A novel KCNQ4 pore-region mutation (p.G296S) causes deafness by impairing cell-surface channel expression

Mutations in the potassium channel gene KCNQ4 underlie DFNA2, a subtype of autosomal dominant progressive, high-frequency hearing loss. Based on a phenotype-guided mutational screening we have identified a novel mutation c.886G>A, leading to the p.G296S substitution in the pore region of KCNQ4 channel. The possible impact of this mutation on total KCNQ4 protein expression, relative surface expression and channel function was investigated. When the G296S mutant was expressed in Xenopus oocytes, electrophysiological recordings did not show voltage-activated K⁺ currents. The p.G296S mutation impaired KCNQ4 channel activity in two manners. It greatly reduced surface expression and, secondarily, abolished channel function. The deficient expression at the cell surface membrane was further confirmed in non-permeabilized NIH-3T3 cells transfected with the mutant KCNQ4 tagged with the hemagglutinin epitope in the extracellular S1–S2 linker. Co-expression of mutant and wild type KCNQ4 in oocytes was performed to mimic the heterozygous condition of the p.G296S mutation in the patients. The results showed that the G296S mutant exerts a strong dominant-negative effect on potassium currents by reducing the wild type KCNQ4 channel expression at the cell surface. This is the first study to identify a trafficking-dependent dominant mechanism for the loss of KCNQ4 channel function in DFNA2.     

Photoprotective responses of Mediterranean and Atlantic trees to the extreme heat-wave of summer 2003 in Southwestern Europe

Summer 2003 was extremely hot in Europe. High light in combination with heat and drought exacerbates the generation of photo-oxidative stress. Under these conditions photoprotective responses can be critical for plant survival. Photoprotection was analysed in 2003 in several Mediterranean and Atlantic woody species. These data were compared with previous summers (1998, 1999 and 2001) to evaluate the potential acclimation for each species. A pattern of changes consisting on a decrease in chlorophyll, ascorbate and Fv/Fm and an increase in tocopherol, xanthophyll cycle pigments (VAZ) and de-epoxidation index was regularly observed. Acclimation potential was measured by the use of the plasticity index for each parameter. Mediterranean species were more plastic than Atlantic ones. The latter were unable to increase antioxidant pools to the same extent or to down-regulate the efficiency of light energy conversion. These results indicate that most Mediterranean species are able to perform an efficient acclimation to heat stress, whilst Atlantic species will be more affected by climate warming.     

Biofilm development by potentially pathogenic non-pigmented rapidly growing mycobacteria

Background  

A study to evaluate the biofilm-development ability in three different media (Middlebrook 7H9, sterile tap water and PBS-5% glucose) was performed with 19 collection strains from 15 different species on non-pigmented rapidly growing mycobacteria (NPRGM). A microtiter plate assay was developed to evaluate the percentage of covered surface of the microtiter plate wells in different days from day 1 to day 69.     

Identification of proteins secreted from leptin stimulated MCF-7 breast cancer cells: a dual proteomic approach

Leptin is an adipocyte-derived hormone that regulates energy expenditure and food intake. A significant role for leptin in breast cancer has also been indicated by the resistance of leptin knockout mice in development of mammary tumors. In vitro, leptin induces proliferation of MCF-7 cells by activating cellular signaling pathways (1, 11, 12, 16, 17, 56). As leptin is emerging as an important factor for tumor growth, and hormones can exert their actions via autocrine/paracrine mechanisms, we hypothesized leptin may act by regulating epithelial-derived proteins. To test this hypothesis, leptin-regulated proteins secreted from MCF-7 mammary tumor cells were identified using proteomics techniques. Treatment of MCF-7 cells with 500 ng/ml leptin for 24 hours resulted in a 40% increase in cell number and a 5-fold increase in protein secretion as compared to controls. Establishing the significance of leptin-induced secreted factors, the addition of conditioned media from leptin-treated MCF-7 cells to synchronized MCF-7 cells resulted in 40% increase in cell number. Identification of leptin-regulated secreted proteins was done by 2D gel electrophoresis coupled with MALDI-TOF mass spectrometry. Proteins identified using Pro Found software and NCBI database included KF10 Collagen Precursor, Serologically Defined Breast Cancer Antigen NY-BR-62 and Cortactin Isoform a. A Human Cytokine Antibody Array system was used to identify low abundant proteins in the media of control and 500 ng/ml leptin-stimulated MCF-7 cells. In leptin treated cells, levels of FGF-9 were increased while IGFBP-3 and TGF-beta3 levels were decreased. Many previous studies have focused on the regulation of distinct cellular proteins by leptin during mammary tumor cell proliferation. However, ours is the first study to identify leptin-regulated secreted proteins, many of which are known to play important roles in cancer. Our data support that leptin can influence mammary tumor growth and progression through regulation of autocrine/paracrine factors and by modulating the extracellular matrix composition.     

