Nitrogen-limited mangrove ecosystems conserve N through dissimilatory nitrate reduction to ammonium

Earlier observations in mangrove sediments of Goa, India have shown denitrification to be a major pathway for N loss1. However, percentage of total nitrate transformed through complete denitrification accounted for <0–72% of the pore water nitrate reduced. Here, we show that up to 99% of nitrate removal in mangrove sediments is routed through dissimilatory nitrate reduction to ammonium (DNRA). The DNRA process was 2x higher at the relatively pristine site Tuvem compared to the anthropogenically-influenced Divar mangrove ecosystem. In systems receiving low extraneous nutrient inputs, this mechanism effectively conserves and re-circulates N minimizing nutrient loss that would otherwise occur through denitrification. In a global context, the occurrence of DNRA in mangroves has important implications for maintaining N levels and sustaining ecosystem productivity. For the first time, this study also highlights the significance of DNRA in buffering the climate by modulating the production of the greenhouse gas nitrous oxide

The Isolation of Nucleic Acids from Fixed, Paraffin-Embedded Tissues–Which Methods Are Useful When?

Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase “blocks” during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims

The dynamic architecture of the metabolic switch in Streptomyces coelicolor

[EN] Background: During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples.Results: Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis.Conclusions: Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological settingSIWe are very grateful to Mervyn Bibb for his generous support with the Affymetrix custom microarray design. We acknowledge the excellent technical help of K. Klein, S. Poths, M. Walter, A. Øverby and E. Hansen. This project was supported by grants of the ERA-NET SySMO Project [GEN2006-27745-E/SYS]: (P-UK-01-11-3i) and the Research Council of Norway [project no. 181840/I30

Characterisation of the Cell Line HC-AFW1 Derived from a Pediatric Hepatocellular Carcinoma

Current treatment of paediatric hepatocellular carcinoma (HCC) is often inefficient due to advanced disease at diagnosis and resistance to common drugs. The aim of this study was to generate a cell line derived from a paediatric HCC in order to expand research in this field. We established the HC-AFW1 cell line from a liver neoplasm of a 4-year-old boy through culturing of primary tumor specimens. The cell line has been stable for over one year of culturing and has a doubling time of 40 h. The tumour cells have an epithelial histology and express HCC-associated proteins such as Alpha-fetoprotein (AFP), Glypican 3, E-cadherin, CD10, CD326, HepPar1 and Vimentin. Forty-nine amino acids in exon 3 of β-Catenin that involve the phosphorylation sites of GSK3 were absent and β-Catenin is detectable in the cell nuclei. Cytogenetic analysis revealed large anomalies in the chromosomal map. Several alterations of gene copy numbers were detected by genome-wide SNP array. Among the different drugs tested, cisplatin and irinotecan showed effective inhibition of tumour cell growth in a proliferation assay at concentrations below 5 µg/ml. Subcutaneous xenotransplantation of HC-AFW1 cells into NOD/SCID mice resulted in fast growing dedifferentiated tumours with high levels of serum AFP. Histological analyses of the primary tumour and xenografts included national and international expert pathological review. Consensus reading characterised the primary tumour and the HC-AFW1-derived tumours as HCC. HC-AFW1 is the first cell line derived from a paediatric HCC without a background of viral hepatitis or cirrhosis and represents a valuable tool for investigating the biology of and therapeutic strategies for childhood HCC

Characterization of active miniature inverted-repeat transposable elements in the peanut genome

Miniature inverted-repeat transposable elements (MITEs), some of which are known as active non-autonomous DNA transposons, are found in the genomes of plants and animals. In peanut (Arachis hypogaea), AhMITE1 has been identified in a gene for fatty-acid desaturase, and possessed excision activity. However, the AhMITE1 distribution and frequency of excision have not been determined for the peanut genome. In order to characterize AhMITE1s, their genomic diversity and transposition ability was investigated. Southern blot analysis indicated high AhMITE1 copy number in the genomes of A. hypogaea, A. magna and A. monticola, but not in A. duranensis. A total of 504 AhMITE1s were identified from the MITE-enriched genomic libraries of A. hypogaea. The representative AhMITE1s exhibited a mean length of 205.5 bp and a GC content of 30.1%, with AT-rich, 9 bp target site duplications and 25 bp terminal inverted repeats. PCR analyses were performed using primer pairs designed against both flanking sequences of each AhMITE1. These analyses detected polymorphisms at 169 out of 411 insertional loci in the four peanut lines. In subsequent analyses of 60 gamma-irradiated mutant lines, four AhMITE1 excisions showed footprint mutations at the 109 loci tested. This study characterizes AhMITE1s in peanut and discusses their use as DNA markers and mutagens for the genetics, genomics and breeding of peanut and its relatives

Directional genetic selection by pulp mill effluent on multiple natural populations of three-spined stickleback (Gasterosteus aculeatus)

Contamination can cause a rapid environmental change which may require populations to respond with evolutionary changes. To evaluate the effects of pulp mill effluents on population genetics, we sampled three-spined sticklebacks (Gasterosteus aculeatus) near four pulp mills and four adjacent reference sites and analyzed Amplified Fragment Length Polymorphism (AFLP) to compare genetic variability. A fine scale genetic structure was detected and samples from polluted sites separated from reference sites in multidimensional scaling plots (P < 0.005, 1000 permutations) and locus-by-locus Analysis of Molecular Variance (AMOVA) further confirmed that habitats are significantly separated (FST = 0.021, P < 0.01, 1023 permutations). The amount of genetic variation between populations did not differ between habitats, and populations from both habitats had similar levels of heterozygosity (polluted sites Nei’s Hs = 0.11, reference sites Nei’s Hs = 0.11). Still, pairwise FST: s between three, out of four, pairs of polluted-reference sites were significant. A FST-outlier analysis showed that 21 (8.4%) loci were statistically different from a neutral distribution at the P < 0.05 level and therefore indicated to be under divergent selection. When removing 13 FST-outlier loci, significant at the P < 0.01 level, differentiation between habitats disappeared in a multidimensional scaling plot. In conclusion, pulp mill effluence has acted as a selective agent on natural populations of G. aculeatus, causing a convergence in genotype composition change at multiple sites in an open environment

