Dam Methylation Participates in the Regulation of PmrA/PmrB and RcsC/RcsD/RcsB Two Component Regulatory Systems in Salmonella enterica Serovar Enteritidis

The absence of Dam in Salmonella enterica serovar Enteritidis causes a defect in lipopolysaccharide (LPS) pattern associated to a reduced expression of wzz gene. Wzz is the chain length regulator of the LPS O-antigen. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. Thus, the expression of rcsB and pmrA was monitored by quantitative real-time RT-PCR and Western blotting using fusions with 3×FLAG tag in wild type (wt) and dam strains of S. Enteritidis. Dam regulated the expression of both rcsB and pmrA genes; nevertheless, the defect in LPS pattern was only related to a diminished expression of RcsB. Interestingly, regulation of wzz in serovar Enteritidis differed from that reported earlier for serovar Typhimurium; RcsB induces wzz expression in both serovars, whereas PmrA induces wzz in S. Typhimurium but represses it in serovar Enteritidis. Moreover, we found that in S. Enteritidis there is an interaction between both wzz regulators: RcsB stimulates the expression of pmrA and PmrA represses the expression of rcsB. Our results would be an example of differential regulation of orthologous genes expression, providing differences in phenotypic traits between closely related bacterial serovars.

Advanced glycation endproduct modified basement membrane attenuates endothelin-1 induced [Ca2+]i signalling and contraction in retinal microvascular pericytes

PURPOSE: To assess the effects of advanced glycation endproduct (AGE) modification of vascular basement membrane (BM) on endothelin-1 (Et-1) induced intracellular [Ca2+] ([Ca2+]i) homeostasis and contraction in retinal microvascular pericytes (RMP). METHODS: RMPs were isolated from bovine retinal capillaries and propagated on AGE modified BM extract (AGE-BM) or non-modified native BM. Cytosolic Ca2+ was estimated using fura-2 microfluorimetry and cellular contraction determined by measurement of planimetric cell surface area. ETA receptor mRNA and protein expression was assessed by real time RT-PCR and western blotting, respectively. RESULTS: Exogenous endothelin-1 (Et-1) evoked rises in [Ca2+]i and contraction in RMPs were found to be mediated entirely through ETA receptor (ETAR) activation. Both peak and plateau phases of the Et-1 induced [Ca2+]i response and contraction were impaired in RMPs propagated on AGE modified BM. ETAR mRNA expression remained unchanged in RMPs exposed to native or AGE-BM, but protein expression for ETAR (66 kDa) was lower in the AGE exposed cells. CONCLUSIONS: These results suggest that substrate derived AGE crosslinks can influence RMP physiology by mechanisms which include disruption of ETA receptor signalling. AGE modification of vascular BMs may contribute to the retinal hemodynamic abnormalities observed during diabetes.

Human dental pulp fibroblasts express the "cold-sensing" transient receptor potential channels TRPA1 and TRPM8.

Abstract
INTRODUCTION:
Transient receptor potential (TRP) channels comprise a group of nonselective calcium-permeable cationic channels, which are polymodal sensors of environmental stimuli such as thermal changes and chemicals. TRPM8 and TRPA1 are cold-sensing TRP channels activated by moderate cooling and noxious cold temperatures, respectively. Both receptors have been identified in trigeminal ganglion neurones, and their expression in nonneuronal cells is now the focus of much interest. The aim of this study was to investigate the molecular and functional expression of TRPA1 and TRPM8 in dental pulp fibroblasts.
METHODS:
Human dental pulp fibroblasts were derived from healthy molar teeth. Gene and protein expression was determined by polymerase chain reaction and Western blotting. Cellular localization was investigated by immunohistochemistry, and TRP functionality was determined by Ca(2+) microfluorimetry.
RESULTS:
Polymerase chain reaction and Western blotting showed gene and protein expression of both TRPA1 and TRPM8 in fibroblast cells in culture. Immunohistochemistry studies showed that TRPA1 and TRPM8 immunoreactivity co-localized with the human fibroblast surface protein. In Ca(2+) microfluorimetry studies designed to determine the functionality of TRPA1 and TRPM8 in pulp fibroblasts, we showed increased intracellular calcium ([Ca(2+)](i)) in response to the TRPM8 agonist menthol, the TRPA1 agonist cinnamaldehyde, and to cool and noxious cold stimuli, respectively. The responses to agonists and thermal stimuli were blocked in the presence of specific TRPA1 and TRPM8 antagonists.
CONCLUSIONS:
Human dental pulp fibroblasts express TRPA1 and TRPM8 at the molecular, protein, and functional levels, indicating a possible role for fibroblasts in mediating cold responses in human teeth.

