In vivo optical imaging of acute myeloid leukemia by green fluorescent protein: time-domain autofluorescence decoupling, fluorophore quantification, and localization

Human xenografts of acute myeloid leukemia (AML) in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice result in disease states of diffuse, nonpalpable tissue infiltrates exhibiting a variable disease course, with some animals not developing a disease phenotype. Thus, disease staging and, more critically, quantification of preclinical therapeutic effect in these models are particularly difficult. In this study, we present the generation of a green fluorescent protein (GFP)-labeled human leukemic cell line, NB4, and validate the potential of a time-domain imager fitted with a 470 nm picosecond pulsed laser diode to decouple GFP fluorescence from autofluorescence on the basis of fluorescence lifetime and thus determine the depth and relative concentration of GFP inclusions in phantoms of homogeneous and heterogeneous optical properties. Subsequently, we developed an optical imageable human xenograft model of NB4-GFP AML and illustrate early disease detection, depth discrimination of leukemic infiltrates, and longitudinal monitoring of disease course employing time-domain optical imaging. We conclude that early disease detection through use of time-domain imaging in this initially slowly progressing AML xenograft model permits accurate disease staging and should aid in future preclinical development of therapeutics for AML.     

Contrasting acclimation responses to elevated CO2 and warming between an evergreen and a deciduous boreal conifer

Rising atmospheric carbon dioxide (CO₂) concentrations may warm northern latitudes up to 8°C by the end of the century. Boreal forests play a large role in the global carbon cycle, and the responses of northern trees to climate change will thus impact the trajectory of future CO₂ increases. We grew two North American boreal tree species at a range of future climate conditions to assess how growth and carbon fluxes were altered by high CO₂ and warming. Black spruce (Picea mariana, an evergreen conifer) and tamarack (Larix laricina, a deciduous conifer) were grown under ambient (407 ppm) or elevated CO₂ (750 ppm) and either ambient temperatures, a 4°C warming, or an 8°C warming. In both species, the thermal optimum of net photosynthesis (T_optA) increased and maximum photosynthetic rates declined in warm‐grown seedlings, but the strength of these changes varied between species. Photosynthetic capacity (maximum rates of Rubisco carboxylation, V_cmax, and of electron transport, J_max) was reduced in warm‐grown seedlings, correlating with reductions in leaf N and chlorophyll concentrations. Warming increased the activation energy for V_cmax and J_max (E_aV and E_aJ, respectively) and the thermal optimum for J_max. In both species, the T_optA was positively correlated with both E_aV and E_aJ, but negatively correlated with the ratio of J_max/V_cmax. Respiration acclimated to elevated temperatures, but there were no treatment effects on the Q₁₀ of respiration (the increase in respiration for a 10°C increase in leaf temperature). A warming of 4°C increased biomass in tamarack, while warming reduced biomass in spruce. We show that climate change is likely to negatively affect photosynthesis and growth in black spruce more than in tamarack, and that parameters used to model photosynthesis in dynamic global vegetation models (E_aV and E_aJ) show no response to elevated CO₂.     

Comparison of Alternative Evidence Summary and Presentation Formats in Clinical Guideline Development: A Mixed-Method Study

Background

Best formats for summarising and presenting evidence for use in clinical guideline development remain less well defined. We aimed to assess the effectiveness of different evidence summary formats to address this gap.

Methods

Healthcare professionals attending a one-week Kenyan, national guideline development workshop were randomly allocated to receive evidence packaged in three different formats: systematic reviews (SRs) alone, systematic reviews with summary-of-findings tables, and ‘graded-entry’ formats (a ‘front-end’ summary and a contextually framed narrative report plus the SR). The influence of format on the proportion of correct responses to key clinical questions, the primary outcome, was assessed using a written test. The secondary outcome was a composite endpoint, measured on a 5-point scale, of the clarity of presentation and ease of locating the quality of evidence for critical neonatal outcomes. Interviews conducted within two months following completion of trial data collection explored panel members’ views on the evidence summary formats and experiences with appraisal and use of research information.

Results

65 (93%) of 70 participants completed questions on the prespecified outcome measures. There were no differences between groups in the odds of correct responses to key clinical questions. ‘Graded-entry’ formats were associated with a higher mean composite score for clarity and accessibility of information about the quality of evidence for critical neonatal outcomes compared to systematic reviews alone (adjusted mean difference 0.52, 95% CI 0.06 to 0.99). There was no difference in the mean composite score between SR with SoF tables and SR alone. Findings from interviews with 16 panelists indicated that short narrative evidence reports were preferred for the improved clarity of information presentation and ease of use.

Conclusions

Our findings suggest that ‘graded-entry’ evidence summary formats may improve clarity and accessibility of research evidence in clinical guideline development.

Trial Registration

Controlled-Trials.com ISRCTN05154264     

Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation,Structural Integrity,and Tolerance to Antibiotics

Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections.     

