Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis

DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog). We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP) from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.

The dual orexin/hypocretin receptor antagonist, almorexant, in the ventral tegmental area attenuates ethanol self-administration

Recent studies have implicated the hypocretin/orexinergic system in reward-seeking behavior. Almorexant, a dual orexin/hypocretin R1 and R2 receptor antagonist, has proven effective in preclinical studies in promoting sleep in animal models and was in Phase III clinical trials for sleep disorders. The present study combines behavioral assays with in vitro biochemical and electrophysiological techniques to elucidate the role of almorexant in ethanol and sucrose intake. Using an operant self-administration paradigm, we demonstrate that systemic administration of almorexant decreased operant selfadministration of both 20% ethanol and 5% sucrose. We further demonstrate that intraventral tegmental area (VTA) infusions, but not intra substantia nigra infusions, of almorexant reduced ethanol self-administration. Extracellular recordings performed in VTA neurons revealed that orexin-A increased firing and this enhancement of firing was blocked by almorexant. The results demonstrate that orexin/hypocretin receptors in distinct brain regions regulate ethanol and sucrose mediated behaviors.

Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment. © 2012 Sieh et al.

Is diurnal temperature range a risk factor for childhood diarrhea?

Background Previous studies have found that high and cold temperatures increase the risk of childhood diarrhea. However, little is known about whether the within-day variation of temperature has any effect on childhood diarrhea. Methods A Poisson generalized linear regression model combined with a distributed lag non-linear model was used to examine the relationship between diurnal temperature range and emergency department admissions for diarrhea among children under five years in Brisbane, from 1st January 2003 to 31st December 2009. Results There was a statistically significant relationship between diurnal temperature range and childhood diarrhea. The effect of diurnal temperature range on childhood diarrhea was the greatest at one day lag, with a 3% (95% confidence interval: 2%–5%) increase of emergency department admissions per 1°C increment of diurnal temperature range. Conclusion Within-day variation of temperature appeared to be a risk factor for childhood diarrhea. The incidence of childhood diarrhea may increase if climate variability increases as predicted.

Maintaining a cognitive map in darkness : the need to fuse boundary knowledge with path integration

Spatial navigation requires the processing of complex, disparate and often ambiguous sensory data. The neurocomputations underpinning this vital ability remain poorly understood. Controversy remains as to whether multimodal sensory information must be combined into a unified representation, consistent with Tolman's "cognitive map", or whether differential activation of independent navigation modules suffice to explain observed navigation behaviour. Here we demonstrate that key neural correlates of spatial navigation in darkness cannot be explained if the path integration system acted independently of boundary (landmark) information. In vivo recordings demonstrate that the rodent head direction (HD) system becomes unstable within three minutes without vision. In contrast, rodents maintain stable place fields and grid fields for over half an hour without vision. Using a simple HD error model, we show analytically that idiothetic path integration (iPI) alone cannot be used to maintain any stable place representation beyond two to three minutes. We then use a measure of place stability based on information theoretic principles to prove that featureless boundaries alone cannot be used to improve localization above chance level. Having shown that neither iPI nor boundaries alone are sufficient, we then address the question of whether their combination is sufficient and - we conjecture - necessary to maintain place stability for prolonged periods without vision. We addressed this question in simulations and robot experiments using a navigation model comprising of a particle filter and boundary map. The model replicates published experimental results on place field and grid field stability without vision, and makes testable predictions including place field splitting and grid field rescaling if the true arena geometry differs from the acquired boundary map. We discuss our findings in light of current theories of animal navigation and neuronal computation, and elaborate on their implications and significance for the design, analysis and interpretation of experiments.

