Technical Note: Could benzalkonium chloride be a suitable alternative to mercuric chloride for preservation of seawater samples?

Instrumental equipment unsuitable or unavailable for fieldwork as well as lack of ship space can necessitate the preservation of seawater samples prior to analysis in a shore-based laboratory. Mercuric chloride (HgCl2) is routinely used for such preservation, but its handling and subsequent disposal incur environmental risks and significant expense. There is therefore a strong motivation to find less hazardous alternatives. Benzalkonium chloride (BAC) has been used previously as microbial inhibitor for freshwater samples. Here, we assess the use of BAC for marine samples prior to the measurement of oxygen-to-argon (O2 / Ar) ratios, as used for the determination of biological net community production. BAC at a concentration of 50 mg dm−3 inhibited microbial activity for at least 3 days in samples tested with chlorophyll a (Chl a) concentrations up to 1 mg m−3. BAC concentrations of 100 and 200 mg dm−3 were no more effective than 50 mg dm−3 . With fewer risks to human health and the environment, and no requirement for expensive waste disposal, BAC could be a viable alternative to HgCl2 for short-term preservation of seawater samples, but is not a replacement for HgCl2 in the case of oxygen triple isotope analysis, which requires storage over weeks to months. In any event, further tests on a case-by-case basis should be undertaken if use of BAC was considered, since its inhibitory activity may depend on concentration and composition of the microbial community

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible

Isolation of viruses responsible for the demise of an Emiliania huxleyi bloom in the English Channel

This study used analytical flow cytometry (AFC) to monitor the abundance of phytoplankton, coccoliths, bacteria and viruses in a transect that crossed a high reflectance area in the western English Channel. The high reflectance area, observed by satellite, was caused by the demise of an Emiliania huxleyi bloom. Water samples were collected from depth profiles at four stations, one station outside and three stations inside the high reflectance area. Plots of transect data revealed very obvious differences between Station 1, outside, and Stations 2–4, inside the high reflectance area. Inside, concentrations of viruses were higher; E. huxleyi cells were lower; coccoliths were higher; bacteria were higher and virus:bacteria ratio was lower than at Station 1, outside the high reflectance area. This data can simply be interpreted as virus-induced lysis of E. huxleyi cells in the bloom causing large concentrations of coccoliths to detach, resulting in the high reflectance observed by satellite imagery. This interpretation was supported by the isolation of two viruses, EhV84 and EhV86, from the high reflectance area that lysed cultures of E. huxleyi host strain CCMP1516. Basic characterization revealed that they were lytic viruses approximately 170 nm–190 nm in diameter with an icosahedral symmetry. Taken together, transect and isolation data suggest that viruses were the major contributor to the demise of the E. huxleyi population in the high reflectance area. Close coupling between microalgae, bacteria and viruses contributed to a large organic carbon input. Consequent cycling influenced the succession of an E. huxleyi-dominated population to a more characteristic mixed summer phytoplankton community

Diel rhythmicity in amino acid uptake by Prochlorococcus

The marine cyanobacterium Prochlorococcus, the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light synchronised axenic Prochlorococcus (PCC9511 strain) culture and 35S-methionine and 3H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >104 times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G2 cell cycle stage were consistently 2.2 times higher than those of cells at the G1 stage. Furthermore, S+G2 cells up-regulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday, and up to 42% at dusk, of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations

Tobacco exposure inhibits SPLUNC1-dependent antimicrobial activity

Background: Tobacco smoke exposure impairs the lung´s innate immune response, leading to an increased risk of chronic infections. SPLUNC1 is a secreted, multifunctional innate defense protein that has antimicrobial activity against Gram negative organisms. We hypothesize that tobacco smoke-induced SPLUNC1 dysfunction contributes to the observed defect in innate immunity in tobacco smokers and that this dysfunction can be used as a potential biomarker of harm. Methods: We collected sputum from never-smokers and otherwise healthy smokers. We performed Western blotting to determine SPLUNC1 levels and determined antimicrobial activity against nontypeable Haemophilus influenzae. An in vitro exposure model was utilized to measure the effect of tobacco exposure on human bronchial epithelial culture (HBEC) antimicrobial activity against H. influenzae. The direct effects of cigarette and little cigar smoke exposure on SPLUNC1 function was determined using 24 h growth measurements and LPS binding assays. Results: H. influenzae growth in cigarette smoker´s sputum was significantly greater compared to never-smokers sputum over 24 h. HBEC supernatants and lysates contained significantly higher numbers of H. influenzae following chronic cigarette and little cigar smoke exposure compared to air-exposed controls. Furthermore, SPLUNC1´s antimicrobial activity and LPS-binding capability against both H. influenzae and P. aeruginosa was attenuated following cigarette and little cigar exposure. Conclusions: These data suggest that cigarette and little cigar exposure impairs SPLUNC1´s antimicrobial ability and that this inhibition may serve as a novel biomarker of harm that can be used to assess the toxicity of commercial tobacco products.Fil: Moore, Patrick J.. University of North Carolina; Estados UnidosFil: Sesma, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; ArgentinaFil: Alexis, Neil E.. University of North Carolina; Estados UnidosFil: Tarran, Robert. University of North Carolina; Estados Unido