Heterogeneity assumptions in the specification of bargaining models: a study of household level trade-offs between commuting time and salary

With many real world decisions being made in conjunction with other decision makers, or single agent decisions having an influence on other members of the decision maker's immediate entourage, there is strong interest in studying the relative weight assigned to different agents in such contexts. In the present paper, we focus on the case of one member of a two person household being asked to make choices affecting the travel time and salary of both members. We highlight the presence of significant heterogeneity across individuals not just in their underlying sensitivities, but also in the relative weight they assign to their partner, and show how this weight varies across attributes. This is in contrast to existing work which uses weights assigned to individual agents at the level of the overall utility rather than for individual attributes. We also show clear evidence of a risk of confounding between heterogeneity in marginal sensitivities and heterogeneity in the weights assigned to each member. We show how this can lead to misleading model results, and argue that this may also explain past results showing bargaining or weight parameters outside the usual [0,1] range in more traditional joint decision making contexts. In terms of substantive results, we find that male respondents place more weight on their partner's travel time, while female respondents place more weight on their partner's salary.

Rapid magmatic processes accompany arc-continent collision: the Western Bismarck arc, Papua New Guinea

New U-Th-Ra, major and trace element, and Sr-Nd-Pb isotope data are presented for young lavas from the New Britain and Western Bismarck arcs in Papua New Guinea. New Britain is an oceanic arc, whereas the latter is the site of an arc-continent collision. Building on a recent study of the Manus Basin, contrasts between the two arcs are used to evaluate the processes and timescales of magma generation accompanying arc-continent collision and possible slab detachment. All three suites share many attributes characteristic of arc lavas that can be ascribed to the addition of a regionally uniform subduction component derived from the subducting altered oceanic crust and sediment followed by dynamic melting of the modified mantle. However, the Western Bismarck arc lavas diverge from the Pb isotope mixing array formed by the New Britain and the Manus Basin lavas toward elevated Pb-208/Pb-204. We interpret this to reflect a second and subsequent addition of sediment melt at crustal depth during collision. U-238 and Ra-226 excesses are preserved in all of the lavas and are greatest in the Western Bismarck arc. High-Mg andesites with high Sr/Y ratios in the westernmost arc are attributed to recent shallow mantle flux melting at the slab edge. Data for two historical rhyolites are also presented. Although these rhyolites formed in quite different tectonic settings and display different geochemical and isotopic compositions, both formed from mafic parents within millennia.

KIF2A silencing inhibits the proliferation and migration of breast cancer cells and correlates with unfavorable prognosis in breast cancer

Background: Kinesin family member 2a (KIF2A), a type of motor protein found in eukaryotic cells, is associated with development and progression of various human cancers. The role of KIF2A during breast cancer tumorigenesis and progression was studied.

Methods: Immunohistochemical staining, real time RT-PCR and western blot were used to examine the expression of KIF2A in cancer tissues and adjacent normal tissues from breast cancer patients. Patients' survival in relation to KIF2A expression was estimated using the Kaplan-Meier survival and multivariate analysis. Breast cancer cell line, MDA-MB-231 was used to study the proliferation, migration and invasion of cells following KIF2A-siRNA transfection.

Results: The expression of KIF2A in cancer tissues was higher than that in normal adjacent tissues from the same patient (P <0.05). KIF2A expression in cancer tissue with lymph node metastasis and HER2 positive cancer were higher than that in cancer tissue without (P <0.05). A negative correlation was found between KIF2A expression levels in breast cancer and the survival time of breast cancer patients (P <0.05). In addition, multivariate analysis indicated that KIF2A was an independent prognostic for outcome in breast cancer (OR: 16.55, 95% CI: 2.216-123.631, P = 0.006). The proliferation, migration and invasion of cancer cells in vitro were suppressed by KIF2A gene silencing (P <0.05).