Proteomics,phylogenetics, and coexpression analyses indicate novel interactions in the plastid CLP chaperone-protease system

The chloroplast chaperone CLPC1 unfolds and delivers substrates to the stromal CLPPRT protease complex for degradation. We previously used an in vivo trapping approach to identify interactors with CLPC1 in Arabidopsis thaliana by expressing a STREPII-tagged copy of CLPC1 mutated in its Walker B domains (CLPC1-TRAP) followed by affinity purification and mass spectrometry. To create a larger pool of candidate substrates, adaptors, or regulators, we carried out a far more sensitive and comprehensive in vivo protein trapping analysis. We identified 59 highly enriched CLPC1 protein interactors, in particular proteins belonging to families of unknown functions (DUF760, DUF179, DUF3143, UVR-DUF151, HugZ/DUF2470), as well as the UVR domain proteins EXE1 and EXE2 implicated in singlet oxygen damage and signaling. Phylogenetic and functional domain analyses identified other members of these families that appear to localize (nearly) exclusively to plastids. In addition, several of these DUF proteins are of very low abundance as determined through the Arabidopsis PeptideAtlas http://www.peptideatlas.org/builds/arabidopsis/ showing that enrichment in the CLPC1-TRAP was extremely selective. Evolutionary rate covariation indicated that the HugZ/DUF2470 family coevolved with the plastid CLP machinery suggesting functional and/or physical interactions. Finally, mRNA-based coexpression networks showed that all 12 CLP protease subunits tightly coexpressed as a single cluster with deep connections to DUF760-3. Coexpression modules for other trapped proteins suggested specific functions in biological processes, e.g., UVR2 and UVR3 were associated with extraplastidic degradation, whereas DUF760-6 is likely involved in senescence. This study provides a strong foundation for discovery of substrate selection by the chloroplast CLP protease system.     

A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.     

The pervasive and multifaceted influence of biocrusts on water in the world's drylands

The capture and use of water are critically important in drylands, which collectively constitute Earth's largest biome. Drylands will likely experience lower and more unreliable rainfall as climatic conditions change over the next century. Dryland soils support a rich community of microphytic organisms (biocrusts), which are critically important because they regulate the delivery and retention of water. Yet despite their hydrological significance, a global synthesis of their effects on hydrology is lacking. We synthesized 2,997 observations from 109 publications to explore how biocrusts affected five hydrological processes (times to ponding and runoff, early [sorptivity] and final [infiltration] stages of water flow into soil, and the rate or volume of runoff) and two hydrological outcomes (moisture storage, sediment production). We found that increasing biocrust cover reduced the time for water to pond on the surface (?40%) and commence runoff (?33%), and reduced infiltration (?34%) and sediment production (?68%). Greater biocrust cover had no significant effect on sorptivity or runoff rate/amount, but increased moisture storage (+14%). Infiltration declined most (?56%) at fine scales, and moisture storage was greatest (+36%) at large scales. Effects of biocrust type (cyanobacteria, lichen, moss, mixed), soil texture (sand, loam, clay), and climatic zone (arid, semiarid, dry subhumid) were nuanced. Our synthesis provides novel insights into the magnitude, processes, and contexts of biocrust effects in drylands. This information is critical to improve our capacity to manage dwindling dryland water supplies as Earth becomes hotter and drier.     

Hydrogen Cycling by the Unicellular Marine Diazotroph Crocosphaera watsonii Strain WH8501

