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Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder ‘click’ reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by 125I) and subsequent accumulation of an 111In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of 111In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using 111In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.

Few treatment options are available for acute myeloid leukemia (AML) patients. DCLL9718A is an antibody-drug conjugate that targets C-type lectin-like molecule-1 (CLL-1). This receptor is prevalent on monocytes, neutrophils, and AML blast cells, and unlike CD33, is not expressed on hematopoietic stem cells, thus providing possible hematopoietic recovery. DCLL9718A comprises an anti-CLL-1 IgG1 antibody (MCLL0517A) linked to a pyrrolobenzodiazepine (PBD) dimer payload, via a cleavable disulfide-labile linker. Here, we characterize the in vitro and in vivo stability, the pharmacokinetics (PK) and pharmacodynamics (PD) of DCLL9718A and MCLL0517A in rodents and cynomolgus monkeys. Three key PK analytes were measured in these studies: total antibody, antibody-conjugated PBD dimer and unconjugated PBD dimer. In vitro, DCLL9718A, was stable with most (> 80%) of the PBD dimer payload remaining conjugated to the antibody over 96 hours. This was recapitulated in vivo with antibody-conjugated PBD dimer clearance estimates similar to DCLL9718A total antibody clearance. Both DCLL9718A and MCLL0517A showed linear PK in the non-binding rodent species, and non-linear PK in cynomolgus monkeys, a binding species. The PK data indicated minimal impact of conjugation on the disposition of DCLL9718A total antibody. Finally, in cynomolgus monkey, MCLL0517A showed target engagement at all doses tested (0.5 and 20 mg/kg) as measured by receptor occupancy, and DCLL9718A (at doses of 0.05, 0.1 and 0.2 mg/kg) showed strong PD activity as evidenced by notable reduction in monocytes and neutrophils.

In Chile, 90% of the fish farms and major natural shellfish beds are located in the region surrounding the Inland Sea, where over the last few decades harmful phytoplankton blooms have often been observed. The onset and recurrence of bloom events are often related to the resuspension and germination of resting cysts that have accumulated in the sediments. The degree of cyst settling, accumulation and germination is highly variable between areas and depends on physical and environmental factors. To learn how differences in oceanographic exposure, amount of river runoff and bathymetry affect dinoflagellate cyst deposition, we examined the diversity and abundance of dinoflagellate resting cysts from two hydrographically contrasting coastal areas (oceanic Guaitecas Archipelago and estuarine Pitipalena Fjord) of the Chilean Inland Sea in September 2006, seven months after a bloom of Alexandrium catenella, a producer of paralytic shellfish toxin. Cyst species diversity consisted of 18 taxa, including A. catenella and the noxious species Protoceratium reticulatum, both of which have caused blooms in the study area. Our results revealed significant differences between the two study sites in terms of the abundance and diversity of resting cysts, suggesting that in the specific case of A. catenella, only Guaitecas stations have potential for cyst accumulation and successful growth of cells. However, there was no evidence of long-term resting cyst beds of A. catenella at either study site.

For antibody-drug conjugates (ADCs) that carry a cytotoxic drug, doses that can be administered in preclinical studies are typically limited by tolerability, leading to a narrow dose range that can be tested. For molecules with non-linear pharmacokinetics (PK), this limited dose range may be insufficient to fully characterize the PK of the ADC and limits translation to humans. Mathematical PK models are frequently used for molecule selection during preclinical drug development and for translational predictions to guide clinical study design. Here, we present a practical approach that uses limited PK and receptor occupancy (RO) data of the corresponding unconjugated antibody to predict ADC PK when conjugation does not alter the non-specific clearance or the antibody-target interaction. We used a 2-compartment model incorporating non-specific and specific (target mediated) clearances, where the latter is a function of RO, to describe the PK of anti-CD33 ADC with dose-limiting neutropenia in cynomolgus monkeys. We tested our model by comparing PK predictions based on the unconjugated antibody to observed ADC PK data that was not utilized for model development. Prospective prediction of human PK was performed by incorporating in vitro binding affinity differences between species for varying levels of CD33 target expression. Additionally, this approach was used to predict human PK of other previously tested anti-CD33 molecules with published clinical data. The findings showed that, for a cytotoxic ADC with non-linear PK and limited preclinical PK data, incorporating RO in the PK model and using data from the corresponding unconjugated antibody at higher doses allowed the identification of parameters to characterize monkey PK and enabled human PK predictions.

