The metalloproteinase ADAM‐12 regulates bronchial epithelial cell proliferation and apoptosis

Abstract. Objectives: The ADAMs (a disintegrin and metalloproteinase) enzymes compose a family of membrane‐bound proteins characterized by their multi‐domain structure and ADAM‐12 expression is elevated in human non‐small cell lung cancers. The aim of this study was to investigate the roles played by ADAM‐12 in critical steps of bronchial cell transformation during carcinogenesis. Materials and methods: To assess the role of ADAM‐12 in tumorigenicity, BEAS‐2B cells were transfected with a plasmid encoding human full‐length ADAM‐12 cDNA, and then the effects of ADAM‐12 overexpression on cell behaviour were explored. Treatment of clones with heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) neutralizing antibodies as well as an EGFR inhibitor allowed the dissection of mechanisms regulating cell proliferation and apoptosis. Results: Overexpression of ADAM‐12 in BEAS‐2B cells promoted cell proliferation. ADAM‐12 overexpressing clones produced higher quantities of HB‐EGF in their culture medium which may rely on membrane‐bound HB‐EGF shedding by ADAM‐12. Targeting HB‐EGF activity with a neutralizing antibody abrogated enhanced cell proliferation in the ADAM‐12 overexpressing clones. In sharp contrast, targeting of amphiregulin, EGF or transforming growth factor‐α failed to influence cell proliferation; moreover, ADAM‐12 transfectants were resistant to etoposide‐induced apoptosis and the use of a neutralizing antibody against HB‐EGF activity restored rates of apoptosis to be similar to controls.Conclusions: ADAM‐12 contributes to enhancing HB‐EGF shedding from plasma membranes leading to increased cell proliferation and reduced apoptosis in this bronchial epithelial cell line.     

Residue Histidine 50 Plays a Key Role in Protecting α-Synuclein from Aggregation at Physiological pH

α-Synuclein (αSyn) aggregation is involved in the pathogenesis of Parkinson disease (PD). Recently, substitution of histidine 50 in αSyn with a glutamine, H50Q, was identified as a new familial PD mutant. Here, nuclear magnetic resonance (NMR) studies revealed that the H50Q substitution causes an increase of the flexibility of the C-terminal region. This finding provides direct evidence that this PD-causing mutant can mediate long range effects on the sampling of αSyn conformations. In vitro aggregation assays showed that substitution of His-50 with Gln, Asp, or Ala promotes αSyn aggregation, whereas substitution with the positively charged Arg suppresses αSyn aggregation. Histidine carries a partial positive charge at neutral pH, and so our result suggests that positively charged His-50 plays a role in protecting αSyn from aggregation under physiological conditions.     

Antlions (Neuroptera,Myrmeleontidae) from ornithological traps on the Curonian Spit: A three-species community containing a new species

A mixed antlion community is recorded at the “Fringilla” Research Station, “Rybachii” Biological Station, Curonian Spit, Baltic Sea, Kaliningrad Province, Russia; the adults were captured using ornithological traps and the larvae were found on sand dunes around. The ratio of the larval numbers in the mixed colonies of Myrmeleon tschernovi sp. n., Myrmeleon formicarius L., and Euroleon nostras (Geoffr.) is 100: 3: 2. The new species is described, the other two are recorded in Kaliningrad Province for the first time. Morphologically, Myrmeleon tschernovi sp. n. is similar to Myrmeleon bore (Tjed.), being its neighbor in the Baltic Region and occupying its econiche. The most characteristic distinctions between these species are found in the male genitalia and in the larval head chaetotaxy and color pattern.     

Identifying a Kinase Network Regulating FGF14:Nav1.6 Complex Assembly Using Split-Luciferase Complementation

