in Mechanism Based Risk Assessment in order to provide a public resource for assessments of variability in baseline gene expression. Data from over 500 Affymetrix microarrays from control rat liver and kidney were collected from 16 different institutions. Thirty-five biological and technical factors were obtained for each animal, describing a wide range of study characteristics, and a subset were evaluated in detail for their contribution to total variability using multivariate statistical and graphical techniques.

Conclusion

The study factors that emerged as key sources of variability included gender, organ section, strain, and fasting state. These and other study factors were identified as key descriptors that should be included in the minimal information about a toxicogenomics study needed for interpretation of results by an independent source. Genes that are the most and least variable, gender-selective, or altered by fasting were also identified and functionally categorized. Better characterization of gene expression variability in control animals will aid in the design of toxicogenomics studies and in the interpretation of their results.
Sequencing and expression of two arsenic resistance operons with different functions in the highly arsenic-resistant strain Ochrobactrum tritici SCII24T
Authors: Branco Rita , Chung Ana-Paula , Morais Paula V
Published: June 2008

Background

Arsenic (As) is a natural metalloid, widely used in anthropogenic activities, that can exist in different oxidation states. Throughout the world, there are several environments contaminated with high amounts of arsenic where many organisms can survive. The most stable arsenical species are arsenate and arsenite that can be subject to chemically and microbiologically oxidation, reduction and methylation reactions. Organisms surviving in arsenic contaminated environments can have a diversity of mechanisms to resist to the harmful effects of arsenical compounds.

Results

The highly metal resistant Ochrobactrum tritici SCII24 was able to grow in media with arsenite (50 mM), arsenate (up to 200 mM) and antimonite (10 mM). This strain contains two arsenic and antimony resistance operons (ars 1 and ars 2), which were cloned and sequenced. Sequence analysis indicated that ars 1 operon contains five genes encoding the following proteins: ArsR, ArsD, ArsA, CBS-domain-containing protein and ArsB. The ars 2 operon is composed of six genes that encode two other ArsR, two ArsC (belonging to different families of arsenate reductases), one ACR3 and one ArsH-like protein. The involvement of ars operons in arsenic resistance was confirmed by cloning both of them in an Escherichia coli ars-mutant. The ars 1 operon conferred resistance to arsenite and antimonite on E. coli cells, whereas the ars 2 operon was also responsible for resistance to arsenite and arsenate. Although arsH was not required for arsenate resistance, this gene seems to be important to confer high levels of arsenite resistance. None of ars 1 genes were detected in the other type strains of genus Ochrobactrum, but sequences homologous with ars 2 operon were identified in some strains.

Conclusion

A new strategy for bacterial arsenic resistance is described in this work. Two operons involved in arsenic resistance, one giving resistance to arsenite and antimonite and the other giving resistance to arsenate were found in the same bacterial strain.
Development of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector
Authors: Monk Ian R , Casey Pat G , Cronin Michael , Gahan Cormac GM , Hill Colin
Published: June 2008

Background

The foodborne, gram-positive pathogen,Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era ofL. monocytogenesresearch, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tagL. monocytogenesstrains, with a particular emphasis on the development of multiple strain competitive index assays.

Results

We have constructed a new site-specific integrative vector, pIMC, based on pPL2, for the selection ofL. monocytogenesfrom complex samples. The pIMC vector was further modified through the incorporation of IPTG inducible markers (antibiotic and phenotypic) to produce a suite of four vectors which allowed the discrimination of multiple strains from a single sample. We were able to perform murine infection studies with up to four EGDe isolates within a single mouse and showed that the tags did not impact upon growth rate or virulence. The system also allowed the identification of subtle differences in virulence between strains ofL. monocytogenescommonly used in laboratory studies.

Conclusion

This study has developed a competitive index assay that can be broadly applied to allL. monocytogenesstrains. Improved statistical robustness of the data was observed, resulting in fewer mice being required for virulence assays. The competitive index assays provide a powerful method to analyse the virulence or fitness ofL. monocytogenesin complex biological samples.
Choline-stabilized orthosilicic acid supplementation as an adjunct to Calcium/Vitamin D3 stimulates markers of bone formation in osteopenic females: a randomized, placebo-controlled trial
Authors: Spector Tim D , Calomme Mario R , Anderson Simon H , Clement Gail , Bevan Liisa , Demeester Nathalie , Swaminathan Rami , Jugdaohsingh Ravin , Berghe Dirk A Vanden , Powell Jonathan J
Published: June 2008

Background

Mounting evidence supports a physiological role for silicon (Si) as orthosilicic acid (OSA, Si(OH)4) in bone formation. The effect of oral choline-stabilized orthosilicic acid (ch-OSA) on markers of bone turnover and bone mineral density (BMD) was investigated in a double-blind placebo-controlled trial.