Conclusions: KIF2A may play an important role in breast cancer progression and is potentially a novel predictive and prognostic marker for breast cancer.

European Economic and Social Constitutionalism after the Treaty of Lisbon

European studies frequently regard the economic and social dimensions of EU integration as diametrically opposed, maintaining that this state of affairs is beyond change. This edited collection challenges this perceived wisdom, focusing on the post-Lisbon constitutional landscape. Taking the multi-layered polity that is Europe today as its central organising theme, it examines how the social and the economic might be reconciled under the Union's different forms of governance. The collection has a clear structure, opening with a theoretical appraisal of its theme, before considering three specific policy fields: migration policy and civic integration, company law and corporate social responsibility and the role of third sector providers in public healthcare. It concludes with three case studies in these fields, illustrating how the argument can be practically applied. Insightful and topical, with a unique interdisciplinary perspective, this is an important contribution to European Union law after the Lisbon Treaty

The Distribution and Adoption of the Byre-house (Longhouse) in Late Medieval Britain

It has long been recognized that the byre-house or longhouse, in which animals and humans lived in the same building and with direct contact, was a distinctive building plan. Earlier interpretations have seen it as a ubiquitous house type found throughout Britain, but gradually replaced by separate buildings for keeping animals and accommodating humans. More recent work has suggested that it was a regional variant of the common late medieval domestic plan. The use of this building type was restricted to parts of Wales, and northern and western areas of England. It is argued that the introduction of the byre-house occurs mainly in the thirteenth century as part of a wider trend to provide accommodation for livestock during the winter months. The byre-house was a one response to this need, and its adoption was not due to climatic or geographical factors. Instead, it is interpreted as reflecting localized cultural attitudes to the relationship between humans and animals.

Inflammatory and cytotoxic effects of acrolein, nicotine, acetylaldehyde and cigarette smoke extract on human nasal epithelial cells

Background: Cigarette smoke induces a pro-inflammatory response in airway epithelial cells but it is not clear which of the various chemicals contained within cigarette smoke (CS) should be regarded as predominantly responsible for these effects. We hypothesised that acrolein, nicotine and acetylaldehyde, important chemicals contained within volatile cigarette smoke in terms of inducing inflammation and causing addiction, have immunomodulatory effects in primary nasal epithelial cell cultures (PNECs).

Methods: PNECs from 19 healthy subjects were grown in submerged cultures and were incubated with acrolein, nicotine or acetylaldehyde prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide (PA LPS). Experiments were repeated using cigarette smoke extract (CSE) for comparison. IL-8 was measured by ELISA, activation of NF-κB by ELISA and Western blotting, and caspase-3 activity by Western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method.

Results: CSE was pro-inflammatory after a 24 h exposure and 42% of cells were apoptotic or necrotic after this exposure time. Acrolein was pro-inflammatory for the PNEC cultures (30 μM exposure for 4 h inducing a 2.0 fold increase in IL-8 release) and also increased IL-8 release after stimulation with PA LPS. In contrast, nicotine had anti-inflammatory properties (0.6 fold IL-8 release after 50 μM exposure to nicotine for 24 h), and acetylaldehyde was without effect. Acrolein and nicotine had cellular stimulatory and anti-inflammatory effects respectively, as determined by NF-κB activation. Both chemicals increased levels of cleaved caspase 3 and induced cell death.

Conclusions: Acrolein is pro-inflammatory and nicotine anti-inflammatory in PNEC cultures. CSE induces cell death predominantly by apoptotic mechanisms. 

