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Marine phytoplankton produce and scavenge Reactive Oxygen Species, to support cellular processes, while limiting damaging reactions. Some prokaryotic picophytoplankton have, however, lost all genes encoding scavenging of hydrogen peroxide. Such losses of metabolic function can only apply to Reactive Oxygen Species which potentially traverse the cell membrane outwards, before provoking damaging intracellular reactions. We hypothesized that cell radius influences which elements of Reactive Oxygen Species metabolism are partially or fully dispensable from a cell. We therefore investigated genomes and transcriptomes from diverse marine eukaryotic phytoplankton, ranging from 0.4 to 44 μm radius, to analyze the genomic allocations encoding enzymes metabolizing Reactive Oxygen Species. Superoxide has high reactivity, short lifetimes and limited membrane permeability. Genes encoding superoxide scavenging are ubiquitous across phytoplankton, but the fractional gene allocation decreased with increasing cell radius, consistent with a nearly fixed set of core genes for scavenging superoxide pools. Hydrogen peroxide has lower reactivity, longer intracellular and extracellular lifetimes and readily crosses cell membranes. Genomic allocations to both hydrogen peroxide production and scavenging decrease with increasing cell radius. Nitric Oxide has low reactivity, long intracellular and extracellular lifetimes and readily crosses cell membranes. Neither Nitric Oxide production nor scavenging genomic allocations changed with increasing cell radius. Many taxa, however, lack the genomic capacity for nitric oxide production or scavenging. The probability of presence of capacity to produce nitric oxide decreases with increasing cell size, and is influenced by flagella and colony formation. In contrast, the probability of presence of capacity to scavenge nitric oxide increases with increasing cell size, and is again influenced by flagella and colony formation.

Polar microalgae face two major challenges: 1- growing at temperatures (-1.7 to 5°C) that limit enzyme kinetics; and 2- surviving and exploiting a wide range of irradiance. The objective of this study is to understand the adaptation of an Arctic diatom to its environment by studying its ability to acclimate to changes in light and temperature. We acclimated the polar diatom Chaetoceros neogracilis to various light levels at two different temperatures and studied its growth and photosynthetic properties using semi-continuous cultures. Rubisco content was high, to compensate for low catalytic rates, but did not change detectably with growth temperature. Contrary to what is observed in temperate species, in C. neogracilis, carbon fixation rate (20 min 14C incorporation) equaled net growth rate (μ) suggesting very low or very rapid (

Prochlorococcus marinus, the smallest picocyanobacterium, comprises multiple clades occupying distinct niches, currently across tropical and sub-tropical oligotrophic ocean regions, including Oxygen Minimum Zones. Ocean warming may open growth-permissive temperatures in new, poleward photic regimes, along with expanded Oxygen Minimum Zones. We used ocean metaproteomic data on current Prochlorococcus marinus niches, to guide testing of Prochlorococcus marinus growth across a matrix of peak irradiances, photoperiods, spectral bands and dissolved oxygen. MED4 from Clade HLI requires greater than 4 h photoperiod, grows at 25 μmol O2 L-1 and above, and exploits high cumulative diel photon doses. MED4, however, relies upon an alternative oxidase to balance electron transport, which may exclude it from growth under our lowest, 2.5 μmol O2 L-1, condition. SS120 from clade LLII/III is restricted to low light under full 250 μmol O2 L-1, shows expanded light exploitation under 25 μmol O2 L-1, but is excluded from growth under 2.5 μmol O2 L-1. Intermediate oxygen suppresses the cost of PSII photoinactivation, and possibly the enzymatic production of H2O2 in SS120, which has limitations on genomic capacity for PSII and DNA repair. MIT9313 from Clade LLIV is restricted to low blue irradiance under 250 μmol O2 L-1, but exploits much higher irradiance under red light, or under lower O2 concentrations, conditions which slow photoinactivation of PSII and production of reactive oxygen species. In warming oceans, range expansions and competition among clades will be governed not only by light levels. Short photoperiods governed by latitude, temperate winters, and depth attenuation of light, will exclude clade HLI (including MED4) from some habitats. In contrast, clade LLII/III (including SS120), and particularly clade LLIV (including MIT9313), may exploit higher light niches nearer the surface, under expanding OMZ conditions, where low O2 relieves the stresses of oxidation stress and PSII photoinhibition.