Methods

Over 12-months, 136 women out of 184 randomized (T-score spine < -1.5) completed the study and received, daily, 1000 mg Ca and 20 μg cholecalciferol (Vit D3) and three different ch-OSA doses (3, 6 and 12 mg Si) or placebo. Bone formation markers in serum and urinary resorption markers were measured at baseline, and after 6 and 12 months. Femoral and lumbar BMD were measured at baseline and after 12 months by DEXA.

Results

Overall, there was a trend for ch-OSA to confer some additional benefit to Ca and Vit D3 treatment, especially for markers of bone formation, but only the marker for type I collagen formation (PINP) was significant at 12 months for the 6 and 12 mg Si dose (vs. placebo) without a clear dose response effect. A trend for a dose-corresponding increase was observed in the bone resorption marker, collagen type I C-terminal telopeptide (CTX-I).

Lumbar spine BMD did not change significantly. Post-hoc subgroup analysis (baseline T-score femur < -1) however was significant for the 6 mg dose at the femoral neck (T-test). There were no ch-OSA related adverse events observed and biochemical safety parameters remained within the normal range.

Conclusion

Combined therapy of ch-OSA and Ca/Vit D3 had a potential beneficial effect on bone collagen compared to Ca/Vit D3 alone which suggests that this treatment is of potential use in osteoporosis. NTR 1029
Safety and tolerability of combination antimalarial therapies for uncomplicated falciparum malaria in Ugandan children
Authors: Maiteki-Sebuguzi Catherine , Jagannathan Prasanna , Yau Vincent M , Clark Tamara D , Njama-Meya Denise , Nzarubara Bridget , Talisuna Ambrose O , Kamya Moses R , Rosenthal Philip J , Dorsey Grant , Staedke Sarah G
Published: June 2008

Background

Combination antimalarial therapy is recommended for the treatment of uncomplicated falciparum malaria in Africa; however, some concerns about the safety and tolerability of new regimens remain. This study compared the safety and tolerability of three combination antimalarial regimens in a cohort of Ugandan children.

Methods

A longitudinal, single-blind, randomized clinical trial of children was conducted between November 2004 and May 2007 in Kampala, Uganda. Upon diagnosis of the first episode of uncomplicated malaria, participants were randomized to treatment with amodiaquine + sulphadoxine-pyrimethamine (AQ+SP), artesunate + amodiaquine (AS+AQ), or artemether-lumefantrine (AL). Once randomized, participants received the same regimen for all subsequent episodes of uncomplicated malaria. Participants were actively monitored for adverse events for the first 14 days after each treatment, and then passively followed until their next study medication treatment, or withdrawal from study. Outcome measures included the risk of adverse events at 14 and 42 days after treatment.

Results

Of 601 enrolled children, 382 were diagnosed with at least one episode of uncomplicated malaria and were treated with study medications. The median age at treatment was 6.3 years (range 1.1 – 12.3 years). At 14 days of follow-up, AQ+SP treatment was associated with a higher risk of anorexia, weakness, and subjective fever than treatment with AL, and a higher risk of weakness, and subjective fever than treatment with AS+AQ. Treatment with AL was associated with a higher risk of elevated temperature. Repeated episodes of neutropaenia associated with AS+AQ were detected in one participant. Considering only children less than five years, those who received AQ+SP were at higher risk of developing moderate or severe anorexia and weakness than those treated with AL (anorexia: RR 3.82, 95% CI 1.59 – 9.17; weakness: RR 5.40, 95% CI 1.86 – 15.7), or AS+AQ (anorexia: RR 2.10, 95% CI 1.04 – 4.23; weakness: RR 2.26, 95% CI 1.01 – 5.05). Extending the analysis to 42 days of follow-up had little impact on the findings.

