Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs.

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing

The JCMT BISTRO Survey: The Magnetic Field Strength in the Orion A Filament

We determine the magnetic field strength in the OMC 1 region of the Orion A filament via a new implementation of the Chandrasekhar-Fermi method using observations performed as part of the James Clerk Maxwell Telescope (JCMT) B-Fields In Star-Forming Region Observations (BISTRO) survey with the POL-2 instrument. We combine BISTRO data with archival SCUBA-2 and HARP observations to find a plane-of-sky magnetic field strength in OMC 1 of B_pos=6.6±4.7 mG, where δB_pos=4.7 mG represents a predominantly systematic uncertainty. We develop a new method for measuring angular dispersion, analogous to unsharp masking. We find a magnetic energy density of ~1.7×10^-7 Jm^-3 in OMC 1, comparable both to the gravitational potential energy density of OMC 1 (~10^-7 Jm^-3), and to the energy density in the Orion BN/KL outflow (~10^-7 Jm^-3). We find that neither the Alfvén velocity in OMC 1 nor the velocity of the super-Alfvénic outflow ejecta is sufficiently large for the BN/KL outflow to have caused large-scale distortion of the local magnetic field in the ~500-year lifetime of the outflow. Hence, we propose that the hour-glass field morphology in OMC 1 is caused by the distortion of a primordial cylindrically-symmetric magnetic field by the gravitational fragmentation of the filament and/or the gravitational interaction of the BN/KL and S clumps. We find that OMC 1 is currently in or near magnetically-supported equilibrium, and that the current large-scale morphology of the BN/KL outflow is regulated by the geometry of the magnetic field in OMC 1, and not vice versa

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

The nitrogen costs of photosynthesis in a diatom under current and future pCO2

With each cellular generation, oxygenic photoautotrophs must accumulate abundant protein complexes that mediate light capture, photosynthetic electron transport and carbon fixation. In addition to this net synthesis, oxygenic photoautotrophs must counter the light-dependent photoinactivation of Photosystem II (PSII), using metabolically expensive proteolysis, disassembly, resynthesis and re-assembly of protein subunits. We used growth rates, elemental analyses and protein quantitations to estimate the nitrogen (N) metabolism costs to both accumulate the photosynthetic system and to maintain PSII function in the diatom Thalassiosira pseudonana, growing at two pCO2 levels across a range of light levels. The photosynthetic system contains c. 15-25% of total cellular N. Under low growth light, N (re)cycling through PSII repair is only c. 1% of the cellular N assimilation rate. As growth light increases to inhibitory levels, N metabolite cycling through PSII repair increases to c. 14% of the cellular N assimilation rate. Cells growing under the assumed future 750 ppmv pCO2 show higher growth rates under optimal light, coinciding with a lowered N metabolic cost to maintain photosynthesis, but then suffer greater photoinhibition of growth under excess light, coincident with rising costs to maintain photosynthesis. We predict this quantitative trait response to light will vary across taxa

Refocusing Seafood Sustainability as a Journey Using the Law of the Minimum

Globally, seafood is an important protein source because it is a nutritious food source produced with relative efficiency compared to other proteins. Because of problems related to overfishing and deleterious environmental impacts, over the last decade, non-governmental organizations (NGOs) have increased their focus on seafood sustainability while businesses have incorporated this issue into their corporate social responsibility (CSR) reporting. Sustainability is a concept that can be addressed in terms of scale of issues considered (narrow <em>vs</em>. broad) as well as the scope of how they are measured (undemanding or demanding). Currently, the message of seafood sustainability is becoming complicated in that the journey toward sustainability is being referred to as having achieved a state of sustainability. In addition, companies making a “sustainable” declaration are often at different points in the “scale/scope” arena. As a result, buyers, retailers and consumers have difficulty differentiating between these products. Furthermore, they often assume that a “sustainable” product has no further need for improvement, when in fact this is rarely the case. This change in reference from a continual process (a journey) to a static point (it is sustainable) limits further advances in seafood sustainability and the drive for continual improvement. Herein, the “Law of the Minimum”, growth toward an end goal will occur until one factor becomes limiting, is adopted as an analogy for sustainability. By refocusing the sustainability discussion on a progressive series of challenges to be met, the discussion will return to the journey as the central point. Doing so will help refresh the dialogue around seafood, and to create new opportunities for improvement

Spectral characteristics of <i>Synechococcus</i> (A, C, E, G, I) and <i>Synechocystis</i> (B, D, F, H, J) over a 120 hour iron depletion time course.

<p>Data were compiled from 6 (<i>Synechococcus</i>) or 5 (<i>Synechocystis</i>) replicate time course experiments. (A, B) ln A₇₅₀ to track optical scattering, a proxy for culture cell suspension density. We fit measurements over the first 72 h with a linear regression to estimate the cell specific growth rates. Dotted lines show 95% confidence intervals on the slope of the regression; Data presented are mean +/− standard error, n = 5 or 6. (C, D) ln (A₆₈₀–A₇₅₀) to track chlorophyll content of the cultures. Dotted lines show 95% confidence intervals on the slope of the regression; Data presented are mean +/− standard error, n = 5 or 6. (E, F) (A₆₈₀–A₇₅₀)/(A₇₅₀) to track chlorophyll per cell. (G, H) The wavelength for the chlorophyll absorbance peak, an optical measure of the accumulation of chlorophyll bound to IsiA. (I, J) (A₆₃₀–A₇₅₀)/(A₆₈₀–A₇₅₀) to track phycobilisome absorbance normalized to chlorophyll absorbance. Data presented are mean +/− standard error, n = 5 or 6.</p

Chlorophyll allocations in <i>Synechococcus</i> (A) and <i>Synechocystis</i> (B) over a 120 hour iron depletion timecourse.

<p>Data were compiled from 6 (<i>Synechococcus</i>) or 5 (<i>Synechocystis</i>) replicate time course experiments. (A, B) Solid bars: IsiA subunit contents per μg of total cellular protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059861#pone-0059861-g001" target="_blank">Figure 1</a>) were multiplied by 12 chlorophyll bound per IsiA monomer; Horizontal cross-hatching: PsaC subunit contents per μg of total cellular protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059861#pone-0059861-g001" target="_blank">Figure 1</a>) were multiplied by 100 chlorophyll bound per PSI monomer; Diagonal cross-hatching: PsbA subunit contents per μg of total cellular protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059861#pone-0059861-g001" target="_blank">Figure 1</a>) were multiplied by 36 per PSII monomer. Data presented are mean +/− standard error, n = 5 or 6.</p

Photosystem II maximum quantum yield (F_v/F_m) (A, B) or Photosystem II quantum yield for electron transport (Φ_PSII) (C, D, E, F) in <i>Synechococcus</i> (A, C, E) or <i>Synechocystis</i> (B, D, F) over a 120 hour iron depletion timecourse.

<p>(A, B) F_v/F_m measured from cells under 0 μmol photons·m⁻²·s⁻¹. (C, D) Φ_PSII measured from cells under the growth light level of 65 μmol photons·m⁻²·s⁻¹. (E, F) Φ_PSII measured from cells under saturating light of 262 μmol photons·m⁻²·s⁻¹, 4X higher than the growth light level. Data were compiled from 6 (<i>Synechococcus</i>) or 5 (<i>Synechocystis</i>) replicate measurements from 6 or 5 separate cyanobacterial cultures. All yield data were captured using blue light excitation of fluorescence. Data presented are mean +/− standard error, n = 5 or 6.</p