Efflux of hydraulically lifted water from mycorrhizal fungal hyphae during imposed drought

Apart from improving plant and soil water status during drought, it has been suggested that hydraulic lift (HL) could enhance plant nutrient capture through the flow of mineral nutrients directly from the soil to plant roots, or by maintaining the functioning of mycorrhizal fungi. We evaluated the extent to which the diel cycle of water availability created by HL covaries with the efflux of HL water from the tips of extramatrical (external) mycorrhizal hyphae, and the possible effects on biogeochemical processes. Phenotypic mycorrhizal fungal variables, such as total and live hyphal lengths, were positively correlated with HL efflux from hyphae, soil water potential (dawn), and plant response variables (foliar ¹⁵N). The efflux of HL water from hyphae was also correlated with bacterial abundance and soil enzyme activity (P), and the moistening of soil organic matter. Such findings indicate that the efflux of HL water from the external mycorrhizal mycelia may be a complementary explanation for plant nutrient acquisition and survival during drought.Key words: hydraulic lift, nitrogen, phosphorus, microbial abundance, mycorrhizal hyphae, QuercusIn environments that experience seasonal or extended drought, plant productivity, resource partitioning, and competition are limited by the availability of water and mineral nutrients. One mechanism that is important to whole plant water balance in these environments is hydraulic lift (HL), a passive process driven by gradients in water potential among soils layers. Soil water is transported upwards from deep moist soils and released into the nutrient-rich upper soil layers by root systems accessing both deep and shallow soil layers.¹ HL water may improve the lifespan and activity of fine roots in a wide variety of plant life forms.²Hydraulic lift may also have a second ecological function in facilitating plant nutrient acquisition.² It been hypothesized that HL water could enhance the supply of nutrients to roots through mass flow or diffusion,³ or trigger episodes of soil biotic activity such as microbe-mediated nutrient transformations^4,5 that are analogous to the increased inflow of nitrogen (N) into roots and flushes of carbon (C) and N mineralization respectively that follow precipitation events.^4,6 However, few data currently exist with which to test these possibilities.Hydraulically lifted water also sustains mycorrhizal fungi,^7,8 a mutualism that enhances the acquisition of water and mineral nutrients in many terrestrial plant species. Mycorrhizal fungal hyphae provide comprehensive exploration and rapid access to small-scale or temporary nutrient flushes that may not be available to plant roots.⁹ This resource flow has often been assumed to be a unidirectional flux whereby resources are moved from source (soil) into the sink (plant) by the fungal hyphae. However, there is now evidence to suggest that the physiological plasticity of the peripheral extramatrical hyphae, and in particular the hyphal tips, permits the exudation, and subsequent reabsorption, of water and solutes.^10,11 Laboratory experiments using pure cultures have demonstrated that water may be exuded from the hyphal tips, especially in fungal species with hydrophobic hyphae, along with a variety of organic molecules, such as free amino acids.^10–13 At the same time, water, mobile minerals, amino acids and other low-molecular weight metabolites may be selectively and actively reabsorbed by mycorrhizal fungal hyphae.¹¹ However, quantitative data on the environmental impact of hyphal exudation and reabsorption is still largely lacking.We ask: could the diel cycle of water availability created by HL produce a water efflux from hyphal tips and if so, would this be sufficient to impact biogeochemical processes? Is there also an opposite rhythm driven by plant transpiration so that any resultant soil solution is pulled towards hyphal tips and consequently, the host plant? By imposing drought on seedlings of Quercus agrifolia Nee (coast live oak; Fagaceae) grown in mesocosms (Fig. 1), we identified a composite of feedbacks that could influence nutrient capture with HL (Fig. 2). Our analyses provide support for the key predictions of the HL-nutrient cycling scenario including the efflux of HL water from the extramatrical hyphae (Fig. 3), moistening of soil organic matter (Figs. 3 and ​and4),4), and the maintenance of soil microbial activity and nutrient capture (N, P; Open in a separate windowFigure 1Quercus mesocosms demonstrating the plant, root, and hyphal compartments. Details of soil conditions, plant inoculation protocol, mycorrhizal fungi and dye injection methods are detailed in previous work (ref. 7) Point 1 (tap root compartment) denotes the region in which fluorescent tracer dyes were injected into the mesocosm at dusk to track the path of HL water. Point 2 (hyphal chamber) denotes spots adjacent to or distant from the mesh screen into which a small volume (200 µl) of fluorescent and ¹⁵N tracers (99% as ¹⁵NH₄¹⁵NO₃) were injected at dawn to measure water and nutrient uptake by the external hyphae.Open in a separate windowFigure 2Path analysis of the influence of different soil and mycorrhizal factors on nutrient capture with HL, and resultant model showing the significant path coefficients among variables in the Q. agrifolia mesocosms. Lines with a single arrow denote possible cause-effect relationships. The partial correlation coefficients adjacent to each line indicate the strength of the association between the individual factors. Thick lines are statistically significant (p < 0.05) whereas thin lines indicate no significant relationship between parameters (p > 0.05) and only significant coefficients are given (p < 0.05).Open in a separate windowFigure 3Fluorescently-labeled structures recovered from the hyphal chamber of Quercus microcosms following 80 days of soil drying and with nocturnal hydraulic lift. Yellow-green fluorescence indicates samples labeled with Lucifer yellow CH (LYCH), blue fluorescence denotes samples labeled with Cascade blue (CB) hydrazide. (A) CB-labeled leaf litter from the soil and (B) soil particle; (C) LYCH-labeled root fragment in the soil mixture with adherent extramatrical hyphae; (D) LYCH tracer dye fluorescence in labeled extramatrical hyphae and in efflux (arrow) from the hyphal tip onto organic matter; (E and F) external hyphae filled with LYCH (influx; arrow) and (G) background fluorescence in non-labeled extramatrical hyphae.Open in a separate windowFigure 4Measurements of hyphal efflux and influx based on the quantitative analysis of LYCH fluorescence intensity in soil solution. Fluorescent intensity values were converted to LYCH concentration using a standard curve generated for the dye since fluorescent intensity correlates with the number of fluorescent molecules in solution. Influx is the uptake of LYCH by hyphae as driven by plant transpiration demands (day), and measured efflux is the passive loss of LYCH from hyphae into the surrounding soil during HL (night). Vertical bars indicate the standard error of the means.