The opioid crisis is a growing public health emergency and increasing resources are being directed towards overdose education. Simulation has emerged as a novel strategy for training overdose response, yet little is known about training non-clinicians in bystander resuscitation. Understanding the perspectives of individuals who are likely to experience or witness opioid overdose is critical to ensure that emergency response is effective. The Surviving Opioid Overdose with Naloxone Education and Resuscitation (SOONER) study evaluates the effectiveness of a novel naloxone education and distribution tool among people who are non-clinicians and likely to witness opioid overdose. Participants' resuscitation skills are evaluated using a realistic overdose simulation as the primary outcome of the trial. The purpose of our study is to describe the experience of participants with the simulation process in the SOONER study. We employed a semi-structured debriefing interview and a follow up qualitative interview to understand the experience of participants with simulation. A qualitative content analysis was performed using data from 21 participants who participated in the SOONER study. Our qualitative analysis identified 5 themes and 17 subthemes which described the experience of participants within the simulation process. These themes included realism, valuing practical experience, improving self-efficacy, gaining new perspective and bidirectional learning. Our analysis found that simulation was a positive and empowering experience for participants in the SOONER trial, most of whom are marginalized in society. Our study supports the notion that expanding simulation-based education to non-clinicians may offer an acceptable and effective way of supplementing current opioid overdose education strategies. Increasing the accessibility of simulation-based education may represent a paradigm shift whereby simulation is transformed from a primarily academic practice into a patient-based community resource.

Marine phytoplankton, and in particular diatoms, are responsible for almost half of all primary production on Earth. Diatom species thrive from polar to tropical waters and across light environments that are highly complex to relatively benign, and so have evolved highly divergent strategies for regulating light capture and utilization. It is increasingly well established that diatoms have achieved such successful ecosystem dominance by regulating excitation energy available for generating photosynthetic energy via highly flexible light harvesting strategies. However, how different light harvesting strategies and downstream pathways for oxygen production and consumption interact to balance excitation pressure remains unknown. We therefore examined the responses of three diatom taxa adapted to inherently different light climates (estuarine Thalassioisira weissflogii, coastal Thalassiosira pseudonana and oceanic Thalassiosira oceanica) during transient shifts from a moderate to high growth irradiance (85 to 1200 μmol photons m-2 s-1). Transient high light exposure caused T. weissflogii to rapidly downregulate PSII with substantial nonphotochemical quenching, protecting PSII from inactivation or damage, and obviating the need for induction of O2 consuming (light-dependent respiration, LDR) pathways. In contrast, T. oceanica retained high excitation pressure on PSII, but with little change in RCII photochemical turnover, thereby requiring moderate repair activity and greater reliance on LDR. T. pseudonana exhibited an intermediate response compared to the other two diatom species, exhibiting some downregulation and inactivation of PSII, but high repair of PSII and induction of reversible PSII nonphotochemical quenching, with some LDR. Together, these data demonstrate a range of strategies for balancing light harvesting and utilization across diatom species, which reflect their adaptation to sustain photosynthesis under environments with inherently different light regimes.

Picocyanobacteria are the numerically dominant photoautotrophs of the oligotrophic regions of Earth's oceans. These organisms are characterized by their small size and highly reduced genomes. Strains partition to different light intensity and nutrient level niches, with differing photosynthetic apparatus stoichiometry, light harvesting machinery and susceptibility to photoinactivation. In this study, we grew three strains of picocyanobacteria: the low light, high nutrient strain Prochlorococcus marinus MIT 9313; the high light, low nutrient Prochlorococcus marinus MED 4; and the high light, high nutrient marine Synechococcus strain WH 8102; under low and high growth light levels. We then performed matched photophysiology, protein and transcript analyses. The strains differ significantly in their rates of Photosystem II repair under high light and in their capacity to remove the PsbA protein as the first step in the Photosystem II repair process. Notably, all strains remove the PsbD subunit at the same rate that they remove PsbA. When grown under low light, MIT 9313 loses active Photosystem II quickly when shifted to high light, but has no measurable capacity to remove PsbA. MED 4 and WH 8102 show less rapid loss of Photosystem II and considerable capacity to remove PsbA. MIT 9313 has less of the FtsH protease thought to be responsible for the removal of PsbA in other cyanobacteria. Furthermore, by transcript analysis the predominant FtsH isoform expressed in MIT 9313 is homologous to the FtsH 4 isoform characterized in the model strain Synechocystis PCC 6803, rather than the FtsH 2 and 3 isoforms thought to be responsible for PsbA degradation. MED 4 on the other hand shows high light inducible expression of the isoforms homologous to FtsH 2 and 3, consistent with its faster rate of PsbA removal. MIT 9313 has adapted to its low light environment by diverting resources away from Photosystem II content and repair.