Abnormal alterations in the Ca2+/CaV1.2/calmodulin/caMKII signaling pathway in a tremor rat model and in cultured hippocampal neurons exposed to Mg2+-free solution

Voltage-dependent calcium channels (VDCCs) are key elements in epileptogenesis. There are several binding-sites linked to calmodulin (CaM) and several potential CaM-dependent protein kinase II (CaMKII)-mediated phosphorylation sites in CaV1.2. The tremor rat model (TRM) exhibits absence‑like seizures from 8 weeks of age. The present study was performed to detect changes in the Ca2+/CaV1.2/CaM/CaMKII pathway in TRMs and in cultured hippocampal neurons exposed to Mg2+‑free solution. The expression levels of CaV1.2, CaM and phosphorylated CaMKII (p‑CaMKII; Thr‑286) in these two models were examined using immunofluorescence and western blotting. Compared with Wistar rats, the expression levels of CaV1.2 and CaM were increased, and the expression of p‑CaMKII was decreased in the TRM hippocampus. However, the expression of the targeted proteins was reversed in the TRM temporal cortex. A significant increase in the expression of CaM and decrease in the expression of CaV1.2 were observed in the TRM cerebellum. In the cultured neuron model, p‑CaMKII and CaV1.2 were markedly decreased. In addition, neurons exhibiting co‑localized expression of CaV1.2 and CaM immunoreactivities were detected. Furthermore, intracellular calcium concentrations were increased in these two models. For the first time, o the best of our knowledge, the data of the present study suggested that abnormal alterations in the Ca2+/CaV1.2/CaM/CaMKII pathway may be involved in epileptogenesis and in the phenotypes of TRMs and cultured hippocampal neurons exposed to Mg2+‑free solution.

LYRIC/AEG-1 is targeted to different subcellular compartments by ubiquitinylation and intrinsic nuclear localization signals

PURPOSE: LYRIC/AEG-1 has been reported to influence breast cancer survival and metastases, and its altered expression has been found in a number of cancers. The cellular function of LYRIC/AEG-1 has previously been related to its subcellular distribution in cell lines. LYRIC/AEG-1 contains three uncharacterized nuclear localization signals (NLS), which may regulate its distribution and, ultimately, function in cells.

EXPERIMENTAL DESIGN: Immunohistochemistry of a human prostate tissue microarray composed of 179 prostate cancer and 24 benign samples was used to assess LYRIC/AEG-1 distribution. Green fluorescent protein-NLS fusion proteins and deletion constructs were used to show the ability of LYRIC/AEG-1 NLS to target green fluorescent protein from the cytoplasm to the nucleus. Immunoprecipitation and Western blotting were used to show posttranslational modification of LYRIC/AEG-1 NLS regions.

RESULTS: Using a prostate tissue microarray, significant changes in the distribution of LYRIC/AEG-1 were observed in prostate cancer as an increased cytoplasmic distribution in tumors compared with benign tissue. These differences were most marked in high grade and aggressive prostate cancers and were associated with decreased survival. The COOH-terminal extended NLS-3 (amino acids 546-582) is the predominant regulator of nuclear localization, whereas extended NLS-1 (amino acids 78-130) regulates its nucleolar localization. Within the extended NLS-2 region (amino acids 415-486), LYRIC/AEG-1 can be modified by ubiquitin almost exclusively within the cytoplasm.

CONCLUSIONS: Changes in LYRIC/AEG-1 subcellular distribution can predict Gleason grade and survival. Two lysine-rich regions (NLS-1 and NLS-3) can target LYRIC/AEG-1 to subcellular compartments whereas NLS-2 is modified by ubiquitin in the cytoplasm.

Survival for oesophageal, stomach and small intestine cancers in Europe 1999-2007: Results from EUROCARE-5