Table 1

Summary of soil, microbial, mycorrhizal and plant parameters in plant or hyphal compartments

Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging

Several human progerias, including Hutchinson-Gilford progeria syndrome (HGPS), are caused by the accumulation at the nuclear envelope of farnesylated forms of truncated prelamin A, a protein that is also altered during normal aging. Previous studies in cells from individuals with HGPS have shown that farnesyltransferase inhibitors (FTIs) improve nuclear abnormalities associated with prelamin A accumulation, suggesting that these compounds could represent a therapeutic approach for this devastating progeroid syndrome. We show herein that both prelamin A and its truncated form progerin/LADelta50 undergo alternative prenylation by geranylgeranyltransferase in the setting of farnesyltransferase inhibition, which could explain the low efficiency of FTIs in ameliorating the phenotypes of progeroid mouse models. We also show that a combination of statins and aminobisphosphonates efficiently inhibits both farnesylation and geranylgeranylation of progerin and prelamin A and markedly improves the aging-like phenotypes of mice deficient in the metalloproteinase Zmpste24, including growth retardation, loss of weight, lipodystrophy, hair loss and bone defects. Likewise, the longevity of these mice is substantially extended. These findings open a new therapeutic approach for human progeroid syndromes associated with nuclear-envelope abnormalities.     