Conclusion

This study confirms the safety and tolerability of AS+AQ and AL in Ugandan children, and suggests that AQ+SP is safe, but less well-tolerated, particularly in younger children. As newer antimalarial regimens are deployed, collecting data on their safety and tolerability will be essential.

Trial registration

Current Controlled Trials Identifier ISRCTN37517549
Molecular profiling of angiogenesis in hypericin mediated photodynamic therapy
Authors: Bhuvaneswari Ramaswamy , Gan Yik Y , Lucky Sasidharan S , Chin William WL , Ali Seyed M , Soo Khee C , Olivo Malini
Published: June 2008

Background

Photodynamic therapy (PDT) involves the administration of a tumor-localizing photosensitizing drug, which is activated by light of specific wavelength in the presence of molecular oxygen thus generating reactive oxygen species that is toxic to the tumor cells. PDT selectively destroys photosensitized tissue leading to various cellular and molecular responses. The present study was designed to examine the angiogenic responses at short (0.5 h) and long (6 h) drug light interval (DLI) hypericin-PDT (HY-PDT) treatment at 24 h and 30 days post treatment in a human bladder carcinoma xenograft model. As short DLI targets tumor vasculature and longer DLI induces greater cellular damage, we hypothesized a differential effect of these treatments on the expression of angiogenic factors.

Results

Immunohistochemistry (IHC) results showed minimal CD31 stained endothelium at 24 h post short DLI PDT indicating extensive vascular damage. Angiogenic proteins such as vascular endothelial growth factor (VEGF), tumor necrosis growth factor-α (TNF-α), interferon-α (IFN-α) and basic fibroblast growth factor (bFGF) were expressed to a greater extent in cellular targeting long DLI PDT compared to vascular mediated short DLI PDT. Gene expression profiling for angiogenesis pathway demonstrated downregulation of adhesion molecules – cadherin 5, collagen alpha 1 and 3 at 24 h post treatment. Hepatocyte growth factor (HGF) and Ephrin-A3 (EFNA3) were upregulated in all treatment groups suggesting a possible activation of c-Met and Ephrin-Eph signaling pathways.

Conclusion

In conclusion, long DLI HY-PDT induces upregulation of angiogenic proteins. Differential expression of genes involved in the angiogenesis pathway was observed in the various groups treated with HY-PDT.
Bioavailable constituents/metabolites of pomegranate (Punica granatum L) preferentially inhibit COX2 activity ex vivo and IL-1beta-induced PGE2 production in human chondrocytes i n vitro
Authors: Shukla Meenakshi , Gupta Kalpana , Rasheed Zafar , Khan Khursheed A , Haqqi Tariq M
Published: June 2008

Several recent studies have documented that supplementation with pomegranate fruit extract inhibits inflammatory symptoms in vivo. However, the molecular basis of the observed effects has not been fully revealed. Although previous studies have documented the inhibition of nitric oxide and cyclooxygenase (COX) activity in vitro by plant and fruit extracts added directly into the culture medium but whether concentrations of bioactive compounds sufficient enough to exert such inhibitory effects in vivo can be achieved through oral consumption has not been reported. In the present study we determined the effect of rabbit plasma obtained after ingestion of a polyphenol rich extract of pomegranate fruit (PFE) on COX enzyme activity ex vivo and the IL-1β-induced production of NO and PGE2 in chondrocytes in vitro. Plasma samples collected before and 2 hr after supplementation with PFE were tested. Plasma samples collected after oral ingestion of PFE were found to inhibit the IL-1β-induced PGE2 and NO production in chondrocytes. These same plasma samples also inhibited both COX-1 and COX-2 enzyme activity ex vivo but the effect was more pronounced on the enzyme activity of COX-2 enzyme. Taken together these results provide additional evidence of the bioavailability and bioactivity of compounds present in pomegranate fruit after oral ingestion. Furthermore, these studies suggest that PFE-derived bioavailable compounds may exert an anti-inflammatory effect by inhibiting the inflammatory cytokine-induced production of PGE2 and NO in vivo.
Automated external cardioversion defibrillation monitoring in cardiac arrest: a randomized trial
Authors: Ali Bakhtiar , Bloom Heather , Veledar Emir , House Dorothy , Norvel Robert , Dudley Samuel C , Zafari A Maziar
Published: June 2008

Background

In-hospital cardiac arrest has a poor prognosis despite active electrocardiography monitoring. The initial rhythm of approximately 25% of in-hospital cardiopulmonary resuscitation (CPR) events is pulseless ventricular tachycardia/ventricular fibrillation (VT/VF). Early defibrillation is an independent predictor of survival in CPR events caused by VT/VF. The automated external cardioverter defibrillator (AECD) is a device attached by pads to the chest wall that monitors, detects, and within seconds, automatically delivers electric countershock to an appropriate tachyarrhythmia.