Kinases play fundamental roles in the brain. Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons. Changes induced by kinases on neuronal excitability, synaptic plasticity and brain connectivity are linked to complex brain disorders, but the molecular mechanisms underlying these cellular events remain for the most part elusive. To further our understanding of brain disease, new methods for rapidly surveying kinase pathways in the cellular context are needed. The bioluminescence-based luciferase complementation assay (LCA) is a powerful, versatile toolkit for the exploration of PPI. LCA relies on the complementation of two firefly luciferase protein fragments that are functionally reconstituted into the full luciferase enzyme by two interacting binding partners. Here, we applied LCA in live cells to assay 12 kinase pathways as regulators of the PPI complex formed by the voltage-gated sodium channel, Nav1.6, a transmembrane ion channel that elicits the action potential in neurons and mediates synaptic transmission, and its multivalent accessory protein, the fibroblast growth factor 14 (FGF14). Through extensive dose-dependent validations of structurally-diverse kinase inhibitors and hierarchical clustering, we identified the PI3K/Akt pathway, the cell-cycle regulator Wee1 kinase, and protein kinase C (PKC) as prospective regulatory nodes of neuronal excitability through modulation of the FGF14:Nav1.6 complex. Ingenuity Pathway Analysis shows convergence of these pathways on glycogen synthase kinase 3 (GSK3) and functional assays demonstrate that inhibition of GSK3 impairs excitability of hippocampal neurons. This combined approach provides a versatile toolkit for rapidly surveying PPI signaling, allowing the discovery of new modular pathways centered on GSK3 that might be the basis for functional alterations between the normal and diseased brain.     

Winter Conditions and Land Cover Structure the Subnivium,A Seasonal Refuge beneath the Snow

In seasonally snow-covered environments, many organisms endure winter by using the subnivium, a below-snow thermally stable seasonal refugium. Because the insulation of snow is dependent on snow depth and density, the stability of temperatures within the subnivium varies across land cover types. Additionally, across much of the Northern Hemisphere snow extent, depth and duration are generally decreasing while snow density is increasing due to climate change. These changes are likely to destabilize the thermal profile of the subnivium, although they have not yet been quantified. To explore the effects of land cover and climate change on the subnivium, we measured snow pack characteristics (depth and density), and ambient and subnivium temperatures from three different land cover types (prairie, deciduous forest, and coniferous forest) and within a micro-greenhouse (2.5 x 2.5 x 2 m) that maintained a temperature of 5°C warmer than outdoor ambient temperatures, and automatically opened during snow events throughout the winter of 2013/14. We found that the mean daily subnivium temperature was significantly colder in the deciduous cover type than the prairie cover type, and that prairie had higher maximum subnivium temperatures than both of the other cover types. Our climate change simulation revealed that, although ambient temperatures within the micro-greenhouse were 5°C warmer than outside the greenhouse, the daily minimum subnivium temperature was significantly lower inside the greenhouse. Our findings suggest that climate change could have considerable effects on the refuge quality of the subnivium, and that some cover types appear to be more susceptible to these effects than others.     

Highly Invasive Listeria monocytogenes Strains Have Growth and Invasion Advantages in Strain Competition

Multiple Listeria monocytogenes strains can be present in the same food sample; moreover, infection with more than one L. monocytogenes strain can also occur. In this study we investigated the impact of strain competition on the growth and in vitro virulence potential of L. monocytogenes. We identified two strong competitor strains, whose growth was not (or only slightly) influenced by the presence of other strains and two weak competitor strains, which were outcompeted by other strains. Cell contact was essential for growth inhibition. In vitro virulence assays using human intestinal epithelial Caco2 cells showed a correlation between the invasion efficiency and growth inhibition: the strong growth competitor strains showed high invasiveness. Moreover, invasion efficiency of the highly invasive strain was further increased in certain combinations by the presence of a low invasive strain. In all tested combinations, the less invasive strain was outcompeted by the higher invasive strain. Studying the effect of cell contact on in vitro virulence competition revealed a complex pattern in which the observed effects depended only partially on cell-contact suggesting that competition occurs at two different levels: i) during co-cultivation prior to infection, which might influence the expression of virulence factors, and ii) during infection, when bacterial cells compete for the host cell. In conclusion, we show that growth of L. monocytogenes can be inhibited by strains of the same species leading potentially to biased recovery during enrichment procedures. Furthermore, the presence of more than one L. monocytogenes strain in food can lead to increased infection rates due to synergistic effects on the virulence potential.     

