ADAM17 Silencing in Mouse Colon Carcinoma Cells: The Effect on Tumoricidal Cytokines and Angiogenesis

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFNγ, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response.     

Impact of Vitamin D Supplementation during Lactation on Vitamin D Status and Body Composition of Mother-Infant Pairs: A MAVID Randomized Controlled Trial

Objective

The optimal vitamin D intake for nursing women is controversial. Deterioration, at least in bone mass, is reported during lactation. This study evaluated whether vitamin D supplementation during lactation enhances the maternal and infant’s vitamin D status, bone mass and body composition.

Design and Methods

After term delivery, 174 healthy mothers were randomized to receive 1200 IU/d (800 IU/d+400 IU/d from multivitamins) or 400 IU/d (placebo+400 IU/d from multivitamins) of cholecalciferol for 6 months while breastfeeding. All infants received 400 IU/d of cholecalciferol. Serum 25-hydroxyvitamin D [25(OH)D], iPTH, calcium, urinary calcium, and densitometry were performed in mother-offspring pairs after delivery, and at 3 and 6 months later.

Results

A total of 137 (79%) (n = 70; 1200 IU/d, n = 67; 400 IU/d) completed the study. 25(OH)D was similar in both groups at baseline (13.7 ng/ml vs. 16.1 ng/ml; P = 0.09) and at 3 months (25.7 ng/ml vs. 24.5 ng/ml; P = 0.09), but appeared higher in the 1200 IU/d group at 6 months of supplementation (25.6 ng/ml vs. 23.1 ng/ml; P = 0.009). The prevalence of 25(OH)D <20 ng/ml was comparable between groups at baseline (71% vs. 64%, P = 0.36) but lower in the 1200 IU/d group after 3 months (9% vs. 25%, P = 0.009) and 6 months (14% vs. 30%, P = 0.03). Maternal and infants’ iPTH, calciuria, bone mass and body composition as well as infants’ 25(OH)D levels were not significantly different between groups during the study. Significant negative correlations were noted between maternal 25(OH)D and fat mass (R = −0.49, P = 0.00001), android fat mass (R = −0.53, P = 0.00001), and gynoid fat mass (R = −0.43, P = 0.00001) after 6 months of supplementation.

Conclusions

Vitamin D supplementation at a dose of 400 IU/d was not sufficient to maintain 25(OH)D >20 ng/ml in nursing women, while 1200 IU/d appeared more effective, but had no effect on breastfed offspring vitamin D status, or changes in the bone mass and the body composition observed in both during breastfeeding.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01506557","term_id":"NCT01506557"}}NCT01506557     

Improving peptide relative quantification in MALDI‐TOF MS for biomarker assessment

Proteomic profiling by MALDI‐TOF MS presents various advantages (speed of analysis, ease of use, relatively low cost, sensitivity, tolerance against detergents and contaminants, and possibility of automation) and is being currently used in many applications (e.g. peptide/protein identification and quantification, biomarker discovery, and imaging MS). Earlier studies by many groups indicated that moderate reproducibility in relative peptide quantification is a major limitation of MALDI‐TOF MS. In the present work, we examined and demonstrate a clear effect, in cases apparently random, of sample dilution in complex samples (urine) on the relative quantification of peptides by MALDI‐TOF MS. Results indicate that in urine relative abundance of peptides cannot be assessed with confidence based on a single MALDI‐TOF MS spectrum. To account for this issue, we developed and propose a novel method of determining the relative abundance of peptides, taking into account that peptides have individual linear quantification ranges in relation to sample dilution. We developed an algorithm that calculates the range of dilutions at which each peptide responds in a linear manner and normalizes the received peptide intensity values accordingly. This concept was successfully applied to a set of urine samples from patients diagnosed with diabetes presenting normoalbuminuria (controls) and macroalbuminuria (cases).     

