Cationic lipids enhance siRNA-mediated interferon response in mice

RNA interference mediated by small interfering RNAs (siRNAs) shows promise as a powerful research tool for gene function studies. However, controversy exists over the potential of siRNA-induced interferon response in vitro and in vivo. In this study, we showed that although intravenous administration of siRNA alone is essentially inert, injection of siRNA complexed with cationic liposomes resulted in a potent induction of both type I and type II interferon responses. Furthermore, i.v. administration of cationic lipid/siRNA complexes led to activation of STAT1. This study suggests caution in data interpretation and the potential toxicity with in vivo use of siRNA, particularly when delivered via a cationic lipid vector. This study also suggests the potential of siRNA as an immunostimulatory agent for immunotherapy.     

CaMKII tethers to L-type Ca2+ channels, establishing a local and dedicated integrator of Ca2+ signals for facilitation

Ca2+-dependent facilitation (CDF) of voltage-gated calcium current is a powerful mechanism for up-regulation of Ca2+ influx during repeated membrane depolarization. CDF of L-type Ca2+ channels (Ca(v)1.2) contributes to the positive force-frequency effect in the heart and is believed to involve the activation of Ca2+/calmodulin-dependent kinase II (CaMKII). How CaMKII is activated and what its substrates are have not yet been determined. We show that the pore-forming subunit alpha(1C) (Ca(v)alpha1.2) is a CaMKII substrate and that CaMKII interaction with the COOH terminus of alpha1C is essential for CDF of L-type channels. Ca2+ influx triggers distinct features of CaMKII targeting and activity. After Ca2+-induced targeting to alpha1C, CaMKII becomes tightly tethered to the channel, even after calcium returns to normal levels. In contrast, activity of the tethered CaMKII remains fully Ca2+/CaM dependent, explaining its ability to operate as a calcium spike frequency detector. These findings clarify the molecular basis of CDF and demonstrate a novel enzymatic mechanism by which ion channel gating can be modulated by activity.     

Cell-surface protein disulfide isomerase is required for transnitrosation of metallothionein by S-nitroso-albumin in intact rat pulmonary vascular endothelial cells

S-nitrosation of the metal binding protein, metallothionein (MT) appears to be a critical link in affecting endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO)-induced changes in cytoplasmic and nuclear labile zinc, respectively. Although low molecular weight S-nitrosothiols also appear to affect this signaling system, less is known about the ability of extracellular protein nitrosothiols to transnitrosate MT. Accordingly, we synthesized fluorescently labeled S-nitroso-albumin (SNO-albumin, a major protein S-nitrosothiol in plasma) and determined, via confocal microscopy in fixed tissue, that it is transported into cultured rat pulmonary vascular endothelial cells in a temperature sensitive fashion. The cells were transfected with an expression vector that encodes human MT-IIa cDNA sandwiched between enhanced cyan (donor) and yellow (acceptor) fluorescent proteins (FRET-MT) that can detect conformational changes in MT through fluorescence resonance energy transfer (FRET). SNO-albumin and the membrane-permeant low molecular weight S-nitroso-l-cysteine ethyl ester (l-SNCEE) caused a conformational change in FRET-MT as ascertained by full spectral laser scanning confocal microscopy in live rat pulmonary vascular endothelial cells, a result which is consistent with transnitrosation of the reporter molecule. Transnitrosation of FRET-MT by SNO-albumin, but not l-SNCEE, was sensitive to antisense oligonucleotide-mediated inhibition of the expression of cell surface protein disulfide isomerase (csPDI). These results extend the original observations of Ramachandran et al. (Ramachandran N, Root P, Jiang XM, Hogg PJ, Mutus B. Proc Natl Acad Sci U S A 98: 9539-9544, 2001) and suggest that csPDI-mediated denitrosation helps to regulate the ability of the major plasma NO carrier (SNO-albumin) to transnitrosate endothelial cell molecular targets (e.g. MT).     

Mass-spectrometry based oxidative lipidomics and lipid imaging: applications in traumatic brain injury

Lipids, particularly phospholipids, are fundamental to CNS tissue architecture and function. Endogenous polyunsaturated fatty acid chains of phospholipids possess cis-double bonds each separated by one methylene group. These phospholipids are very susceptible to free-radical attack and oxidative modifications. A combination of analytical methods including different versions of chromatography and mass spectrometry allows detailed information to be obtained on the content and distribution of lipids and their oxidation products thus constituting the newly emerging field of oxidative lipidomics. It is becoming evident that specific oxidative modifications of lipids are critical to a number of cellular functions, disease states and responses to oxidative stresses. Oxidative lipidomics is beginning to provide new mechanistic insights into traumatic brain injury which may have significant translational potential for development of therapies in acute CNS insults. In particular, selective oxidation of a mitochondria-specific phospholipid, cardiolipin, has been associated with the initiation and progression of apoptosis in injured neurons thus indicating new drug discovery targets. Furthermore, imaging mass-spectrometry represents an exciting new opportunity for correlating maps of lipid profiles and their oxidation products with structure and neuropathology. This review is focused on these most recent advancements in the field of lipidomics and oxidative lipidomics based on the applications of mass spectrometry and imaging mass spectrometry as they relate to studies of phospholipids in traumatic brain injury.     

Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances

The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 +/- 1.2 per thousand, 32.5 +/- 0.6 per thousand, and 35.6 +/- 1.4 per thousand for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 +/- 4.2 per thousand) was similar to that produced during hydroxylamine oxidation (33.5 +/- 1.2 per thousand) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 +/- 1.7 per thousand), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (-0.6 +/- 1.9 per thousand and -0.5 +/- 1.9 per thousand, respectively) were similar to those during nitrate reduction (-0.5 +/- 1.9 per thousand and -0.5 +/- 0.6 per thousand, respectively), indicating no influence of either substrate on site preference. Site preferences of approximately 33 per thousand and approximately 0 per thousand are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.     

Jelly-falls historic and recent observations: a review to drive future research directions

The biological pump describes the transport of particulate matter from the sea surface to the ocean’s interior including the seabed. The contribution by gelatinous zooplankton bodies as particulate organic matter (POM) vectors (“jelly-falls”) has been neglected owing to technical and spatiotemporal sampling limitations. Here, we assess the existing evidence on jelly-falls from early ocean observations to present times. The seasonality of jelly-falls indicates that they mostly occur after periods of strong upwelling and/or spring blooms in temperate/subpolar zones and during late spring/early summer. A conceptual model helps to define a jelly-fall based on empirical and field observations of biogeochemical and ecological processes. We then compile and discuss existing strategic and observational oceanographic techniques that could be implemented to further jelly-falls research. Seabed video- and photography-based studies deliver the best results, and the correct use of fishing techniques, such as trawling, could provide comprehensive regional datasets. We conclude by considering the possibility of increased gelatinous biomasses in the future ocean induced by upper ocean processes favouring their populations, thus increasing jelly-POM downward transport. We suggest that this could provide a “natural compensation” for predicted losses in pelagic POM with respect to fuelling benthic ecosystems.     

Biological and genetic characterisation of Phoma macrostoma isolates with bioherbicidal activity

The fungus Phoma macrostoma Mont. isolate 94-44B was registered as a bioherbicide for control of broadleaved weeds in Canada and the USA in 2011 and 2012, respectively. To obtain the registrations, the fungus had to be characterised both biologically and genetically. The objectives of this study were to demonstrate that bioherbicidal activity was associated with specific genetic markers and to determine whether bioherbicidal activity was a general trait of the species or only selected isolates. A collection of 64 isolates of P. macrostoma was established. A greenhouse bioassay and bioherbicidal-specific primers were used to determine bioherbicidal activity of all isolates. Only isolates originating from Canada thistle demonstrated the ability to reduce dandelion seedlings and display the 853 bp amplicon for the bioherbicidal-specific primer. Bioherbicidal isolates were consistently differentiated from all other isolates with two main genotypic groupings (I and II) arising from internal transcribed spacer (ITS) and amplified fragment length polymorphisms (AFLP) sequence analyses. Using AFLP, two biotypes of bioherbicidal isolates were also differentiated by the presence or absence of an AFLP marker at a single polymorphic locus. The genetic divergence among the bioherbicidal and nonbioherbicidal isolates of P. macrostoma was only 2.21% which was lower than that reported for other related Phoma sp. Other than the bioherbicidal trait, there was no apparent affiliation of the genetics with known varietal types, host or geographic origin. ITS sequence analysis and AFLP fingerprinting may be used as tools to detect bioherbicidal isolates of P. macrostoma.     

The missing wetlands: using local ecological knowledge to find cryptic ecosystems

Small, temporally dynamic, biologically diverse isolated wetlands are among the most imperiled ecosystems, yet their conservation is hindered by lack of protective legislation and mapping. As part of an effort to better understand isolated wetland ecology in an area undergoing dramatic land use change, we mapped isolated wetlands in South Carolina’s Piedmont and Blue Ridge regions using remote sensing and local ecological knowledge (LEK). Remote detection of isolated wetlands was limited by digital resource resolution, topography, and wetland size. LEK was the most useful tool for locating small isolated wetlands. We sampled 10% of the study area using LEK and discovered 44 wetlands with “isolated” characteristics, none of which had been identified by remote sensing. Only 8 of 44 wetlands found through LEK could be identified using remote sensing after their discovery. LEK fills a gap in cryptic ecosystem detection when adequate remotely sensed data are not available. Though effective, using LEK is neither as rapid nor as repeatable as remote sensing. We suggest a two-pronged approach for finding cryptic ecosystems: remote sensing coupled with LEK where data resolution is inadequate. For remote detection of isolated wetlands, we suggest a minimum resolution of 0.33 m for Color Infrared, leaf-off, high-water photography. Despite great advances in remote sensing, data are not uniformly available worldwide and LEK may serve as an effective tool for locating cryptic resources for biodiversity conservation. Mapping cryptic resources will allow for more accurate resource and biodiversity conservation planning under current and future climate scenarios.