While measurement of serum prostate specific antigen (PSA) is an important screening tool for prostate cancer, new biomarkers are necessary for better discrimination between presence and absence of disease. The MIL-38 monoclonal antibody is specific for the membrane glycoprotein glypican 1 (GPC-1) and binds to prostate cancer tissue. Urine is known to be a source of cellular material. Thus, we hypothesized that detection of GPC-1 in urine cellular material may identify individuals with prostate cancer. Urine samples from patients with prostate cancer, benign prostatic hyperplasia (BPH), or normal controls were collected and cell sediments prepared. GPC-1-positive cells were detected using a MIL-38 immunofluorescence assay (IFA) and samples were classed positive or negative for GPC-1 expressing cells. Assay sensitivity and specificity, stratified by PSA, was reported. A total of 125 patient samples were analyzed (N = 41 prostate cancer; N = 37 BPH; N = 47 normal controls). The use of MIL-38 to detect GPC-1 by IFA discriminated between prostate cancer and BPH urine specimens with a sensitivity and specificity of 71% and 76%, respectively. Assay specificity increased with increasing PSA, with the highest specificity (89%) for patients with PSA ≥4 ng/ml. At lower PSA (

Nitrogen stress is an important control on the growth of phytoplankton and varying responses to this common condition among taxa may affect their relative success within phytoplankton communities. We analyzed photosynthetic responses to nitrogen (N) stress in two classes of phytoplankton that often dominate their respective size ranges, diatoms and prasinophytes, selecting species of distinct niches within each class. Changes in photosynthetic structures appeared similar within each class during N stress, but photophysiological and growth responses were more species- or niche-specific. In the coastal diatom Thalassiosira pseudonana and the oceanic diatom T. weissflogii, N starvation induced large declines in photosynthetic pigments and Photosystem II (PSII) quantity and activity as well as increases in the effective absorption cross-section of PSII photochemistry (σ'PSII). These diatoms also increased photoprotection through energy-dependent non-photochemical quenching (NPQ) during N starvation. Resupply of N in diatoms caused rapid recovery of growth and relaxation of NPQ, while recovery of PSII photochemistry was slower. In contrast, the prasinophytes Micromonas sp., an Arctic Ocean species, and Ostreococcus tauri, a temperate coastal eutrophile, showed little change in photosynthetic pigments and structures and a decline or no change, respectively, in σ'PSII with N starvation. Growth and PSII function recovered quickly in Micromonas sp. after resupply of N while O. tauri failed to recover N-replete levels of electron transfer from PSII and growth, possibly due to their distinct photoprotective strategies. O. tauri induced energy-dependent NPQ for photoprotection that may suit its variable and nutrient-rich habitat. Micromonas sp. relies upon both energy-dependent NPQ and a sustained, energy-independent NPQ mechanism. A strategy in Micromonas sp. that permits photoprotection with little change in photosynthetic structures is consistent with its Arctic niche, where low temperatures and thus low biosynthetic rates create higher opportunity costs to rebuild photosynthetic structures.

