The Interaction of Large Amplitude Internal Seiches with a Shallow Sloping Lakebed: Observations of Benthic Turbulence in Lake Simcoe,Ontario, Canada

Observations of the interactions of large amplitude internal seiches with the sloping boundary of Lake Simcoe, Canada show a pronounced asymmetry between up- and downwelling. Data were obtained during a 42-day period in late summer with an ADCP and an array of four thermistor chains located in a 5 km line at the depths where the thermocline intersects the shallow slope of the lakebed. The thermocline is located at depths of 12–14 m during the strongly stratified period of late summer. During periods of strong westerly winds the thermocline is deflected as much as 8 m vertically and interacts directly with the lakebed at depth between 14–18 m. When the thermocline was rising at the boundary, the stratification resembles a turbulent bore that propagates up the sloping lakebed with a speed of 0.05–0.15 m s⁻¹ and a Froude number close to unity. There were strong temperature overturns associated with the abrupt changes in temperature across the bore. Based on the size of overturns in the near bed stratification, we show that the inferred turbulent diffusivity varies by up to two orders of magnitude between up- and downwellings. When the thermocline was rising, estimates of turbulent diffusivity were high with K_Z ∼10⁻⁴ m²s⁻¹, whereas during downwelling events the near-bed stratification was greatly increased and the turbulence was reduced. This asymmetry is consistent with previous field observations and underlines the importance of shear-induced convection in benthic bottom boundary layers of stratified lakes.     

Extracellular Alkaline pH Leads to Increased Metastatic Potential of Estrogen Receptor Silenced Endocrine Resistant Breast Cancer Cells

Introduction

Endocrine resistance in breast cancer is associated with enhanced metastatic potential and poor clinical outcome, presenting a significant therapeutic challenge. We have established several endocrine insensitive breast cancer lines by shRNA induced depletion of estrogen receptor (ER) by transfection of MCF-7 cells which all exhibit enhanced expression profile of mesenchymal markers with reduction of epithelial markers, indicating an epithelial to mesenchymal transition. In this study we describe their behaviour in response to change in extracellular pH, an important factor controlling cell motility and metastasis.

Methods

Morphological changes associated with cell exposure to extracellular alkaline pH were assessed by live cell microscopy and the effect of various ion pumps on this behavior was investigated by pretreatment with chemical inhibitors. The activity and expression profile of key signaling molecules was assessed by western blotting. Cell motility and invasion were examined by scratch and under-agarose assays respectively. Total matrix metalloproteinase (MMP) activity and specifically of MMP2/9 was assessed in conditioned medium in response to brief alkaline pH exposure.

Results

Exposure of ER –ve but not ER +ve breast cancer cells to extracellular alkaline pH resulted in cell shrinkage and spherical appearance (termed contractolation); this was reversed by returning the pH back to 7.4. Contractolation was blocked by targeting the Na⁺/K⁺ and Na⁺/H⁺ pumps with specific chemical inhibitors. The activity and expression profile of key signaling molecules critical for cell adhesion were modulated by the exposure to alkaline pH. Brief exposure to alkaline pH enhanced MMP2/9 activity and the invasive potential of ER –ve cells in response to serum components and epithelial growth factor stimulation without affecting unhindered motility.

Conclusions

Endocrine resistant breast cancer cells behave very differently to estrogen responsive cells in alkaline pH, with enhanced invasive potential; these studies emphasise the crucial influence of extracellular pH and caution against indiscriminate application of alkalinising drug therapy.     

Genetic Diversity in Exon 2 of the Major Histocompatibility Complex Class II DQB1 Locus in Nigerian Goats

The DQB1 locus is located in the major histocompatibility complex (MHC) class II region and involved in immune response. We identified 20 polymorphic sites in a 228 bp fragment of exon 2, one of the most critical regions of the MHC DQB1 gene, in 60 Nigerian goats. Four sites are located in the peptide binding region, and 10 amino acid substitutions are peculiar to Nigerian goats, compared with published sequences. A significantly higher ratio of nonsynonymous/synonymous substitutions (d _N/d _S) suggests that allelic sequence evolution is driven by balancing selection (P < 0.01). In silico functional analysis using PANTHER predicted that substitution P56R, with a subPSEC score of ?4.00629 (P_deleterious = 0.73229), is harmful to protein function. The phylogenetic tree from consensus sequences placed the two northern breeds closer to each other than either was to the southern goats. This first report of sequence diversity at the DQB1 locus for any African goat breed may be useful in the search for disease-resistant genotypes.     

Transforming Growth Factor-β1 (TGF-β1)-stimulated Fibroblast to Myofibroblast Differentiation Is Mediated by Hyaluronan (HA)-facilitated Epidermal Growth Factor Receptor (EGFR) and CD44 Co-localization in Lipid Rafts

Fibroblast to myofibroblast differentiation drives effective wound healing and is largely regulated by the cytokine transforming growth factor-β1 (TGF-β1). Myofibroblasts express α-smooth muscle actin and are present in granulation tissue, where they are responsible for wound contraction. Our previous studies show that fibroblast differentiation in response to TGF-β1 is dependent on and mediated by the linear polysaccharide hyaluronan (HA). Both the HA receptor, CD44, and the epidermal growth factor receptor (EGFR) are involved in this differentiation response. The aim of this study was to understand the mechanisms linking HA-, CD44-, and EGFR-regulated TGF-β1-dependent differentiation. CD44 and EGFR co-localization within membrane-bound lipid rafts was necessary for differentiation, and this triggered downstream mitogen-activated protein kinase (MAPK/ERK) and Ca²⁺/calmodulin kinase II (CaMKII) activation. We also found that ERK phosphorylation was upstream of CaMKII phosphorylation, that ERK activation was necessary for CaMKII signaling, and that both kinases were essential for differentiation. In addition, HA synthase-2 (HAS2) siRNA attenuated both ERK and CaMKII signaling and sequestration of CD44 into lipid rafts, preventing differentiation. In summary, the data suggest that HAS2-dependent production of HA facilitates TGF-β1-dependent fibroblast differentiation through promoting CD44 interaction with EGFR held within membrane-bound lipid rafts. This induces MAPK/ERK, followed by CaMKII activation, leading to differentiation. This pathway is synergistic with the classical TGF-β1-dependent SMAD-signaling pathway and may provide a novel opportunity for intervention in wound healing.     

