Respiratory responses of three Antarctic ascidians and a sea pen to increased sediment concentrations

Glacial retreat and subglacial bedrock erosion are consequences of rapid regional warming on the West Antarctic Peninsula. Sedimentation of fine-grained eroded particles can impact the physiology of filter-feeding benthic organisms. We investigated the effect of increasing concentrations of sediment on the oxygen consumption of suspension feeding species, the ascidians Molgula pedunculata, Cnemidocarpa verrucosa, Ascidia challengeri, and the pennatulid Malacobelemnon daytoni in Potter Cove (South Shetland Islands, Antarctica). In A. challengeri and C. verrucosa, oxygen consumption increased gradually up to a critical sediment concentration (C _crit) where species oxygen consumption was maximal (O _max in mg O₂?g^?1dm?day^?1) and further addition of sediments decreased respiration. C _crit was 200?mg?L^?1 for A. challengeri (O _max of 0.651?±?0.238) and between 100 and 200?mg?L^?1 for C. verrucosa (O _max of 0.898?±?0.582). Oxygen consumption of M. pedunculata increased significantly even at low sediment concentrations (15–50?mg sediment?L^?1). Contrary to the ascidians, sediment exposure did not affect oxygen consumption of the sea pen. The tiered response to sedimentation in the four species corroborates recent field observations that detected a reduction in the abundance of the sensitive ascidian M. pedunculata from areas strongly affected by glacial sediment discharge, whereas sea pens are increasing in abundance. Our investigation relates consequences (population shifts in filter-feeder communities) to causes (glacial retreat) and is of importance for modelling of climate change effects in Antarctic shallow coastal areas.     

A consistent phylogenetic backbone for the fungi

The kingdom of fungi provides model organisms for biotechnology, cell biology, genetics, and life sciences in general. Only when their phylogenetic relationships are stably resolved, can individual results from fungal research be integrated into a holistic picture of biology. However, and despite recent progress, many deep relationships within the fungi remain unclear. Here, we present the first phylogenomic study of an entire eukaryotic kingdom that uses a consistency criterion to strengthen phylogenetic conclusions. We reason that branches (splits) recovered with independent data and different tree reconstruction methods are likely to reflect true evolutionary relationships. Two complementary phylogenomic data sets based on 99 fungal genomes and 109 fungal expressed sequence tag (EST) sets analyzed with four different tree reconstruction methods shed light from different angles on the fungal tree of life. Eleven additional data sets address specifically the phylogenetic position of Blastocladiomycota, Ustilaginomycotina, and Dothideomycetes, respectively. The combined evidence from the resulting trees supports the deep-level stability of the fungal groups toward a comprehensive natural system of the fungi. In addition, our analysis reveals methodologically interesting aspects. Enrichment for EST encoded data-a common practice in phylogenomic analyses-introduces a strong bias toward slowly evolving and functionally correlated genes. Consequently, the generalization of phylogenomic data sets as collections of randomly selected genes cannot be taken for granted. A thorough characterization of the data to assess possible influences on the tree reconstruction should therefore become a standard in phylogenomic analyses.     

Impaired ability of burn patient neutrophils to stimulate β-defensin production by keratinocytes

Immunosuppressive neutrophils (PMN-II) appearing in association with burn injury have a role on the increased susceptibility of burn patients to various infections. In the present study, the role of PMN-II on the production of human β-defensins (HBDs), important molecules on host antimicrobial innate immunities, by human keratinocytes was studied. Normal human epidermal keratinocytes (NHEKs) were cultured with neutrophils (PMNs) isolated from burn patients or healthy volunteers in dual-chamber transwells. Culture fluids harvested 24?h after cultivation were assayed for HBDs using enzyme-linked immunosorbent assay. Also, these culture fluids were assayed for their antimicrobial activities by a standard colony-counting method using Pseudomonas aeruginosa. In the results, PMNs isolated from peripheral blood of burn patients were confirmed as PMN-II, because these cells produced CC-chemokine ligand 2 (CCL2), but not interleukin (IL)-12 and CC-chemokine ligand 3 (CCL3). Culture fluids of NHEK transwell-cultured with healthy PMNs exhibited strong killing activities against P. aeruginosa (96% inhibition), however, the growth of bacteria was not dramatically inhibited by the culture fluids of NHEK transwell-cultured with burn-patient PMNs (36% inhibition). IL-12 and CCL3 containing culture fluids of healthy PMNs stimulated with the bacterial antigen or rCCL3 and rIL-12 enhanced the production of HBD2 and HBD3 by NHEK, whereas CCL2 containing culture fluids of burn-patient PMN stimulated with the antigen or rCCL2 inhibited the HBD production by NHEK. These results indicate that PMN-II appearing in association with burn injury contribute to the decreased production of HBDs in thermally injured patients.     