The effect of Tenofovir on vitamin D metabolism in HIV-infected adults is dependent on sex and ethnicity

Background: Tenofovir has been associated with renal phosphate wasting, reduced bone mineral density, and higher parathyroid hormone levels. The aim of this study was to carry out a detailed comparison of the effects of tenofovir versus non-tenofovir use on calcium, phosphate and, vitamin D, parathyroid hormone (PTH), and bone mineral density. Methods: A cohort study of 56 HIV-1 infected adults at a single centre in the UK on stable antiretroviral regimes comparing biochemical and bone mineral density parameters between patients receiving either tenofovir or another nucleoside reverse transcriptase inhibitor. Principal Findings: In the unadjusted analysis, there was no significant difference between the two groups in PTH levels (tenofovir mean 5.9 pmol/L, 95% confidence intervals 5.0 to 6.8, versus non-tenofovir; 5.9, 4.9 to 6.9; p = 0.98). Patients on tenofovir had significantly reduced urinary calcium excretion (median 3.01 mmol/24 hours) compared to non-tenofovir users (4.56; p,0.0001). Stratification of the analysis by age and ethnicity revealed that non-white men but not women, on tenofovir had higher PTH levels than non-white men not on tenofovir (mean difference 3.1 pmol/L, 95% CI 5.3 to 0.9; p = 0.007). Those patients with optimal 25-hydroxyvitamin D (.75 nmol/L) on tenofovir had higher 1,25-dihydroxyvitamin D [1,25(OH)2D] (median 48 pg/mL versus 31; p = 0.012), fractional excretion of phosphate (median 26.1%, versus 14.6;p = 0.025) and lower serum phosphate (median 0.79 mmol/L versus 1.02; p = 0.040) than those not taking tenofovir. Conclusions: The effects of tenofovir on PTH levels were modified by sex and ethnicity in this cohort. Vitamin D status also modified the effects of tenofovir on serum concentrations of 1,25(OH)2D and phosphate.

Miscarriage, preterm delivery, and stillbirth: Large variations in rates within a cohort of Australian women

Objectives We aimed to use simple clinical questions to group women and provide their specific rates of miscarriage, preterm delivery, and stillbirth for reference. Further, our purpose was to describe who has experienced particularly low or high rates of each event. Methods Data were collected as part of the Australian Longitudinal Study on Women's Health, a national prospective cohort. Reproductive histories were obtained from 5806 women aged 31–36 years in 2009, who had self-reported an outcome for one or more pregnancy. Age at first birth, number of live births, smoking status, fertility problems, use of in vitro fertilisation (IVF), education and physical activity were the variables that best separated women into groups for calculating the rates of miscarriage, preterm delivery, and stillbirth. Results Women reported 10,247 live births, 2544 miscarriages, 1113 preterm deliveries, and 113 stillbirths. Miscarriage was correlated with stillbirth (r = 0.09, P<0.001). The calculable rate of miscarriage ranged from 11.3 to 86.5 miscarriages per 100 live births. Women who had high rates of miscarriage typically had fewer live births, were more likely to smoke and were more likely to have tried unsuccessfully to conceive for ≥12 months. The highest proportion of live preterm delivery (32.2%) occurred in women who had one live birth, had tried unsuccessfully to conceive for ≥12 months, had used IVF, and had 12 years education or equivalent. Women aged 14–19.99 years at their first birth and reported low physical activity had 38.9 stillbirths per 1000 live births, compared to the lowest rate at 5.5 per 1000 live births. Conclusion Different groups of women experience vastly different rates of each adverse pregnancy event. We have used simple questions and established reference data that will stratify women into low- and high-rate groups, which may be useful in counselling those who have experienced miscarriage, preterm delivery, or stillbirth, plus women with fertility intent.

Ca2+ regulates the Drosophila Stoned-A and Stoned-B proteins interaction with the C2B domain of Synaptotagmin-1.

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the µ-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca(2+). The Ca(2+)-dependent interaction between the µ-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca(2+) binding loop region that modulate the Ca(2+)-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca(2+)-binding loop region of C2B domain. The results indicate that Ca(2+)-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca(2+)-bound Synaptotagmin-1 associated synaptic vesicles.