Diatoms are marine primary producers that sink in part due to the density of their silica frustules. Sinking of these phytoplankters is crucial for both the biological pump that sequesters carbon to the deep ocean and for the life strategy of the organism. Sinking rates have been previously measured through settling columns, or with fluorimeters or video microscopy arranged perpendicularly to the direction of sinking. These side-view techniques require large volumes of culture, specialized equipment and are difficult to scale up to multiple simultaneous measures for screening. We established a method for parallel, large scale analysis of multiple phytoplankton sinking rates through top-view monitoring of chlorophyll a fluorescence in microtitre well plates. We verified the method through experimental analysis of known factors that influence sinking rates, including exponential versus stationary growth phase in species of different cell sizes; Thalassiosira pseudonana CCMP1335, chain-forming Skeletonema marinoi RO5A and Coscinodiscus radiatus CCMP312. We fit decay curves to an algebraic transform of the decrease in fluorescence signal as cells sank away from the fluorometer detector, and then used minimal mechanistic assumptions to extract a sinking rate (m d-1) using an RStudio script, SinkWORX. We thereby detected significant differences in sinking rates as larger diatom cells sank faster than smaller cells, and cultures in stationary phase sank faster than those in exponential phase. Our sinking rate estimates accord well with literature values from previously established methods. This well plate-based method can operate as a high throughput integrative phenotypic screen for factors that influence sinking rates including macromolecular allocations, nutrient availability or uptake rates, chain-length or cell size, degree of silification and progression through growth stages. Alternately the approach can be used to phenomically screen libraries of mutants.

The marine picocyanobacteria Synechococcus and Prochlorococcus numerically dominate open ocean phytoplankton. Although evolutionarily related they are ecologically distinct, with different strategies to harvest, manage and exploit light. We grew representative strains of Synechococcus and Prochlorococcus and tracked their susceptibility to photoinactivation of Photosystem II under a range of light levels. As expected blue light provoked more rapid photoinactivation than did an equivalent level of red light. The previous growth light level altered the susceptibility of Synechococcus, but not Prochlorococcus, to this photoinactivation. We resolved a simple linear pattern when we expressed the yield of photoinactivation on the basis of photons delivered to Photosystem II photochemistry, plotted versus excitation pressure upon Photosystem II, the balance between excitation and downstream metabolism. A high excitation pressure increases the generation of reactive oxygen species, and thus increases the yield of photoinactivation of Photosystem II. Blue photons, however, retained a higher baseline photoinactivation across a wide range of excitation pressures. Our experiments thus uncovered the relative influences of the direct photoinactivation of Photosystem II by blue photons which dominates under low to moderate blue light, and photoinactivation as a side effect of reactive oxygen species which dominates under higher excitation pressure. Synechococcus enjoyed a positive metabolic return upon the repair or the synthesis of a Photosystem II, across the range of light levels we tested. In contrast Prochlorococcus only enjoyed a positive return upon synthesis of a Photosystem II up to 400 μmol photons m-2 s-1. These differential cost-benefits probably underlie the distinct photoacclimation strategies of the species.

Iron is an essential component in many protein complexes involved in photosynthesis, but environmental iron availability is often low as oxidized forms of iron are insoluble in water. To adjust to low environmental iron levels, cyanobacteria undergo numerous changes to balance their iron budget and mitigate the physiological effects of iron depletion. We investigated changes in key protein abundances and photophysiological parameters in the model cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 over a 120 hour time course of iron deprivation. The iron stress induced protein (IsiA) accumulated to high levels within 48 h of the onset of iron deprivation, reaching a molar ratio of ~42 IsiA : Photosystem I in Synechococcus PCC 7942 and ~12 IsiA : Photosystem I in Synechocystis PCC 6803. Concomitantly the iron-rich complexes Cytochrome b6f and Photosystem I declined in abundance, leading to a decrease in the Photosystem I : Photosystem II ratio. Chlorophyll fluorescence analyses showed a drop in electron transport per Photosystem II in Synechococcus, but not in Synechocystis after iron depletion. We found no evidence that the accumulated IsiA contributes to light capture by Photosystem II complexes.