Chromogranins A and B are key proteins in amine accumulation, but the catecholamine secretory pathway is conserved without them

Chromogranins are the main soluble proteins in the large dense core secretory vesicles (LDCVs) found in aminergic neurons and chromaffin cells. We recently demonstrated that chromogranins A and B each regulate the concentration of adrenaline in chromaffin granules and its exocytosis. Here we have further studied the role played by these proteins by generating mice lacking both chromogranins. Surprisingly, these animals are both viable and fertile. Although chromogranins are thought to be essential for their biogenesis, LDCVs were evident in these mice. These vesicles do have a somewhat atypical appearance and larger size. Despite their increased size, single-cell amperometry recordings from chromaffin cells showed that the amine content in these vesicles is reduced by half. These data demonstrate that although chromogranins regulate the amine concentration in LDCVs, they are not completely essential, and other proteins unrelated to neurosecretion, such as fibrinogen, might compensate for their loss to ensure that vesicles are generated and the secretory pathway conserved.     

Study of the influence of sporulation conditions on heat resistance of Geobacillus stearothermophilus used in the development of biological indicators for steam sterilization

Biological indicators are important tools in infection control via sterilization process monitoring. The use of a standardized spore crop with a well-defined heat resistance will guarantee the quality of a biological indicator. Ambient factors during sporulation can affect spore characteristics and properties, including heat resistance. The aim of this study is to evaluate the main sporulation factors responsible for heat resistance in Geobacillus stearothermophilus, a useful biological indicator for steam sterilization. A sequence of a three-step optimization of variables (initial pH, nutrient concentration, tryptone, peptone, beef extract, yeast extract, manganese sulfate, magnesium sulfate, calcium chloride and potassium phosphate) was carried out to screen those that have a significant influence on heat resistance of produced spores. The variable exerting greatest influence on G. stearothermophilus heat resistance during sporulation was found to be the initial pH. Lower nutrient concentration and alkaline pH around 8.5 tended to enhance decimal reduction time at 121?°C (D_121°C). A central composite design enabled a fourfold enhancement in heat resistance, and the model obtained accurately describes positive pH and negative manganese sulfate concentration influence on spore heat resistance.     

The antibiotic Furvina? targets the P-site of 30S ribosomal subunits and inhibits translation initiation displaying start codon bias

Furvina®, also denominated G1 (MW 297), is a synthetic nitrovinylfuran [2-bromo-5-(2-bromo-2-nitrovinyl)-furan] antibiotic with a broad antimicrobial spectrum. An ointment (Dermofural®) containing G1 as the only active principle is currently marketed in Cuba and successfully used to treat dermatological infections. Here we describe the molecular target and mechanism of action of G1 in bacteria and demonstrate that in vivo G1 preferentially inhibits protein synthesis over RNA, DNA and cell wall synthesis. Furthermore, we demonstrate that G1 targets the small ribosomal subunit, binds at or near the P-decoding site and inhibits its function interfering with the ribosomal binding of fMet-tRNA during 30S initiation complex (IC) formation ultimately inhibiting translation. Notably, this G1 inhibition displays a bias for the nature (purine vs. pyrimidine) of the 3′-base of the codon, occurring efficiently only when the mRNA directing 30S IC formation and translation contains the canonical AUG initiation triplet or the rarely found AUA triplet, but hardly occurs when the mRNA start codon is either one of the non-canonical triplets AUU or AUC. This codon discrimination by G1 is reminiscent, though of opposite type of that displayed by IF3 in its fidelity function, and remarkably does not occur in the absence of this factor.     

Allopregnanolone Potentiates the Glutamate-Mediated Seizures Induced by 4-Aminopyridine in Rat Hippocampus in vivo

Excitatory and inhibitory neurotransmission in the central nervous system can be modulated by neurosteroids. We previously found that in rat hippocampal slices allopregnanolone (3α-hydroxy-5α-pregnan-20-one), a positive GABA_A receptor modulator, suppresses the epileptic discharges induced by 4-aminopyridine (4-AP), a convulsant K⁺ channel blocker that stimulates glutamate release. Here, we tested the action of allopregnanolone on the epileptogenic and excitotoxic effects of the intrahippocampal administration of 4-AP in vivo. Drugs were perfused by a microdialysis cannula-electrode in the dorsal hippocampus and the EEG was recorded. Extracellular levels of aspartate, glutamate and GABA were analyzed by HPLC in the microdialysis fractions, and 24 h after the experiment the hippocampus was studied histologically. 4-AP induced intense epileptic discharges, increased the extracellular levels of aspartate, glutamate, and GABA by 383, 420, and 245%, respectively, and produced a notable neurodegeneration in CA1 and CA3 areas. Allopregnanolone administration alone did not affect the electrical activity, amino acids levels or cellular morphology, but when co-infused with 4-AP incremented 55–77% the duration of the epileptic discharges, and potentiated 32–49% the release of glutamate in comparison with 4-AP alone. The 4-AP-induced neurodegeneration was not modified by allopregnanolone. The NMDA receptor antagonist MK-801 protected against the epilepsy and neurodegeneration produced by 4-AP, and allopregnanolone did not affect this protection. We conclude that, differently from the observations in vitro, allopregnanolone potentiated the stimulatory effect of 4-AP on glutamate release and that this may explain the potentiation of the epileptogenic effect of 4-AP in vivo.     