Epidemiology of a <Emphasis Type="Italic">Daphnia</Emphasis> brood parasite and its implications on host life-history traits

Parasites influence host life-history traits and therefore might crucially shape host populations in natural systems. In a series of laboratory experiments, we studied the impact of an oomycete brood parasite on its Daphnia (waterflea) host. We asked whether Daphnia dump the infected brood and subsequently are able to reproduce again as was occasionally observed in a preliminary study. No viable offspring developed from infected clutches, but 78% of the infected females produced healthy offspring after releasing the infected brood while molting. Neither those offsprings’ development success nor their mothers’ reproductive potential was affected by the brood parasite. However, infected Daphnia had a reduced life-span and suffered an increased susceptibility to another parasite, an unidentified bacterium. Additionally, we studied the prevalence of this brood parasite and the unidentified bacterium in a natural Daphnia assemblage in a pre-alpine lake, across changing demographic and environmental conditions. The brood parasite epidemic seemed to be host-density dependent. Our results show that the brood parasite’s impact on the host population is enhanced when combined with the unidentified bacterium.     

A relationship between Pseudomonal growth behaviour and cystic fibrosis patient lung function identified in a metabolomic investigation

Chronic polymicrobial lung infections in adult cystic fibrosis patients are typically dominated by high levels of Pseudomonas aeruginosa. Determining the impact of P. aeruginosa growth on airway secretion composition is fundamental to understanding both the behaviour of this pathogen in vivo, and its relationship with other potential colonising species. We hypothesised that the marked differences in the phenotypes of clinical isolates would be reflected in the metabolite composition of spent culture media. ¹H NMR spectroscopy was used to characterise the impact of P. aeruginosa growth on a synthetic medium as part of an in vitro CF lower airways model system. Comparisons of 15 CF clinical isolates were made and four distinct metabolomic clusters identified. Highly significant relationships between P. aeruginosa isolate cluster membership and both patient lung function (FEV₁) and spent culture pH were identified. This link between clinical isolate growth behaviour and FEV₁ indicates characterisation of P. aeruginosa growth may find application in predicting patient lung function while the significant divergence in metabolite production and consumption observed between CF clinical isolates suggests dominant isolate characteristics have the potential to play both a selective role in microbiota composition and influence pseudomonal behaviour in vivo.     

Inactivation of Glucocorticoid Receptor in Noradrenergic System Influences Anxiety- and Depressive-Like Behavior in Mice

The aim of this study was to investigate whether conditional inactivation of the glucocorticoid receptors (GRs) in noradrenergic neurons affects animal behavior in mice. Selective ablation of GRs in the noradrenergic system was achieved using the Cre/loxP approach. We crossed transgenic mice expressing the Cre recombinase under the dopamine beta-hydroxylase (DBH) promoter with animals harboring the floxed GR gene. The resulting GR^DBHCre mutant mice exhibited no alterations in terms of normal cage behavior, weight gain, spatial memory or spontaneous locomotor activity, regardless of gender. To assess depressive- and anxiety-like behaviors we performed the Tail Suspension Test and the Light-Dark Box Test. While male mutant animals did not show any alternations in both tests, female GR^DBHCre mutants displayed depressive- and anxiety-like behavior. Additionally, male GR^DBHCre mice were exposed to chronic restraint stress but still exhibited immobility times and anxiety statuses similar to those of non-stressed animals while stressed control mice clearly revealed depressive- and anxiety-like phenotype. Thus, in males the effects of the mutation were precipitated only after chronic restraint stress procedure. Our data reveal a possible gender-dependent role of GRs in the noradrenergic system in anxiety- and depressive-like behavior in mice.     

Influence of the separation of the charged groups and aromatic ring on interaction of tyrosine and phenylalanine analogues and derivatives with beta-cyclodextrin

Interactions of tyrosine and phenylalanine analogues with beta-cyclodextrin have been examined in terms of structural features of the ligand such as the separation of the charged amino group and aromatic ring, the presence of additional functional group attached to the amino or phenyl ring, and the presence of a charge on amino or carboxyl group, and steric effects using steady-state and time-resolved fluorescence spectroscopy and microcalorimetry. The studied aromatic amino acids possess low binding constant to beta-cyclodextrin, diversified with respect to the presence or absence of a substituent in para position of the phenyl ring. However, calculated, based on the global analysis of the fluorescence intensity decays, binding constants do not allow to estimate unequivocally the influence of the distance between the charged groups and phenol/phenyl ring on the inclusion complex stability because of their low diversification.     