Patterns in the abundance of kangaroo populations in arid Australia are consistent with the exploitation ecosystems hypothesis

The exploitation ecosystems hypothesis (EEH) makes predictions about trophic interactions along gradients of primary productivity. The EEH has been shown to apply to a wide range of terrestrial environments but its applicability to arid environments has received little attention. One reason for this is that arid environments may not satisfy the assumptions of the EEH because dearth of water may limit biological activity in both temporal and spatial contexts. The EEH predicts that herbivore biomass should increase linearly with primary productivity in the absence of predators; but when predators are present herbivore biomass will remain relatively constant due to top down regulation. We tested this prediction in an arid environment using rainfall as a proxy of primary productivity and an index of the abundance of the dominant herbivores (kangaroos Macropus spp.). We compared an index of kangaroo abundance at 18 areas situated along a gradient of mean annual rainfall in areas where a top predator (the dingo Canis lupus dingo) was rare and common. We also explored the relationship between the density of artificial water points (AWPs) and kangaroo abundance to investigate if the resource subsidy provided by AWPs allows kangaroos to persist in high numbers. Consistent with the EEH, kangaroo abundance showed a weak relationship with mean annual rainfall in the presence of dingoes but increased with increasing annual rainfall in the absence of dingoes. The density of AWPs was a poor predictor of kangaroo abundance. Our analysis of macro‐ecological patterns suggests that kangaroo populations are primarily top down regulated in the presence of dingoes, but are bottom up regulated in the absence of dingoes. Our findings provide evidence that top down regulation can prevail over bottom up regulation of herbivore populations in arid ecosystems and highlights the usefulness of the EEH as a predictor of macro‐ecological patterns of species abundance.     

Oxidative Stress, NF-κB and the Ubiquitin Proteasomal Pathway in the Pathology of Calpainopathy

The neuromuscular disorder, calpainopathy (LGMD 2A), is a major muscular dystrophy classified under limb girdle muscular dystrophies. Genetic mutations of the enzyme calpain 3 cause LGMD 2A. Calpainopathy is phenotypically observed as progressive muscle wasting and weakness. Pathomechanisms of muscle wasting of calpainopathy remain poorly understood. Oxidative stress, NF-κB and the ubiquitin proteasomal pathway underlie the pathology of several muscle wasting conditions but their role in calpainopathic dystrophy is not known. Oxidative and nitrosative stress, the source of reactive oxygen species, NF-κB signaling and protein ubiquitinylation were studied in 15 calpainopathic and 8 healthy control human muscle biopsies. Oxidative stress and NF-κB/IKK β signaling were increased in calpainopathic muscle and may contribute to increased protein ubiquitinylation and muscle protein loss. Preventing oxidative stress or inhibition of NF-κB signaling could be considered for treatment of LGMD 2A.     

Lipophilic Drug Transfer Between Liposomal and Biological Membranes: What Does It Mean for Parenteral and Oral Drug Delivery?

This review presents the current knowledge on the interaction of lipophilic, poorly water soluble drugs with liposomal and biological membranes. The center of attention will be on drugs having the potential to dissolve in a lipid membrane without perturbing them too much. The degree of interaction is described as solubility of a drug in phospholipid membranes and the kinetics of transfer of a lipophilic drug between membranes. Finally, the consequences of these two factors on the design of lipid-based carriers for oral, as well as parenteral use, for lipophilic drugs and lead selection of oral lipophilic drugs is described. Since liposomes serve as model-membranes for natural membranes, the assessment of lipid solubility and transfer kinetics of lipophilic drug using liposome formulations may additionally have predictive value for bioavailability and biodistribution and the pharmacokinetics of lipophilic drugs after parenteral as well as oral administration.     

2B4+ CD8+ T cells play an inhibitory role against constrained HIV epitopes

Cytotoxic T cells play a critical role in the control of HIV and the progression of infected individuals to AIDS. 2B4 (CD244) is a member of the SLAM family of receptors that regulate lymphocyte development and function. The expression of 2B4 on CD8⁺ T cells was shown to increase during AIDS disease progression. However, the functional role of 2B4⁺ CD8⁺ T cells against HIV infection is not known. Here, we have examined the functional role of 2B4⁺ CD8⁺ T cells during and after stimulation with HLA B14 or B27 restricted HIV epitopes. Interestingly, IFN-γ secretion and cytotoxic activity of 2B4⁺ CD8⁺ T cells stimulated with HIV peptides were significantly decreased when compared to influenza peptide stimulated 2B4⁺ CD8⁺ T cells. The expression of the signaling adaptor molecule SAP was downregulated in 2B4⁺ CD8⁺ T cells upon HIV peptide stimulation. These results suggest that 2B4⁺ CD8⁺ T cells play an inhibitory role against constrained HIV epitopes underlying the inability to control the virus during disease progression.