Structure-based functional inference of hypothetical proteins from Mycoplasma hyopneumoniae

Enzootic pneumonia caused by Mycoplasma hyopneumoniae is a major constraint to efficient pork production throughout the world. This pathogen has a small genome with 716 coding sequences, of which 418 are homologous to proteins with known functions. However, almost 42% of the 716 coding sequences are annotated as hypothetical proteins. Alternative methodologies such as threading and comparative modeling can be used to predict structures and functions of such hypothetical proteins. Often, these alternative methods can answer questions about the properties of a model system faster than experiments. In this study, we predicted the structures of seven proteins annotated as hypothetical in M. hyopneumoniae, using the structure-based approaches mentioned above. Three proteins were predicted to be involved in metabolic processes, two proteins in transcription and two proteins where no function could be assigned. However, the modeled structures of the last two proteins suggested experimental designs to identify their functions. Our findings are important in diminishing the gap between the lack of annotation of important metabolic pathways and the great number of hypothetical proteins in the M. hyopneumoniae genome

The proteasome inhibitor MG-132 sensitizes PC-3 prostate cancer cells to ionizing radiation by a DNA-PK-independent mechanism

BACKGROUND: By modulating the expression levels of specific signal transduction molecules, the 26S proteasome plays a central role in determining cell cycle progression or arrest and cell survival or death in response to stress stimuli, including ionizing radiation. Inhibition of proteasome function by specific drugs results in cell cycle arrest, apoptosis and radiosensitization of many cancer cell lines. This study investigates whether there is also a concomitant increase in cellular radiosensitivity if proteasome inhibition occurs only transiently before radiation. Further, since proteasome inhibition has been shown to activate caspase-3, which is involved in apoptosis, and caspase-3 can cleave DNA-PKcs, which is involved in DNA-double strand repair, the hypothesis was tested that caspase-3 activation was essential for both apoptosis and radiosensitization following proteasome inhibition. METHODS: Prostate carcinoma PC-3 cells were treated with the reversible proteasome inhibitor MG-132. Cell cycle distribution, apoptosis, caspase-3 activity, DNA-PKcs protein levels and DNA-PK activity were monitored. Radiosensitivity was assessed using a clonogenic assay. RESULTS: Inhibition of proteasome function caused cell cycle arrest and apoptosis but this did not involve early activation of caspase-3. Short-time inhibition of proteasome function also caused radiosensitization but this did not involve a decrease in DNA-PKcs protein levels or DNA-PK activity. CONCLUSION: We conclude that caspase-dependent cleavage of DNA-PKcs during apoptosis does not contribute to the radiosensitizing effects of MG-132

Feedforward and recurrent inhibitory receptive fields of principal cells in the cat’s dorsal lateral geniculate nucleus

Principal cells in the dorsal lateral geniculate nucleus receive both feedforward and recurrent inhibition. Despite many years of study, the receptive field structure of these inhibitory mechanisms has not been determined. Here, we have used intracellular recordings in vivo to differentiate between the two types of inhibition and map their respective receptive fields. The feedforward inhibition of a principal cell originates from the same type of retinal ganglion cells as its excitation, while the recurrent inhibition is provided by both on- and off-centre cells. Both inhibitory effects are strongest at the centre of the excitatory receptive field. The diameter of the feedforward inhibitory field is two times larger, and the recurrent two to four times larger than the excitatory field centre. The inhibitory circuitry is similar for X and Y principal cells

Comprehensive in vivo Mapping of the Human Basal Ganglia and Thalamic Connectome in Individuals Using 7T MRI

Basal ganglia circuits are affected in neurological disorders such as Parkinson's disease (PD), essential tremor, dystonia and Tourette syndrome. Understanding the structural and functional connectivity of these circuits is critical for elucidating the mechanisms of the movement and neuropsychiatric disorders, and is vital for developing new therapeutic strategies such as deep brain stimulation (DBS). Knowledge about the connectivity of the human basal ganglia and thalamus has rapidly evolved over recent years through non-invasive imaging techniques, but has remained incomplete because of insufficient resolution and sensitivity of these techniques. Here, we present an imaging and computational protocol designed to generate a comprehensive in vivo and subject-specific, three-dimensional model of the structure and connections of the human basal ganglia. High-resolution structural and functional magnetic resonance images were acquired with a 7-Tesla magnet. Capitalizing on the enhanced signal-to-noise ratio (SNR) and enriched contrast obtained at high-field MRI, detailed structural and connectivity representations of the human basal ganglia and thalamus were achieved. This unique combination of multiple imaging modalities enabled the in-vivo visualization of the individual human basal ganglia and thalamic nuclei, the reconstruction of seven white-matter pathways and their connectivity probability that, to date, have only been reported in animal studies, histologically, or group-averaged MRI population studies. Also described are subject-specific parcellations of the basal ganglia and thalamus into sub-territories based on their distinct connectivity patterns. These anatomical connectivity findings are supported by functional connectivity data derived from resting-state functional MRI (R-fMRI). This work demonstrates new capabilities for studying basal ganglia circuitry, and opens new avenues of investigation into the movement and neuropsychiatric disorders, in individual human subjects