Heterogeneity of the digestible insoluble fiber of selected forages in situ

Long-term in situ incubations were performed to verify the likelihood of the heterogeneity concept of the potentially digestible fraction of the insoluble fiber (NDFom) by fitting both heterogeneous and homogeneous potentially digestible NDFom versions of a generalized compartmental model of digestion (GCMD). Corn silage and eleven tropical grasses and alfalfa hay were studied. Data were gathered from a study in which forage samples in nylon bags were incubated in rumen cannulated steers so that three profiles per forage were generated. The incubation endpoint was used to form sets of time profiles. The original set consisted of profiles ending at 1440 h, and the other two were formed by using 96 and 240 h as the incubation endpoints, respectively. The indigestible residue was estimated using nonlinear least squares or by assuming it to be 2.4 times lignin determined by the sulphuric acid method (Lignin (sa)). Therefore, eight different models were evaluated by combining end points of digestion, and the homogeneous and heterogeneous versions of GCMD with the two ways of estimating the indigestible residue. The likelihood of the models was assessed by computing Akaike information criteria. The effects of forage, model, and their interaction were analyzed by taking model as a repeated measurement. Heterogeneity of the potentially degradable fraction for NDFom was detected with long-term incubation trials (up to 1440 h) for some forages, and the introduction of the 2.4×Lignin (sa) as a direct measure of the indigestible residue improved the likelihood of the heterogeneous version of GCMD. The forage by model interaction was significant for many comparable parameter estimates, which means that specific and inconsistent results for models within forages were produced depending on the definition of the incubation end-point. The indigestible residue was overestimated with short-term incubation profiles, but the overestimation was lower for the profiles ending at 240 h whether compared to profiles ending at 96 h. Given the likelihood of the heterogeneous version of GCMD fitted to profiles ending at 1440 h and at 240 h for some forages, the heterogeneity concept should be investigated whenever the research interest relies on estimating the kinetic attributes of the degradation profiles of the NDFom in situ.     

Multifunctional roles for the N-terminal basic motif of Alfalfa mosaic virus coat protein: nucleolar/cytoplasmic shuttling, modulation of RNA-binding activity, and virion formation

In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved in the regulation of replication and translation of viral RNAs, and in cell-to-cell and systemic movement of the virus. An intriguing feature of the AMV CP is its nuclear and nucleolar accumulation. Here, we identify an N-terminal lysine-rich nucleolar localization signal (NoLS) in the AMV CP required to both enter the nucleus and accumulate in the nucleolus of infected cells, and a C-terminal leucine-rich domain which might function as a nuclear export signal. Moreover, we demonstrate that AMV CP interacts with importin-α, a component of the classical nuclear import pathway. A mutant AMV RNA 3 unable to target the nucleolus exhibited reduced plus-strand RNA synthesis and cell-to-cell spread. Moreover, virion formation and systemic movement were completely abolished in plants infected with this mutant. In vitro analysis demonstrated that specific lysine residues within the NoLS are also involved in modulating CP-RNA binding and CP dimerization, suggesting that the NoLS represents a multifunctional domain within the AMV CP. The observation that nuclear and nucleolar import signals mask RNA-binding properties of AMV CP, essential for viral replication and translation, supports a model in which viral expression is carefully modulated by a cytoplasmic/nuclear balance of CP accumulation.     

Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage

Mitochondria divide and fuse continuously, and the balance between these two processes regulates mitochondrial shape. Alterations in mitochondrial dynamics are associated with neurodegenerative diseases. Here we investigate the physiological and cellular functions of mitochondrial division in postmitotic neurons using in vivo and in vitro gene knockout for the mitochondrial division protein Drp1. When mouse Drp1 was deleted in postmitotic Purkinje cells in the cerebellum, mitochondrial tubules elongated due to excess fusion, became large spheres due to oxidative damage, accumulated ubiquitin and mitophagy markers, and lost respiratory function, leading to neurodegeneration. Ubiquitination of mitochondria was independent of the E3 ubiquitin ligase parkin in Purkinje cells lacking Drp1. Treatment with antioxidants rescued mitochondrial swelling and cell death in Drp1KO Purkinje cells. Moreover, hydrogen peroxide converted elongated tubules into large spheres in Drp1KO fibroblasts. Our findings suggest that mitochondrial division serves as a quality control mechanism to suppress oxidative damage and thus promote neuronal survival.