Intracellular nitrate of marine diatoms as a driver of anaerobic nitrogen cycling in sinking aggregates

Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly in concentrations > 60 mmol L-1 both as free-living cells and associated to aggregates. Intracellular nitrate concentrations exceeded extracellular concentrations by three orders of magnitude. Intracellular nitrate was used up within 2-3 days after shifting diatom-bacteria aggregates to dark and anoxic conditions. Thirty-one percent of the diatom-derived nitrate was converted to nitrogen gas, indicating that a substantial fraction of the intracellular nitrate pool of S. marinoi becomes available to the aggregate-associated bacterial community. Only 5% of the intracellular nitrate was reduced to ammonium, while 59% was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before the aggregates have settled onto the seafloor could fuel benthic nitrogen transformations

Law Libraries and Laboratories: The Legacies of Langdell and His Metaphor

Law Librarians and others have often referred to Harvard Law School Dean C.C. Langdell’s statements that the law library is the lawyer’s laboratory. Professor Danner examines the context of what Langdell through his other writings, the educational environment at Harvard in the late nineteenth century, and the changing perceptions of university libraries generally. He then considers how the “laboratory metaphor” has been applied by librarians and legal scholars during the twentieth century and into the twenty-first. The article closes with thoughts on Langdell’s legacy for law librarians and the usefulness of the laboratory metaphor

Coupling of alpha(1)-Adrenoceptors to ERK1/2 in the Human Prostate

Introduction: alpha(1)-Adrenoceptors are considered critical for the regulation of prostatic smooth muscle tone. However, previous studies suggested further alpha(1)-adrenoceptor functions besides contraction. Here, we investigated whether alpha(1)-adrenoceptors in the human prostate may activate extracellular signal-regulated kinases (ERK1/2). Methods: Prostate tissues from patients undergoing radical prostatectomy were stimulated in vitro. Activation of ERK1/2 was assessed by Western blot analysis. Expression of ERK1/2 was studied by immunohistochemistry. The effect of ERK1/2 inhibition by U0126 on phenylephrine-induced contraction was studied in organ-bath experiments. Results: Stimulation of human prostate tissue with noradrenaline (30 mu M) or phenylephrine (10 mu M) resulted in ERK activation. This was reflected by increased levels of phosphorylated ERK1/2. Expression of ERK1/2 in the prostate was observed in smooth muscle cells. Incubation of prostate tissue with U0126 (30 mu M) resulted in ERK1/2 inhibition. Dose-dependent phenylephrine-induced contraction of prostate tissue was not modulated by U0126. Conclusions: alpha(1)-Adrenoceptors in the human prostate are coupled to ERK1/2. This may partially explain previous observations suggesting a role of alpha(1)-adrenoceptors in the regulation of prostate growth. Copyright (C) 2011 S. Karger AG, Base

Genome‐wide off‐target analyses of CRISPR/Cas9‐mediated T‐cell receptor engineering in primary human T cells

Objectives Exploiting the forces of human T cells for treatment has led to the current paradigm of emerging immunotherapy strategies. Genetic engineering of the T-cell receptor (TCR) redirects specificity, ablates alloreactivity and brings significant progress and off-the-shelf options to emerging adoptive T-cell transfer (ACT) approaches. Targeted CRISPR/Cas9-mediated double-strand breaks in the DNA enable knockout or knock-in engineering. Methods Here, we perform CRISPR/Cas9-mediated TCR knockout using a therapeutically relevant ribonucleoprotein (RNP) delivery method to assess the safety of genetically engineered T-cell products. Whole-genome sequencing was performed to analyse whether CRISPR/Cas9-mediated DNA double-strand break at the TCR locus is associated with off-target events in human primary T cells. Results TCRα chain and TCRβ chain knockout leads to high on-target InDel frequency and functional knockout. None of the predicted off-target sites could be confirmed experimentally, whereas whole-genome sequencing and manual Integrative Genomics Viewer (IGV) review revealed 9 potential low-frequency off-target events genome-wide. Subsequent amplification and targeted deep sequencing in 7 of 7 evaluable loci did not confirm these low-frequency InDels. Therefore, off-target events are unlikely to be caused by the CRISPR/Cas9 engineering. Conclusion The combinatorial approach of whole-genome sequencing and targeted deep sequencing confirmed highly specific genetic engineering using CRISPR/Cas9-mediated TCR knockout without potentially harmful exonic off-target effects

Fixed-Nitrogen Loss Associated with Sinking Zooplankton Carcasses in a Coastal Oxygen Minimum Zone (Golfo Dulce, Costa Rica)