Using pot plants to clean indoor air

Polluted indoor air, air contaminated by Volatile Organic Compounds (VOCs), are a major cause of headaches, nausea, concentration loss and other `building-related illnesses. Previous laboratory research by the Plants and Environmental Quality Group at the University of Technology, Sydney (UTS) has shown that the `pot plant system (plants-and-potting-mix combination) can daily eliminate several times the Australian maximum exposure concentrations of the common VOCs benzene and n-hexane

Expression of gain-of-function CFTR in cystic fibrosis airway cells restores epithelial function better than wild-type or codon-optimized CFTR

Class Ia/b cystic fibrosis transmembrane regulator (CFTR) variants cause severe lung disease in 10% of cystic fibrosis (CF) patients and are untreatable with small-molecule pharmaceuticals. Genetic replacement of CFTR offers a cure, but its effectiveness is limited in vivo. We hypothesized that enhancing protein levels (using codon optimization) and/or activity (using gain-of-function variants) of CFTR would more effectively restore function to CF bronchial epithelial cells. Three different variants of the CFTR protein were tested: codon optimized (high codon adaptation index [hCAI]), a gain-of-function (GOF) variant (K978C), and a combination of both (hˆK978C). In human embryonic kidney (HEK293T) cells, initial results showed that hCAI and hˆK978C produced greater than 10-fold more CFTR protein and displayed ∼4-fold greater activity than wild-type (WT) CFTR. However, functionality was profoundly different in CF bronchial epithelial cells. Here, K978C CFTR more potently restored essential epithelial functions (anion transport, airway surface liquid height, and pH) than WT CFTR. hCAI and hˆK978C CFTRs had limited impact because of mislocalization in the cell. These data provide a proof of principle showing that GOF variants may be more effective than codon-optimized forms of CFTR for CF gene therapy. Video abstract: [Video presented

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor

CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR’s function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR’s PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs

Nutrient regimes control phytoplankton ecophysiology in the South Atlantic

Fast Repetition Rate fluorometry (FRRf) measurements of phytoplankton photophysiology from an across-basin South Atlantic cruise (as part of the GEOTRACES programme) characterised two dominant ecophysiological regimes which were interpreted on the basis of nutrient limitation. South of the South Subtropical Convergence (SSTC) in the northern sub-Antarctic sector of the Antarctic Circumpolar Current (ACC) in the Eastern Atlantic Basin, waters are characterised by elevated chlorophyll concentrations, a dominance by larger phytoplankton cells, and low apparent photochemical efficiency (<i>F</i>_v / <i>F</i>_m). Shipboard 24 h iron (Fe) addition incubation experiments confirmed that Fe stress was primarily responsible for the low <i>F</i>_v / <i>F</i>_m, with Fe addition to these waters, either within the artificial bottle additions or naturally occurring downstream enrichment from Gough Island, significantly increasing <i>F</i>_v / <i>F</i>_m values. To the north of the SSTC at the southern boundary of the South Atlantic Gyre, phytoplankton are characterised by high values of <i>F</i>_v / <i>F</i>_m which, coupled with the low macronutrient concentrations and increased presence of picocyanobacteria, are interpreted as conditions of Fe replete, balanced macronutrient-limited growth. Spatial correlation was found between <i>F</i>_v / <i>F</i>_m and Fe:nitrate ratios, supporting the suggestion that the relative supply ratios of these two nutrients can control patterns of limitation and consequently the ecophysiology of phytoplankton in subtropical gyre and ACC regimes

Seasonal phosphorus and carbon dynamics in a temperate shelf sea (Celtic Sea)

The seasonal cycle of resource availability in shelf seas has a strong selective pressure on phytoplankton diversity and the biogeochemical cycling of key elements, such as carbon (C) and phosphorus (P). Shifts in carbon consumption relative to P availability, via changes in cellular stoichiometry for example, can lead to an apparent ‘excess’ of carbon production. We made measurements of inorganic P (Pi) uptake, in parallel to C-fixation, by plankton communities in the Celtic Sea (NW European Shelf) in spring (April 2015), summer (July 2015) and autumn (November 2014). Short-term (<8 h) Pi-uptake coupled with dissolved organic phosphorus (DOP) release, in parallel to net (24 h) primary production (NPP), were all measured across an irradiance gradient designed to typify vertically and seasonally varying light conditions. Rates of Pi-uptake were highest during spring and lowest in the low light conditions of autumn, although biomass-normalised Pi-uptake was highest in the summer. The release of DOP was highest in November and declined to low levels in July, indicative of efficient utilization and recycling of the low levels of Pi available. Examination of daily turnover times of the different particulate pools, including estimates of phytoplankton and bacterial carbon, indicated a differing seasonal influence of autotrophs and heterotrophs in P-dynamics, with summer conditions associated with a strong bacterial influence and the early spring period with fast growing phytoplankton. These seasonal changes in autotrophic and heterotrophic influence, coupled with changes in resource availability (Pi, light) resulted in seasonal changes in the stoichiometry of NPP to daily Pi-uptake (C:P ratio); from relatively C-rich uptake in November and late April, to P-rich uptake in early April and July. Overall, these results highlight the seasonally varying influence of both autotrophic and heterotrophic components of shelf sea ecosystems on the relative uptake of C and P