The hydrogen (H₂) cycle associated with the dinitrogen (N₂) fixation process was studied in laboratory cultures of the marine cyanobacterium Crocosphaera watsonii. The rates of H₂ production and acetylene (C₂H₂) reduction were continuously measured over the diel cycle with simultaneous measurements of fast repetition rate fluorometry and dissolved oxygen. The maximum rate of H₂ production was coincident with the maximum rates of C₂H₂ reduction. Theoretical stoichiometry for N₂ fixation predicts an equimolar ratio of H₂ produced to N₂ fixed. However, the maximum rate of net H₂ production observed was 0.09 nmol H₂ μg chlorophyll a (chl a)⁻¹ h⁻¹ compared to the N₂ fixation rate of 5.5 nmol N₂ μg chl a⁻¹ h⁻¹, with an H₂ production/N₂ fixation ratio of 0.02. The 50-fold discrepancy between expected and observed rates of H₂ production was hypothesized to be a result of H₂ reassimilation by uptake hydrogenase. This was confirmed by the addition of carbon monoxide (CO), a potent inhibitor of hydrogenase, which increased net H₂ production rates ∼40-fold to a maximum rate of 3.5 nmol H₂ μg chl a⁻¹ h⁻¹. We conclude that the reassimilation of H₂ by C. watsonii is highly efficient (>98%) and hypothesize that the tight coupling between H₂ production and consumption is a consequence of fixing N₂ at nighttime using a finite pool of respiratory carbon and electrons acquired from daytime solar energy capture. The H₂ cycle provides unique insight into N₂ fixation and associated metabolic processes in C. watsonii.The biological production of hydrogen (H₂) can occur as a by-product of photosynthesis, fermentation, and N₂ fixation (22). Of these three metabolic pathways, N₂ fixation remains a particularly enigmatic process, and to date there is no clear explanation for why H₂ evolves during the reduction of N₂ (11). The unfavorable energy cost of N₂ fixation can be mitigated by reassimilating the released H₂ via uptake hydrogenase enzyme activity (30). The coupled production and consumption of H₂ during cellular nitrogenase activity creates a H₂ cycle that can be hidden from measurements of ambient environmental H₂ concentrations and fluxes, depending upon the overall efficiency of H₂ assimilation (Fig. ​(Fig.11).Open in a separate windowFIG. 1.H₂ is formed during N₂ fixation by the binding of a N₂ molecule to the molybdenum-iron protein of the nitrogenase enzyme complex, prior to the reduction of N₂ to ammonia (11, 15). The most energetically favorable theoretical in vivo stoichiometry predicts that one mole of H₂ is produced for every mole of N₂ reduced: N₂ + 8H⁺ + 8e⁻ + 16ATP → 2NH₃ + H₂ + 16ADP + 16P_i. The production of H₂ consumes 25% of the electron flux through nitrogenase and diazotrophs mitigate this loss of potential energy by reassimilating the H₂ via uptake hydrogenase (21, 30). The electrons produced by uptake hydrogenase either generate reductant or ATP with simultaneous consumption of O₂ (3). (Adapted from reference 32a.)For most cultures of phototrophic marine diazotrophs grown under optimal conditions, complete reassimilation of H₂ is not achieved, and the excess H₂ is lost to the surrounding environment. This excess H₂ equates to the net production of H₂ and is expressed as the ratio of H₂ formed to N₂ fixed or the H₂/N₂ ratio. To date, H₂/N₂ ratios have mainly been measured on filamentous, colony-forming diazotrophs such as Anabaena spp. and Trichodesmium spp. with H₂ production rates of up to 20 nmol H₂ μg chlorophyll a (chl a)⁻¹ h⁻¹ and H₂/N₂ ratios ranging from 0.01 to 0.48 (3, 20, 24). H₂ production has also been quantified in unicellular diazotrophs (12, 16, 17, 32), although the H₂ measurements have rarely been performed in conjunction with rates of N₂ fixation. However, recent H₂ measurements of two N₂-fixing unicellular cyanobacteria species reached a maximum of 1.38 nmol H₂ μg chl a⁻¹ h⁻¹, with H₂/N₂ ratios ranging from 0.003 to 0.05, indicating an effective reassimilation of H₂ can occur under certain conditions (34).H₂ cycling in marine diazotrophs has important ecological implications both for the cell and for the marine H₂ cycle. Surface waters of low-latitude oceans are typically 200 to 300% supersaturated in dissolved H₂ with respect to atmospheric concentrations (25), implying a sustained localized production of H₂. The source of the dissolved H₂ is thought to be biological N₂ fixation (7); however, the relative contributions of diverse diazotrophic communities and in situ controls on H₂/N₂ ratios are not well constrained. N₂ fixation is performed by a suite of diazotrophs typically identified by their nitrogenase gene (nifH) sequences amplified directly from oceanic water samples (35). The importance of unicellular diazotrophs, including Crocosphaera spp., in marine N₂ fixation has recently become widely recognized (36). Size-fractionated rates of N₂ fixation indicate that in the oligotrophic ocean, <10-μm microorganisms, which include the unicellular cyanobacteria, make a substantial contribution to the daily N₂ fixation (9, 18). Correlating the species-specific production of H₂ with the activity and biomass of diazotrophs will help elucidate dissolved H₂ cycling in the upper ocean.We examined the cycling of H₂ in cultures of Crocosphaera watsonii strain WH8501, a marine unicellular diazotroph, and correlated it with other metabolic parameters, including N₂ fixation measured via acetylene (C₂H₂) reduction, O₂ production and consumption, and photosynthetic efficiency. Carbon monoxide (CO) was used as an inhibitor of intracellular H₂ reassimilation to reveal the H₂ cycling that can occur in conjunction with nitrogenase activity. H₂ reassimilation by C. watsonii was shown to be very efficient in our laboratory experiments, which is considered to be a consequence of the temporal separation between daytime photosynthetic activity and nighttime N₂ fixation. Therefore, the present study not only reveals the cell''s H₂ cycle but also provides insight into the metabolism of nitrogenase in C. watsonii.     

Information is a fitness enhancing resource

Here we provide a biologically relevant proof of the well‐known result from economics and statistical decision theory that having more information never reduces the expected payoff to a decision‐making agent. We then go on to illustrate this with an ecologically motivated example based on a model of growth under uncertain predation risk. Throughout we use the central result that the fitness (reproductive) value of information can never be negative to highlight conceptual inconsistencies in the ecological literature on information use.