Oxygen minimum zones (OMZs) in the ocean are of key importance for pelagic fixed-nitrogen loss (N-loss) through microbial denitrification and anaerobic ammonium oxidation (anammox). Recent studies document that zooplankton is surprisingly abundant in and around OMZs and that the microbial community associated with carcasses of a large copepod species mediates denitrification. Here, we investigate the complex N-cycling associated with sinking zooplankton carcasses exposed to the steep O2 gradient in a coastal OMZ (Golfo Dulce, Costa Rica). 15N-stable-isotope enrichment experiments revealed that the carcasses of abundant copepods and ostracods provide anoxic microbial hotspots in the pelagic zone by hosting intense anaerobic N-cycle activities even in the presence of ambient O2. Carcass-associated anaerobic N-cycling was clearly dominated by dissimilatory nitrate reduction to ammonium (DNRA) at up to 30.8 nmol NH+4 individual−1 d−1, followed by denitrification (up to 10.8 nmol N2-N individual−1 d−1), anammox (up to 1.6 nmol N2-N individual−1 d−1), and N2O production (up to 1.2 nmol N2O-N individual−1 d−1). In contrast, anaerobic N-cycling mediated by free-living bacteria proceeded mainly through anammox and denitrification in the anoxic bottom water, which underpins the distinctive microbial metabolism associated with zooplankton carcasses. Pelagic N-loss is potentially enhanced by zooplankton carcasses both directly through N2 and N2O production, and indirectly through NH+4 production that may fuel free-living anammox bacteria. We estimate that in the hypoxic water layer of Golfo Dulce, carcass-associated N2 and N2O production enhance N-loss as much as 1.4-fold at a relative carcass abundance of 36%. In the anoxic bottom water, however, N-loss is likely enhanced only marginally due to high ambient rates and low zooplankton abundance. Thus, zooplankton carcasses may enhance N-loss mainly at the hypoxic boundaries of OMZs which are usually more extensive in open-ocean than in coastal settings. Notably, these contributions by zooplankton carcasses to pelagic N-loss remain undetected by conventional, incubation-based rate measurements.Danish National Research Council/[0602-02276B]/DNRC/DinamarcaOXYGEN/[267233]/OXYGEN/DinamarcaEuropean Research Council/[669947]/ERC/FranciaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Ciencias del Mar y Limnología (CIMAR

Fixed-Nitrogen Loss Associated with Sinking Zooplankton Carcasses in a Coastal Oxygen Minimum Zone (Golfo Dulce, Costa Rica)

Oxygen minimum zones (OMZs) in the ocean are of key importance for pelagic fixed-nitrogen loss (N-loss) through microbial denitrification and anaerobic ammonium oxidation (anammox). Recent studies document that zooplankton is surprisingly abundant in and around OMZs and that the microbial community associated with carcasses of a large copepod species mediates denitrification. Here, we investigate the complex N-cycling associated with sinking zooplankton carcasses exposed to the steep O2 gradient in a coastal OMZ (Golfo Dulce, Costa Rica). 15N-stable-isotope enrichment experiments revealed that the carcasses of abundant copepods and ostracods provide anoxic microbial hotspots in the pelagic zone by hosting intense anaerobic N-cycle activities even in the presence of ambient O2. Carcass-associated anaerobic N-cycling was clearly dominated by dissimilatory nitrate reduction to ammonium (DNRA) at up to 30.8 nmol NH4+ individual−1 d−1, followed by denitrification (up to 10.8 nmol N2-N individual−1 d−1), anammox (up to 1.6 nmol N2-N individual−1 d−1), and N2O production (up to 1.2 nmol N2O-N individual−1 d−1). In contrast, anaerobic N-cycling mediated by free-living bacteria proceeded mainly through anammox and denitrification in the anoxic bottom water, which underpins the distinctive microbial metabolism associated with zooplankton carcasses. Pelagic N-loss is potentially enhanced by zooplankton carcasses both directly through N2 and N2O production, and indirectly through NH4+ production that may fuel free-living anammox bacteria. We estimate that in the hypoxic water layer of Golfo Dulce, carcass-associated N2 and N2O production enhance N-loss as much as 1.4-fold at a relative carcass abundance of 36%. In the anoxic bottom water, however, N-loss is likely enhanced only marginally due to high ambient rates and low zooplankton abundance. Thus, zooplankton carcasses may enhance N-loss mainly at the hypoxic boundaries of OMZs which are usually more extensive in open-ocean than in coastal settings. Notably, these contributions by zooplankton carcasses to pelagic N-loss remain undetected by conventional, incubation-based rate measurements

Infrared molecular fingerprinting of blood-based liquid biopsies for the detection of cancer

Recent omics analyses of human biofluids provide opportunities to probe selected species of biomolecules for disease diagnostics. Fourier-transform infrared (FTIR) spectroscopy investigates the full repertoire of molecular species within a sample at once. Here, we present a multi-institutional study in which we analysed infrared fingerprints of plasma and serum samples from 1639 individuals with different solid tumours and carefully matched symptomatic and non-symptomatic reference individuals. Focusing on breast, bladder, prostate, and lung cancer, we find that infrared molecular fingerprinting is capable of detecting cancer: training a support vector machine algorithm allowed us to obtain binary classification performance in the range of 0.78–0.89 (area under the receiver operating characteristic curve [AUC]), with a clear correlation between AUC and tumour load. Intriguingly, we find that the spectral signatures differ between different cancer types. This study lays the foundation for high-throughput onco-IR-phenotyping of four common cancers, providing a cost-effective, complementary analytical tool for disease recognition

The Eyes Have It: Sex and Sexual Orientation Differences in Pupil Dilation Patterns

Recent research suggests profound sex and sexual orientation differences in sexual response. These results, however, are based on measures of genital arousal, which have potential limitations such as volunteer bias and differential measures for the sexes. The present study introduces a measure less affected by these limitations. We assessed the pupil dilation of 325 men and women of various sexual orientations to male and female erotic stimuli. Results supported hypotheses. In general, self-reported sexual orientation corresponded with pupil dilation to men and women. Among men, substantial dilation to both sexes was most common in bisexual-identified men. In contrast, among women, substantial dilation to both sexes was most common in heterosexual-identified women. Possible reasons for these differences are discussed. Because the measure of pupil dilation is less invasive than previous measures of sexual response, it allows for studying diverse age and cultural populations, usually not included in sexuality research