Using spin-label electron paramagnetic resonance (EPR) to discriminate and characterize the cholesterol bilayer domain

Electron paramagnetic resonance (EPR) spin-labeling methods make it possible not only to discriminate the cholesterol bilayer domain (CBD) but also to obtain information about the organization and dynamics of cholesterol molecules in the CBD. The abilities of spin-label EPR were demonstrated for Chol/POPC (cholesterol/1-palmitoyl-2-oleoylphosphatidylcholine) membranes, with a Chol/POPC mixing ratio that changed from 0 to 3. Using the saturation-recovery (SR) EPR approach with cholesterol analogue spin labels, ASL and CSL, and oxygen or NiEDDA relaxation agents, it was confirmed that the CBD was present in all membrane suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST). Conventional EPR spectra of ASL and CSL in the CBD were similar to those in the surrounding POPC bilayer (which is saturated with cholesterol), indicating that in both domains, cholesterol exists in the lipid-bilayer-like structures. The behavior of ASL and CSL (and, thus, the behavior of cholesterol molecules) in the CBD was compared with that in the surrounding POPC-cholesterol domain (PCD). In the CBD, ASL and CSL molecules are better ordered than in the surrounding PCD. This difference is small and can be compared to that induced in the surrounding domain by an ∼10 °C decrease in temperature. Thus, cholesterol molecules are unexpectedly dynamic in the CBD, which should enhance their interaction with the surrounding domain. The polarity of the water/membrane interface of the CBD is significantly greater than that of the surrounding PCD, which significantly enhances penetration of the water-soluble relaxation agent, NiEDDA, into that region. Hydrophobicity measured in the centers of both domains is similar. The oxygen transport parameter (oxygen diffusion-concentration product) measured in the center of the CBD is about ten times smaller than that measured in the center of the surrounding domain. Thus, the CBD can form a significant barrier to oxygen transport. The results presented here point out similarities between the organization and dynamics of cholesterol molecules in the CBD and in the surrounding PCD, as well as significant differences between CBDs and cholesterol crystals.     

Size Distribution of Air Bubbles Entering the Brain during Cardiac Surgery

Background

Thousands of air bubbles enter the cerebral circulation during cardiac surgery, but whether high numbers of bubbles explain post-operative cognitive decline is currently controversial. This study estimates the size distribution of air bubbles and volume of air entering the cerebral arteries intra-operatively based on analysis of transcranial Doppler ultrasound data.

Methods

Transcranial Doppler ultrasound recordings from ten patients undergoing heart surgery were analysed for the presence of embolic signals. The backscattered intensity of each embolic signal was modelled based on ultrasound scattering theory to provide an estimate of bubble diameter. The impact of showers of bubbles on cerebral blood-flow was then investigated using patient-specific Monte-Carlo simulations to model the accumulation and clearance of bubbles within a model vasculature.

Results

Analysis of Doppler ultrasound recordings revealed a minimum of 371 and maximum of 6476 bubbles entering the middle cerebral artery territories during surgery. This was estimated to correspond to a total volume of air ranging between 0.003 and 0.12 mL. Based on analysis of a total of 18667 embolic signals, the median diameter of bubbles entering the cerebral arteries was 33 μm (IQR: 18 to 69 μm). Although bubble diameters ranged from ~5 μm to 3.5 mm, the majority (85%) were less than 100 μm. Numerous small bubbles detected during cardiopulmonary bypass were estimated by Monte-Carlo simulation to be benign. However, during weaning from bypass, showers containing large macro-bubbles were observed, which were estimated to transiently affect up to 2.2% of arterioles.

Conclusions

Detailed analysis of Doppler ultrasound data can be used to provide an estimate of bubble diameter, total volume of air, and the likely impact of embolic showers on cerebral blood flow. Although bubbles are alarmingly numerous during surgery, our simulations suggest that the majority of bubbles are too small to be harmful.