Study Objectives

• To evaluate safety of AECD monitoring in hospitalized patients.

• To evaluate whether AECDs provide earlier defibrillation than hospital code teams.

Methods

The study is a prospective trial randomizing patients admitted to the telemetry ward to standard CPR (code team) or standard CPR plus AECD monitoring (PowerHeart CRM). The AECD is programmed to deliver one 150 J biphasic shock to patients in sustained VT/VF. Data is collected using the Utstein criteria for cardiac arrest. The primary endpoint is time-to-defibrillation; secondary outcomes include neurological status and survival to discharge, with 3-year follow-up.

Results

To date, 192 patients have been recruited in the time period between 10/10/2006 to 7/20/2007. A total of 3,655 hours of telemetry data have been analyzed in the AECD arm. The AECD has monitored ambulatory telemetry patients in sinus rhythm, sinus tachycardia, supraventricular tachycardia, atrial flutter or fibrillation, with premature ventricular complexes and non-sustained VT without delivery of inappropriate shocks. One patient experienced sustained VT during AECD monitoring, who was successfully defibrillated (17 seconds after meeting programmed criteria). There are no events to report in the control arm. The patient survived the event without neurological complications. During the same time period, mean time to shock for VT/VF cardiac arrest occurring outside the telemetry ward was 230 ± 50 seconds.

Conclusion

AECD monitoring is safe and likely results in earlier defibrillation than standard telemetry monitoring.

Trial Registration

National Institutes of Health registration ID: NCT00382928
Systems analysis of quantitative shRNA-library screens identifies regulators of cell adhesion
Authors: Huang XiaoDong , Wang Jean YJ , Lu Xin
Published: June 2008

Background

High throughput screens with RNA interference technology enable loss-of-function analyses of gene activities in mammalian cells. While the construction of genome-scale shRNA libraries has been successful, results of large-scale screening of those libraries can be difficult to analyze because of the relatively high noise levels and the fact that not all shRNAs in a library are equally effective in silencing gene expression.

Results

We have screened a library consisting of 43,828 shRNAs directed against 8,500 human genes for functions that are necessary in cell detachment induced by a constitutively activated c-Abl tyrosine kinase. To deal with the issues of noise and uncertainty of knockdown efficiencies, we employed an analytical strategy that combines quantitative data analysis with biological knowledge, i.e. Gene Ontology and pathway information, to increase the power of the RNAi screening technique. Using this strategy we found 16 candidate genes to be involved in Abl-induced disruption of cell adhesion, and verified that the knockdown of IL6ST is associated with enhanced cell attachment.

Conclusion

Our results suggest that the power of genome-wide quantitative shRNA screens can be significantly increased when analyzed using a systems biology-based approach to identify functional gene networks.
Whole genome assessment of the retinal response to diabetes reveals a progressive neurovascular inflammatory response
Authors: Brucklacher Robert M , Patel Kruti M , VanGuilder Heather D , Bixler Georgina V , Barber Alistair J , Antonetti David A , Lin Cheng-Mao , LaNoue Kathryn F , Gardner Thomas W , Bronson Sarah K , Freeman Willard M
Published: June 2008

Background

Despite advances in the understanding of diabetic retinopathy, the nature and time course of molecular changes in the retina with diabetes are incompletely described. This study characterized the functional and molecular phenotype of the retina with increasing durations of diabetes.

Results

Using the streptozotocin-induced rat model of diabetes, levels of retinal permeability, caspase activity, and gene expression were examined after 1 and 3 months of diabetes. Gene expression changes were identified by whole genome microarray and confirmed by qPCR in the same set of animals as used in the microarray analyses and subsequently validated in independent sets of animals. Increased levels of vascular permeability and caspase-3 activity were observed at 3 months of diabetes, but not 1 month. Significantly more and larger magnitude gene expression changes were observed after 3 months than after 1 month of diabetes. Quantitative PCR validation of selected genes related to inflammation, microvasculature and neuronal function confirmed gene expression changes in multiple independent sets of animals.