We studied the interactive effects of pCO(2) and growth light on the coastal marine diatom Thalassiosira pseudonana CCMP 1335 growing under ambient and expected end-of-the-century pCO(2) (750 ppmv), and a range of growth light from 30 to 380 µmol photons·m(-2)·s(-1). Elevated pCO(2) significantly stimulated the growth of T. pseudonana under sub-saturating growth light, but not under saturating to super-saturating growth light. Under ambient pCO(2) susceptibility to photoinactivation of photosystem II (σ(i)) increased with increasing growth rate, but cells growing under elevated pCO(2) showed no dependence between growth rate and σ(i), so under high growth light cells under elevated pCO(2) were less susceptible to photoinactivation of photosystem II, and thus incurred a lower running cost to maintain photosystem II function. Growth light altered the contents of RbcL (RUBISCO) and PsaC (PSI) protein subunits, and the ratios among the subunits, but there were only limited effects on these and other protein pools between cells grown under ambient and elevated pCO(2).

DNA content and cell volume have both been hypothesized as controls on metabolic rate and other physiological traits. We use cultures of two cryptic species of Ditylum brightwellii (West) Grunow with an approximately two-fold difference in genome size and a small and large culture of each clone obtained by isolating small and large cells to compare the physiological consequences of size changes due to differences in DNA content and reduction in cell size following many generations of asexual reproduction. We quantified the growth rate, the functional absorption cross-section of photosystem II (PSII), susceptibility of PSII to photoinactivation, PSII repair capacity, and PSII reaction center proteins D1 (PsbA) and D2 (PsbD) for each culture at a range of irradiances. The species with the smaller genome has a higher growth rate and, when acclimated to growth-limiting irradiance, has higher PSII repair rate capacity, PSII functional optical absorption cross-section, and PsbA per unit protein, relative to the species with the larger genome. By contrast, cell division rates vary little within clonal cultures of the same species despite significant differences in average cell volume. Given the similarity in cell division rates within species, larger cells within species have a higher demand for biosynthetic reductant. As a consequence, larger cells within species have higher numbers of PSII per unit protein (PsbA), since PSII photochemically generates the reductant to support biosynthesis. These results suggest that DNA content, as opposed to cell volume, has a key role in setting the differences in maximum growth rate across diatom species of different size while PSII content and related photophysiological traits are influenced by both growth rate and cell size.

Reef corals are heterotrophic coelenterates that achieve high productivity through their photosynthetic dinoflagellate symbionts. Excessive seawater temperature destabilises this symbiosis and causes corals to "bleach," lowering their photosynthetic capacity. Bleaching poses a serious threat to the persistence of coral reefs on a global scale. Despite expanding research on the causes of bleaching, the mechanisms remain a subject of debate.This study determined how light and food availability modulate the effects of temperature stress on photosynthesis in two reef coral species. We quantified the activities of Photosystem II, Photosystem I and whole chain electron transport under combinations of normal and stressful growth temperatures, moderate and high light levels and the presence or absence of feeding of the coral hosts. Our results show that PS1 function is comparatively robust against temperature stress in both species, whereas PS2 and whole chain electron transport are susceptible to temperature stress. In the symbiotic dinoflagellates of Stylophora pistillata the contents of chlorophyll and major photosynthetic complexes were primarily affected by food availability. In Turbinaria reniformis growth temperature was the dominant influence on the contents of the photosynthetic complexes. In both species feeding the host significantly protected photosynthetic function from high temperature stress.Our findings support the photoinhibition model of coral bleaching and demonstrate that PS1 is not a major site for thermal damage during bleaching events. Feeding mitigates bleaching in two scleractinian corals, so that reef responses to temperature stresses will likely be influenced by the coinciding availabilities of prey for the host.

Prochlorococcus and Synechococcus picocyanobacteria are dominant contributors to marine primary production over large areas of the ocean. Phytoplankton cells are entrained in the water column and are thus often exposed to rapid changes in irradiance within the upper mixed layer of the ocean. An upward fluctuation in irradiance can result in photosystem II photoinactivation exceeding counteracting repair rates through protein turnover, thereby leading to net photoinhibition of primary productivity, and potentially cell death. Here we show that the effective cross-section for photosystem II photoinactivation is conserved across the picocyanobacteria, but that their photosystem II repair capacity and protein-specific photosystem II light capture are negatively correlated and vary widely across the strains. The differences in repair rate correspond to the light and nutrient conditions that characterize the site of origin of the Prochlorococcus and Synechococcus isolates, and determine the upward fluctuation in irradiance they can tolerate, indicating that photoinhibition due to transient high-light exposure influences their distribution in the ocean.