Real-time measurement of F-Actin remodelling during exocytosis using lifeact-EGFP transgenic animals

F-actin remodelling is essential for a wide variety of cell processes. It is important in exocytosis, where F-actin coats fusing exocytic granules. The purpose of these F-actin coats is unknown. They may be important in stabilizing the fused granules, they may play a contractile role and promote expulsion of granule content and finally may be important in endocytosis. To elucidate these functions of F-actin remodelling requires a reliable method to visualize F-actin dynamics in living cells. The recent development of Lifeact-EGFP transgenic animals offers such an opportunity. Here, we studied the characteristics of exocytosis in pancreatic acinar cells obtained from the Lifeact-EGFP transgenic mice. We show that the time-course of agonist-evoked exocytic events and the kinetics of each single exocytic event are the same for wild type and Lifeact-EGFP transgenic animals. We conclude that Lifeact-EGFP animals are a good model to study of exocytosis and reveal that F-actin coating is dependent on the de novo synthesis of F-actin and that development of actin polymerization occurs simultaneously in all regions of the granule. Our insights using the Lifeact-EGFP mice demonstrate that F-actin coating occurs after granule fusion and is a granule-wide event.

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) slows cancer development but increases metastases in TRAMP prostate cancer prone mice

Macrophage inhibitory cytokine-1 (MIC-1/GDF15), a divergent member of the TGF-β superfamily, is over-expressed by many common cancers including those of the prostate (PCa) and its expression is linked to cancer outcome. We have evaluated the effect of MIC-1/GDF15 overexpression on PCa development and spread in the TRAMP transgenic model of spontaneous prostate cancer. TRAMP mice were crossed with MIC-1/GDF15 overexpressing mice (MIC-1fms) to produce syngeneic TRAMPfmsmic-1 mice. Survival rate, prostate tumor size, histopathological grades and extent of distant organ metastases were compared. Metastasis of TC1-T5, an androgen independent TRAMP cell line that lacks MIC-1/GDF15 expression, was compared by injecting intravenously into MIC-1fms and syngeneic C57BL/6 mice. Whilst TRAMPfmsmic-1 survived on average 7.4 weeks longer, had significantly smaller genitourinary (GU) tumors and lower PCa histopathological grades than TRAMP mice, more of these mice developed distant organ metastases. Additionally, a higher number of TC1-T5 lung tumor colonies were observed in MIC-1fms mice than syngeneic WT C57BL/6 mice. Our studies strongly suggest that MIC-1/GDF15 has complex actions on tumor behavior: it limits local tumor growth but may with advancing disease, promote metastases. As MIC-1/GDF15 is induced by all cancer treatments and metastasis is the major cause of cancer treatment failure and cancer deaths, these results, if applicable to humans, may have a direct impact on patient care.

Surveillance of dengue fever virus: a review of epidemiological models and early warning systems

Dengue fever is one of the world’s most important vector-borne diseases. The transmission area of this disease continues to expand due to many factors including urban sprawl, increased travel and global warming. Current preventative techniques are primarily based on controlling mosquito vectors as other prophylactic measures, such as a tetravalent vaccine are unlikely to be available in the foreseeable future. However, the continually increasing dengue incidence suggests that this strategy alone is not sufficient. Epidemiological models attempt to predict future outbreaks using information on the risk factors of the disease. Through a systematic literature review, this paper aims at analyzing the different modeling methods and their outputs in terms of accurately predicting disease outbreaks. We found that many previous studies have not sufficiently accounted for the spatio-temporal features of the disease in the modeling process. Yet with advances in technology, the ability to incorporate such information as well as the socio-environmental aspect allowed for its use as an early warning system, albeit limited geographically to a local scale.