Conclusion

These changes in permeability, apoptosis, and gene expression provide further evidence of progressive retinal malfunction with increasing duration of diabetes. The specific gene expression changes confirmed in multiple sets of animals indicate that pro-inflammatory, anti-vascular barrier, and neurodegenerative changes occur in tandem with functional increases in apoptosis and vascular permeability. These responses are shared with the clinically documented inflammatory response in diabetic retinopathy suggesting that this model may be used to test anti-inflammatory therapeutics.
Identification of motifs that function in the splicing of non-canonical introns
Authors: Murray Jill I , Voelker Rodger B , Henscheid Kristy L , Warf M Bryan , Berglund J Andrew
Published: June 2008

Background

While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors. The diversity of human intronic sequences suggests the existence of novel recognition pathways for non-canonical introns. This study addresses the recognition and splicing of human introns that lack a canonical PY tract. The PY tract is a uridine-rich region at the 3' end of introns that acts as a binding site for U2AF65, a key factor in splicing machinery recruitment.

Results

Human introns were classified computationally into low- and high-scoring PY tracts by scoring the likely U2AF65 binding site strength. Biochemical studies confirmed that low-scoring PY tracts are weak U2AF65 binding sites while high-scoring PY tracts are strong U2AF65 binding sites. A large population of human introns contains weak PY tracts. Computational analysis revealed many families of motifs, including C-rich and G-rich motifs, that are enriched upstream of weak PY tracts. In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts.

Conclusion

The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif.
Transmembrane TNF-α mediates “forward” and “reverse” signaling, inducing cell death or survival via the NF-κB pathway in Raji Burkitt lymphoma cells
Authors: Zhang Hailong , Yan Dan , Shi Xu , Liang Huifang , Pang Yan , Qin Nalin , Chen Hui , Wang Jing , Yin Bingjiao , Jiang Xiaodan , Feng Wei , Zhang Wenjie , Zhou Muxiang , Li Zhuoya
Published: June 2008

Interestingly, some lymphoma cells, expressing high levels of transmembrane (tm)TNF-α, are resistant to secretory (s)TNF-α-induced necrosis but sensitive to tmTNF-α-mediated apoptosis. As tmTNF-α mediates “forward” as well as “reverse” signaling, we hypothesize that a balanced signaling between forward and reverse directions may play a critical role in determining the fate of cells bearing tmTNF-α. Using Raji cells as a model, we first added exogenous tmTNF-α on fixed, transfected NIH3T3 cells onto Raji cells to examine tmTNF-α forward signaling and its effects, showing that constitutive NF-κB activity and cellular inhibitor-of-apoptosis protein 1 transcription were down-regulated, paralleled with Raji cell death. As Raji cells express tmTNF-α, an inhibition of their tmTNF-α expression by antisense oligonucleotide caused down-regulation of NF-κB activity. Conversely, increasing tmTNF-α expression by suppressing expression of TNF-α-converting enzyme that cleaves tmTNF-α led to an enhanced activation of NF-κB, indicating that tmTNF-α, but not sTNF-α, contributes to constitutive NF-κB activation. We next transfected Raji cells with a mutant tmTNF-α lacking the intracellular domain to competitively suppress reverse signaling via tmTNF-α; as expected, constitutive NF-κB activity was decreased. In contrast, treating Raji cells with sTNFR2 to stimulate reverse signaling via tmTNF-α ehanced NF-κB activation. We conclude that tmTNF-α, when highly expressed on tumor cells and acting as a receptor, promotes NF-κB activation through reverse signaling, which is helpful to maintain tumor cell survival. On the contrary, tmTNF-α, when acting as a ligand, inhibits NF-κB activity through forward signaling, which is inclined to induce tumor cell death.
Prolactin Secretion Sites Contain Syntaxin-1 and Differ from Ganglioside Monosialic Acid Rafts in Rat Lactotrophs
Authors: Gonçalves Paula P. , Stenovec Matjaž , Chowdhury Helena H. , Grilc Sonja , Kreft Marko , Zorec Robert
Published: June 2008