Gene Polymorphisms and Pharmacogenetics in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic, chronic and inflammatory disease of unknown etiology with genetic predisposition. The advent of new biological agents, as well as the more traditional disease-modifying antirheumatic drugs, has resulted in highly efficient therapies for reducing the symptoms and signs of RA; however, not all patients show the same level of response in disease progression to these therapies. These variations suggest that RA patients may have different genetic regulatory mechanisms. The extensive polymorphisms revealed in non-coding gene-regulatory regions in the immune system, as well as genetic variations in drug-metabolizing enzymes, suggest that this type of variation is of functional and evolutionary importance and may provide clues for developing new therapeutic strategies. Pharmacogenetics is a rapidly advancing area of research that holds the promise that therapies will soon be tailored to an individual patient’s genetic profile.     

Carbon Pools and Emissions from Deforestation in Extra-Tropical Forests of Northern Argentina Between 1900 and 2005

We estimated carbon pools and emissions from deforestation in northern Argentine forests between 1900 and 2005, based on forest inventories, deforestation estimates from satellite images and historical data on forests and agriculture. Carbon fluxes were calculated using a book-keeping model. We ran 1000 simulations for a 105-year period with different combinations of values of carbon stocks (Mg C ha⁻¹), soil carbon in the top 0.2 m, and annual deforestation series. The 1000 combinations of parameters were performed as a sensitivity analysis that for each run, randomly selected the values of each variable within a predefined range of values and probability distributions. Using the simulation outputs, we calculated the accumulated C emissions due to deforestation from 1900 to 2005 and the annual emission as the average of the 1000 simulations, and uncertainties of our estimates as the standard deviation. We found that northern Argentine forests contain an estimated 4.54 Pg C (2.312 Pg C in biomass and 2.233 Pg C in soil). Between 1900 and 2005 approximately 30% of the forests were deforested, yielding carbon emissions of 0.945 (SD = 0.270) Pg C. Estimated average annual carbon emissions between 1996 and 2005, mostly from deforestation of the Chaco dry forests, were 20,875 (SD = 6,156) Gg C y⁻¹ (1 Gg = 10⁻⁶ Pg). These values represent the largest source of carbon from land-cover change in the extra-tropical southern hemisphere, between 0.9 and 2.7% of the global carbon emissions from deforestation, and approximately 10% of carbon emissions from the Brazilian Amazon. Deforestation, which has accelerated during the last decades as a result of modern agriculture expansion, represents a major national source of greenhouse gases and the second emission source, after fossil fuel consumption by fixed sources. We conclude that Argentine forests are an important carbon pool and emission source that need more attention for accurate global estimates, and seasonally dry forest deforestation is a key component of the Argentine carbon cycle. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CAIcal: A combined set of tools to assess codon usage adaptation

 

A recent systematic survey suggested that the YRG (or YawG/YlqF) family with the G4-G5-G1-G2-G3 order of the conserved GTPase motifs represents the only possible circularly permuted variation of the canonical GTPase structure. Here we show that a different circularly permuted GTPase domain actually does exist, conforming to the pattern G3-G4-G5-G1-G2. The domain, dubbed cpRAS, is a variant of RAS family GTPases and occurs in two types of larger proteins, either inserted into a region homologous to a bacterial group of proteins classified as COG2373 and potentially related to the alpha-2-macroglobulin family (so far a single protein in Dictyostelium) or in combination with a von Willebrand factor type A (VWA) domain. For the latter protein type, which was found in a few metazoans and several distantly related protists, existence in the common ancestor of opisthokonts, Amoebozoa and excavates followed by at least eight independent losses may be inferred. Our findings thus bring further evidence for the importance of parallel reduction of ancestral complexity in the eukaryotic evolution.

Reviewers

This article was reviewed by Lakshminarayan Iyer and Fyodor Kondrashov. For the full reviews, please go to the Reviewers' comments section.