Modulation of the Leptin Receptor Mediates Tumor Growth and Migration of Pancreatic Cancer Cells

Obesity has been implicated as a significant risk factor for development of pancreatic cancer. In the setting of obesity, a systemic chronic inflammatory response is characterized by alterations in the production and secretion of a wide variety of growth factors. Leptin is a hormone whose level increases drastically in the serum of obese patients. High fat diet induced obesity in mice leads to an overall increased body weight, pancreatic weight, serum leptin, and pancreatic tissue leptin levels. Here we report the contribution of obesity and leptin to pancreatic cancer growth utilizing an in vivo orthotopic murine pancreatic cancer model, which resulted in increased tumor proliferation with concomitant increased tumor burden in the diet induced obese mice compared to lean mice. Human and murine pancreatic cancer cell lines were found to express the short as well as the long form of the leptin receptor and functionally responded to leptin induced activation through an increased phosphorylation of AKT473. In vitro, leptin stimulation increased cellular migration which was blocked by addition of a PI3K inhibitor. In vivo, depletion of the leptin receptor through shRNA knockdown partially abrogated increased orthotopic tumor growth in obese mice. These findings suggest that leptin contributes to pancreatic tumor growth through activation of the PI3K/AKT pathway, which promotes pancreatic tumor cell migration.     

Real-Time Visualization of Joint Cavitation

Cracking sounds emitted from human synovial joints have been attributed historically to the sudden collapse of a cavitation bubble formed as articular surfaces are separated. Unfortunately, bubble collapse as the source of joint cracking is inconsistent with many physical phenomena that define the joint cracking phenomenon. Here we present direct evidence from real-time magnetic resonance imaging that the mechanism of joint cracking is related to cavity formation rather than bubble collapse. In this study, ten metacarpophalangeal joints were studied by inserting the finger of interest into a flexible tube tightened around a length of cable used to provide long-axis traction. Before and after traction, static 3D T1-weighted magnetic resonance images were acquired. During traction, rapid cine magnetic resonance images were obtained from the joint midline at a rate of 3.2 frames per second until the cracking event occurred. As traction forces increased, real-time cine magnetic resonance imaging demonstrated rapid cavity inception at the time of joint separation and sound production after which the resulting cavity remained visible. Our results offer direct experimental evidence that joint cracking is associated with cavity inception rather than collapse of a pre-existing bubble. These observations are consistent with tribonucleation, a known process where opposing surfaces resist separation until a critical point where they then separate rapidly creating sustained gas cavities. Observed previously in vitro, this is the first in-vivo macroscopic demonstration of tribonucleation and as such, provides a new theoretical framework to investigate health outcomes associated with joint cracking.     

Rapid Screening of Gene Function by Systemic Delivery of Morpholino Oligonucleotides to Live Mouse Embryos

Traditional gene targeting methods in mice are complex and time consuming, especially when conditional deletion methods are required. Here, we describe a novel technique for assessing gene function by injection of modified antisense morpholino oligonucleotides (MOs) into the heart of mid-gestation mouse embryos. After allowing MOs to circulate through the embryonic vasculature, target tissues were explanted, cultured and analysed for expression of key markers. We established proof-of-principle by partially phenocopying known gene knockout phenotypes in the fetal gonads (Stra8, Sox9) and pancreas (Sox9). We also generated a novel double knockdown of Gli1 and Gli2, revealing defects in Leydig cell differentiation in the fetal testis. Finally, we gained insight into the roles of Adamts19 and Ctrb1, genes of unknown function in sex determination and gonadal development. These studies reveal the utility of this method as a means of first-pass analysis of gene function during organogenesis before committing to detailed genetic analysis.     

Testing the Role of Climate Change in Species Decline: Is the Eastern Quoll a Victim of a Change in the Weather?

To conserve a declining species we first need to diagnose the causes of decline. This is one of the most challenging tasks faced by conservation practitioners. In this study, we used temporally explicit species distribution models (SDMs) to test whether shifting weather can explain the recent decline of a marsupial carnivore, the eastern quoll (Dasyurus viverrinus). We developed an SDM using weather variables matched to occurrence records of the eastern quoll over the last 60 years, and used the model to reconstruct variation through time in the distribution of climatically suitable range for the species. The weather model produced a meaningful prediction of the known distribution of the species. Abundance of quolls, indexed by transect counts, was positively related to the modelled area of suitable habitat between 1990 and 2004. In particular, a sharp decline in abundance from 2001 to 2003 coincided with a sustained period of unsuitable weather over much of the species’ distribution. Since 2004, abundance has not recovered despite a return to suitable weather conditions, and abundance and area of suitable habitat have been uncorrelated. We suggest that fluctuations in weather account for the species’ recent decline, but other unrelated factors have suppressed recovery.