Background: European regional variation in cancer survival was reported in the EUROCARE-4 study for patients diagnosed in 1995-1999. Relative survival (RS) estimates are here updated for patients diagnosed with cancer of the oesophagus, stomach and small intestine from 2000 to 2007. Trends in RS from 1999-2001 to 2005-2007 are presented to monitor and discuss improvements in patient survival in Europe. Materials and methods: EUROCARE-5 data from 29 countries (87 cancer registries) were used to investigate 1- and 5-year RS. Using registry-specific life-tables stratified by age, gender and calendar year, age-standardised 'complete analysis' RS estimates by country and region were calculated for Northern, Southern, Eastern and Central Europe, and for Ireland and United Kingdom (UK). Survival trends of patients in periods 1999-2001, 2002-2004 and 2005-2007 were investigated using the 'period' RS approach. We computed the 5-year RS conditional on surviving the first year (5-year conditional survival), as the ratio of age-standardised 5-year RS to 1-year RS. Results Oesophageal cancer 1- and 5-year RS (40% and 12%, respectively) remained poor in Europe. Patient survival was worst in Eastern (8%), Northern (11%) and Southern Europe (10%). Europe-wide, there was a 3% improvement in oesophageal cancer 5-year survival by 2005-2007, with Ireland and the UK (3%), and Central Europe (4%) showing large improvements. Europe-wide, stomach cancer 5-year RS was 25%. Ireland and UK (17%) and Eastern Europe (19%) had the poorest 5-year patient survival. Southern Europe had the best 5-year survival (30%), though only showing an improvement of 2% by 2005-2007. Small intestine cancer 5-year RS for Europe was 48%, with Central Europe having the best (54%), and Ireland and UK the poorest (37%). Five-year patient survival improvement for Europe was 8% by 2005-2007, with Central, Southern and Eastern Europe showing the greatest increases (≥9%). Conclusions Survival for these cancer sites, particularly oesophageal cancer, remains poor in Europe with wide variation. Further investigation into the wide variation, including analysis by histology and anatomical sub-site, will yield insights to better monitor and explain the improvements in survival observed over time.

Biometric gonioscopy and the effects of age, race, and sex on the anterior chamber angle.

AIM:

To utilise a novel method for making measurements in the anterior chamber in order to compare the anterior chamber angles of people of European, African, and east Asian descent aged 40 years and over.

METHODS:

A cross sectional study on 15 people of each sex from each decade from the 40s to the 70s, from each of three racial groups-black, white, and Chinese Singaporeans. Biometric gonioscopy (BG) utilises a slit lamp mounted reticule to make measurements from the apparent iris insertion to Schwalbe's line through a Goldmann one mirror goniolens. The main outcome measures were BG measurements of the anterior chamber angle as detailed above.

RESULTS:

There was no significant difference in angle measurement between black, white, and Chinese races in this study. However, at younger ages people of Chinese race appeared to have deeper angles than white or black people, whereas the angles of older Chinese were significantly narrower (p = 0.004 for the difference in slope of BG by age between Chinese and both black and white people).

CONCLUSION:

The failure to detect a difference in angle measurements between these groups was surprising, given the much higher prevalence of angle closure among Chinese. It appears that the overall apparent similarity of BG means between Chinese and Western populations may mask very different trends with age. The apparently more rapid decline in angle width measurements with age among Chinese may be due to the higher prevalence of cataract or "creeping angle closure." However, longitudinal inferences from cross sectional data are problematic, and this may represent a cohort phenomenon caused by the increasing prevalence of myopia in the younger Singaporean population.

Prevalence of cataract and pseudophakia/aphakia among adults in the United States.

OBJECTIVES:

To determine the prevalence of cataract and pseudophakia/aphakia in the United States and to project the expected change in these prevalence figures by 2020.

METHODS:

Summary prevalence estimates of cataract and of pseudophakia/aphakia were prepared separately for black, white, and Hispanic persons (for whom only cataract surgery data were available) in 5-year age intervals starting at 40 years for women and men. The estimates were based on a standardized definition of various types of cataract: cortical, greater than 25% of the lens involved; posterior subcapsular, present according to the grading system used in each study; and nuclear, greater than or equal to the penultimate grade in the system used. Data were collected from major population-based studies in the United States, and, where appropriate, Australia, Barbados, and Western Europe. The age-, gender-, and race/ethnicity-specific rates were applied to 2000 US Census data, and projected population figures for 2020, to obtain overall estimates.

RESULTS:

An estimated 20.5 million (17.2%) Americans older than 40 years have cataract in either eye, and 6.1 million (5.1%) have pseudophakia/aphakia. Women have a significantly (odds ratio = 1.37; 95% confidence interval, 1.26-1.50) higher age-adjusted prevalence of cataract than men in the United States. The total number of persons who have cataract is estimated to rise to 30.1 million by 2020; and for those who are expected to have pseudophakia/aphakia, to 9.5 million.

CONCLUSION:

The number of Americans affected by cataract and undergoing cataract surgery will dramatically increase over the next 20 years as the US population ages.