In neuroendocrine cells, discharge of hormones follows the fusion of exocytotic vesicles with the plasma membrane at confined sites; however, the molecular nature of these distinct sites remains poorly understood. We studied intact pituitary lactotrophs and plasma membrane lawns by confocal microscopy in conjunction with antibodies against rat prolactin (rPRL), soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins (syntaxin-1 and synaptobrevin-2,) and fluorescent cholera toxin subunit B (CT-B), a marker of ganglioside monosialic acid (GM1) lipid rafts, to examine 1) whether rPRL vesicles discharge cargo at GM1 rafts, 2) whether discharging rPRL vesicles interact with SNAREs, and 3) to examine the overlap of GM1 rafts, rPRL, and syntaxin-1 sites in plasma membrane lawns. In intact cells, immunofluorescently labeled rPRL poorly colocalized (<6%) with CT-B. In conditions favoring endocytotic trafficking, vesicle SNARE synaptobrevin-2 modestly colocalized (35%) with CT-B, whereas it highly colocalized (58%) with retrieved rPRL. Although partial mixing between rPRL and CT-B intracellular trafficking pathways is likely, our results indicated that rPRL discharge involves interactions with plasma membrane SNAREs, but not with GM1 rafts. In support of this, the plasma membrane SNARE syntaxin-1 poorly colocalized with CT-B (<5%), whereas it highly colocalized (75%) with rPRL in inside-out plasma membrane lawns. Spontaneous and stimulated rPRL discharge in live lactotrophs is thus associated with plasma membrane sites enriched with SNARE proteins, however, spatially confined to plasma membrane areas other than GM1 rafts.
Brain Glucagon-Like Peptide 1 Signaling Controls the Onset of High-Fat Diet-Induced Insulin Resistance and Reduces Energy Expenditure
Authors: Knauf Claude , Cani Patrice D. , Ait-Belgnaoui Afifa , Benani Alexandre , Dray Cédric , Cabou Cendrine , Colom André , Uldry Marc , Rastrelli Sophie , Sabatier Eric , Godet Natacha , Waget Aurélie , Pénicaud Luc , Valet Philippe , Burcelin Rémy
Published: June 2008

Glucagon-like peptide-1 (GLP-1) is a peptide released by the intestine and the brain. We previously demonstrated that brain GLP-1 increases glucose-dependent hyperinsulinemia and insulin resistance. These two features are major characteristics of the onset of type 2 diabetes. Therefore, we investigated whether blocking brain GLP-1 signaling would prevent high-fat diet (HFD)-induced diabetes in the mouse. Our data show that a 1-month chronic blockage of brain GLP-1 signaling by exendin-9 (Ex9), totally prevented hyperinsulinemia and insulin resistance in HFD mice. Furthermore, food intake was dramatically increased, but body weight gain was unchanged, showing that brain GLP-1 controlled energy expenditure. Thermogenesis, glucose utilization, oxygen consumption, carbon dioxide production, muscle glycolytic respiratory index, UCP2 expression in muscle, and basal ambulatory activity were all increased by the exendin-9 treatment. Thus, we have demonstrated that in response to a HFD, brain GLP-1 signaling induces hyperinsulinemia and insulin resistance and decreases energy expenditure by reducing metabolic thermogenesis and ambulatory activity.
Hepatic Expression of the SPOT 14 (S14) Paralog S14-Related (Mid1 Interacting Protein) Is Regulated by Dietary Carbohydrate
Authors: Tsatsos Nikolas G. , Augustin Lance B. , Anderson Grant W. , Towle Howard C. , Mariash Cary N.
Published: June 2008