Muscle, skin and core temperature after −110°C cold air and 8°C water treatment

The aim of this investigation was to elucidate the reductions in muscle, skin and core temperature following exposure to −110°C whole body cryotherapy (WBC), and compare these to 8°C cold water immersion (CWI). Twenty active male subjects were randomly assigned to a 4-min exposure of WBC or CWI. A minimum of 7 days later subjects were exposed to the other treatment. Muscle temperature in the right vastus lateralis (n = 10); thigh skin (average, maximum and minimum) and rectal temperature (n = 10) were recorded before and 60 min after treatment. The greatest reduction (P<0.05) in muscle (mean ± SD; 1 cm: WBC, 1.6±1.2°C; CWI, 2.0±1.0°C; 2 cm: WBC, 1.2±0.7°C; CWI, 1.7±0.9°C; 3 cm: WBC, 1.6±0.6°C; CWI, 1.7±0.5°C) and rectal temperature (WBC, 0.3±0.2°C; CWI, 0.4±0.2°C) were observed 60 min after treatment. The largest reductions in average (WBC, 12.1±1.0°C; CWI, 8.4±0.7°C), minimum (WBC, 13.2±1.4°C; CWI, 8.7±0.7°C) and maximum (WBC, 8.8±2.0°C; CWI, 7.2±1.9°C) skin temperature occurred immediately after both CWI and WBC (P<0.05). Skin temperature was significantly lower (P<0.05) immediately after WBC compared to CWI. The present study demonstrates that a single WBC exposure decreases muscle and core temperature to a similar level of those experienced after CWI. Although both treatments significantly reduced skin temperature, WBC elicited a greater decrease compared to CWI. These data may provide information to clinicians and researchers attempting to optimise WBC and CWI protocols in a clinical or sporting setting.

Malnutrition in a sample of community-dwelling people with Parkinson’s disease

Objective: Malnutrition results in poor health outcomes, and people with Parkinson’s disease may be more at risk of malnutrition. However, the prevalence of malnutrition in Parkinson’s disease is not yet well defined. The aim of this study is to provide an estimate of the extent of malnutrition in community-dwelling people with Parkinson’s disease. Methods: This is a cross-sectional study of people with Parkinson’s disease residing within a 2 hour driving radius of Brisbane, Australia. The Subjective Global Assessment (SGA) and scored Patient Generated Subjective Global Assessment (PG-SGA) were used to assess nutritional status. Body weight, standing or knee height, mid-arm circumference and waist circumference were measured. Results: Nineteen (15%) of the participants were moderately malnourished (SGA-B). The median PG-SGA score of the SGA-B group was 8 (4 – 15), significantly higher than the SGA-A group, U=1860.5,p<.05. The symptoms most influencing intake were loss of appetite, constipation, early satiety and problems swallowing. Conclusions: As with other populations, malnutrition remains under-recognised and undiagnosed in people with Parkinson’s disease. Regular screening of nutritional status in people with Parkinson’s disease by health professionals with whom they have regular contact should occur to identify those who may benefit from further nutrition assessment and intervention.

Productivity and time use during occupational therapy and nutrition/dietetics clinical education : a cohort study

Background: Currently in the Australian higher education sector higher productivity from allied health clinical education placements are a contested issue. This paper will report results of a study that investigated output changes associated with occupational therapy and nutrition/dietetics clinical education placements in Queensland, Australia. Supervisors’ and students’ time use during placements and how this changes for supervisors compared to when students are not present in the workplace is also presented. Methodology/Principal Findings: A cohort design was used with students from four Queensland universities, and their supervisors employed by Queensland Health. There was an increasing trend in the number of occasions of service delivered when the students were present, and a statistically significant increase in the daily mean length of occasions of service delivered during the placement compared to pre-placement levels. For project-based placements that were not directly involved in patient care, supervisors’ project activity time decreased during placements, with students undertaking considerably more time in project activities. Conclusions/Significance: A novel method for estimating productivity and time use changes during clinical education programs for allied health disciplines has been applied. During clinical education placements there was a net increase in outputs, suggesting supervisors engage in longer consultations with patients for the purpose of training students, while maintaining patient numbers. Other activities are reduced. This paper is the first time these data have been shown and form a good basis for future assessments of the economic impact of student placements for allied health disciplines.

Stroma regulates increased epithelial lateral cell adhesion in 3D culture : a role for actin/cadherin dynamics

BACKGROUND: Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures. METHODOLOGY/PRINCIPAL FINDINGS: The prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability. CONCLUSIONS/SIGNIFICANCE: In 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.