The Spot 14 (S14) gene is rapidly up-regulated by signals that induce lipogenesis such as enhanced glucose metabolism and thyroid hormone administration. Previous studies in S14 null mice show that S14 is required for normal lipogenesis in the lactating mammary gland, but not the liver. We speculated that the lack of a hepatic phenotype was due to the expression of a compensatory gene. We recently reported that this gene is likely an S14 paralog that we named S14-Related (S14-R). S14-R is expressed in the liver, but not in the mammary gland. If S14-R compensates for the absence of S14 in the liver, we hypothesized that, like S14, S14-R expression should be glucose responsive. Here, we report that hepatic S14-R mRNA levels increase with high-carbohydrate feeding in mice or within 2 h of treating cultured hepatocytes with elevated glucose. A potential carbohydrate response element (ChoRE) was identified at position −458 of the S14-R promoter. Deletion of or point mutations within the putative S14-R ChoRE led to 50–95% inhibition of the glucose response. Gel-shift analysis revealed that the glucose-activated transcription complex carbohydrate responsive element-binding protein/Max-like protein X (Mlx) binds to the S14-R ChoRE. Finally, S14-R glucose induction is completely blocked when a dominant-negative form of Mlx is overexpressed in primary hepatocytes. In conclusion, our results indicate that the S14-R gene is a glucose-responsive target of carbohydrate responsive element-binding protein/Mlx and suggest that the S14-R protein is a compensatory factor, at least partially responsible for the normal liver lipogenesis observed in the S14 null mouse.
Angiotensin II AT1 Receptor Blockade Decreases Lipopolysaccharide-Induced Inflammation in the Rat Adrenal Gland
Authors: Sanchez-Lemus Enrique , Murakami Yuki , Larrayoz-Roldan Ignacio M. , Moughamian Armen J. , Pavel Jaroslav , Nishioku Tsuyoshi , Saavedra Juan M.
Published: June 2008

Peripheral administration of bacterial endotoxin [lipopolysaccharide (LPS)] to rodents produces an innate immune response and hypothalamic-pituitary-adrenal axis stimulation. Renin-angiotensin-aldosterone system inhibition by angiotensin II AT1 receptor blockade has antiinflammatory effects in the vasculature. We studied whether angiotensin II receptor blockers (ARBs) prevent the LPS response. We focused on the adrenal gland, one organ responsive to LPS and expressing a local renin-angiotensin-aldosterone system. LPS (50 μg/kg, ip) produced a generalized inflammatory response with increased release of TNF-α and IL-6 to the circulation, enhanced adrenal aldosterone synthesis and release, and enhanced adrenal cyclooxygenase-2, IL-6, and TNF-α gene expression. ACTH and corticosterone release were also increased by LPS. Pretreatment with the ARB candesartan (1 mg/kg·d, sc for 3 d before the LPS administration) decreased LPS-induced cytokine release to the circulation, adrenal aldosterone synthesis and release, and cyclooxygenase-2 and IL-6 gene expression. Candesartan did not prevent the LPS-induced ACTH and corticosterone release. Our results suggest that AT1 receptors are essential for the development of the full innate immune and stress responses to bacterial endotoxin. The ARB decreased the general peripheral inflammatory response to LPS, partially decreased the inflammatory response in the adrenal gland, prevented the release of the pro-inflammatory hormone aldosterone, and protected the antiinflammatory effects of glucocorticoid release. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that the ARBs may have therapeutic effects on inflammatory conditions.
Leptin Enhances Human β-Defensin-2 Production in Human Keratinocytes
Authors: Kanda Naoko , Watanabe Shinichi
Published: June 2008

Leptin, an adipocyte-derived cytokine/hormone, modulates innate and adaptive immunity. Human β-defensin-2 (hBD-2) produced by epidermal keratinocytes promotes cutaneous antimicrobial defense, inflammation, and wound repair. We examined the in vitro effects of leptin on hBD-2 production in human keratinocytes. hBD-2 secretion and mRNA expression were analyzed by ELISA and RT-PCR, respectively. Although leptin alone was ineffective, it enhanced IL-1β-induced hBD-2 secretion and mRNA expression in keratinocytes. IL-1β- and IL-1β plus leptin-induced hBD-2 production both were suppressed by antisense oligonucleotides against nuclear factor-κB (NF-κB) p50 and p65; the latter was also suppressed by antisense signal transducer and activator of transcription (STAT)1 and STAT3. IL-1β enhanced the transcriptional activity of NF-κB, whereas leptin enhanced STAT1 and STAT3 activity. The p38 MAPK inhibitor SB202190 suppressed IL-1β- and IL-1β plus leptin-induced hBD-2 production, IL-1β-induced NF-κB activity, and leptin-induced STAT1 and STAT3 activity; contrastingly, the Janus kinase (JAK) 2 inhibitor AG490 suppressed IL-1β plus leptin-induced hBD-2 production and leptin-induced STAT1 and STAT3 activity. IL-1β induced serine phosphorylation of inhibitory κBα, STAT1, and STAT3. Leptin induced tyrosine and serine phosphorylation of STAT1 and STAT3, both of which were suppressed by AG490, and serine phosphorylation was also suppressed by SB202190. IL-1β or leptin individually induced threonine/tyrosine phosphorylation of p38 MAPK, whereas only leptin induced tyrosine phosphorylation of JAK2, suggesting that leptin may enhance hBD-2 production in keratinocytes by activating STAT1 and STAT3 via JAK2 and p38 MAPK in cooperation with NF-κB, which is activated by IL-1β. Leptin may promote cutaneous antimicrobial defense, inflammation, and wound repair via hBD-2.
Stem Cell Contribution to Ovarian Development, Function, and Disease
Authors: Tilly Jonathan L. , Rueda Bo R.
Published: June 2008

By virtue of the fact that oocytes not only serve to produce embryos after fertilization but also can effectively reprogram adult somatic cell nuclei to a pluripotent state, much of the interest in the role of stem cells in ovarian biology has been focused on the germline. However, very recent studies have revealed that somatic stem cells may also be of considerable relevance to the study of normal ovarian function. Furthermore, stem cell dysfunction may underlie or contribute to disease states such as ovarian cancer and polycystic ovary syndrome. Our objective is to explore these concepts in greater detail, with the hope of stimulating further research efforts into understanding what role stem cells may play in the physiology and pathology of the mammalian female gonads.
Osteoimmunology: Interactions of the Bone and Immune System
Authors: Lorenzo Joseph , Horowitz Mark , Choi Yongwon
Published: June 2008

Bone and the immune system are both complex tissues that respectively regulate the skeleton and the body’s response to invading pathogens. It has now become clear that these organ systems often interact in their function. This is particularly true for the development of immune cells in the bone marrow and for the function of bone cells in health and disease. Because these two disciplines developed independently, investigators in each don’t always fully appreciate the significance that the other system has on the function of the tissue they are studying. This review is meant to provide a broad overview of the many ways that bone and immune cells interact so that a better understanding of the role that each plays in the development and function of the other can develop. It is hoped that an appreciation of the interactions of these two organ systems will lead to better therapeutics for diseases that affect either or both.
Signal Transducer and Activator of Transcription 5b, c-Src, and Epidermal Growth Factor Receptor Signaling Play Integral Roles in Estrogen-Stimulated Proliferation of Estrogen Receptor-Positive Breast Cancer Cells
Authors: Fox Emily M. , Bernaciak Teresa M. , Wen Jie , Weaver Amanda M. , Shupnik Margaret A. , Silva Corinne M.
Published: June 2008

17β-Estradiol (E2) acts through the estrogen receptor α (ERα) to stimulate breast cancer proliferation. Here, we investigated the functional relationship between ERα and signal transducer and activator of transcription (STAT)5b activity in ER+ MCF-7 and T47D human breast cancer cells after specific knockdown of STAT5b. STAT5b small interfering RNA (siRNA) inhibited E2-induced bromodeoxyuridine (BrdU) incorporation in both cell lines, as well as the E2-induced increase in MCF-7 cell number, cyclin D1 and c-myc mRNA, and cyclin D1 protein expression, indicating that STAT5b is required for E2-stimulated breast cancer proliferation. E2 treatment stimulated STAT5b tyrosine phosphorylation at the activating tyrosine Y699, resulting in increased STAT5-mediated transcriptional activity, which was inhibited by a Y669F STAT5b mutant. E2-induced STAT5-mediated transcriptional activity was inhibited by overexpressing a kinase-defective epidermal growth factor receptor (EGFR), or the EGFR tyrosine kinase inhibitor tyrphostin AG1478, indicating a requirement for EGFR kinase activity. Both E2-induced STAT5b tyrosine phosphorylation and STAT5-mediated transcription were also inhibited by the ER antagonist ICI 182,780 and the c-Src inhibitor PP2, indicating additional requirements for the ER and c-Src kinase activity. EGFR and c-Src kinase activities were also required for E2-induced cyclin D1 and c-myc mRNA. Together, these studies demonstrate positive cross talk between ER, c-Src, EGFR, and STAT5b in ER+ breast cancer cells. Increased EGFR and c-Src signaling is associated with tamoxifen resistance in ER+ breast cancer cells. Here we show that constitutively active STAT5b not only increased basal DNA synthesis, but also conferred tamoxifen resistance. Because STAT5b plays an integral role in E2-stimulated proliferation and tamoxifen resistance, it may be an effective therapeutic target in ER+ breast tumors.