Benthic dinoflagellates of the genus Gambierdiscus produce ciguatoxins, compounds that when metabolized in fish and consumed by humans cause ciguatera poisoning (CP). This syndrome, which is widespread in tropical and subtropical regions, has recently been reported also in subtropical-temperate latitudes such as the Canary Islands where CP events have been regularly detected since 2004. This study examined the effect of temperature on the growth of Gambierdiscus isolated from Canary waters: G. australes, G. caribaeus, G. carolinianus, G. excentricus, and G. silvae. From the temperature vs. growth curves, the maximum growth (µm), optimum temperature range for growth (Topt), and the temperature yielding maximum growth (Tm) were estimated for each species. The results revealed temperature-dependent differences in the growth parameters. G. caribaeus had the highest Tm and Topt, followed by G. australes, G. carolinianus, G. silvae, and G excentricus. G. australes tolerated the widest range of temperatures (from 15 ◦C to 29 ◦C), which may explain its broader geographic distribution, both worldwide and across the Canary archipelago. Neither G. excentricus nor G. silvae survived at 29 ◦C whereas G. caribaeus reached mean growth rates (± standard deviation) up to 0.19 ± 0.01 div.day− 1 at that temperature, followed by G. australes (0.16 ± 0.01 div.day− 1) and G. carolinianus (0.14 ± 0.04 div.day− 1). G. caribaeus showed no measurable growth at 19◦C, whereas G. excentricus and G. silvae along with G. australes appeared as the species better adapted to lower temperatures. In an intraspecific variability study of 12 strains of G. australes, the mean (± standard deviation) of µm and Tm were 0.17 ± 0.01 div.day− 1 and 27.7 ± 0.5 ◦C, respectively. An analysis of the shapes and position of the cell nuclei at the different temperatures showed that nuclei characteristic of vegetative cells appeared mainly at 26 ◦C but extreme temperatures resulted in nuclei with a more variable morphology. The presence of putative zygotes at extreme temperatures (17 ◦C, 19 ◦C and 29 ◦C) suggests that sexual reproduction is promoted as an adaptive strategy which could play an important role in the expansion of geographic distribution of Gambierdiscus species., SI

Marine parasites of the genus Parvilucifera have been described as endoparasitoids of dinoflagellates. Recently, the species Parvilucifera corolla was described, but its host range was not examined. Here, the host selectivity of P. corolla was screened, including 110 strains of dinoflagellates (24 genera) and other microalgal groups as potential hosts. Infections and the full life cycle of the parasitoid were observed in 73 strains (16 genera) of dinoflagellates. Parvilucifera corolla did not infect most chlorophytes, cryptophytes, chrysophytes, diatoms, haptophytes and raphidophytes but one strain of Pyramimonas(chlorophyte) was infected, although without viable sporangia. In Symbiodinium natans, a transition to the coccoid stage was induced above a certain parasite:host ratio. These results confirm P. corolla as a generalist parasitoid of dinoflagellates, with important differences in host range regarding other species of the genus., Sí

Elucidation of the cell cycle of dinoflagellates is essential to understand the processes leading to their massive proliferations, known as harmful algal blooms. In this study, we used imaging flow cytometry (IFC) to monitor the changes in DNA content and nuclear and cell morphology that occur during clonal growth of the toxic species Alexandrium minutum Halim. Our results indicate that the population was in S phase (C→2C DNA content) during the light period, whereas haploid cells with a C DNA content peaked only during a short interval of the dark period. The timing of the phases, identified based on the nuclear morphology and cytoplasmic-to-nuclear (CNR) ratio of the cells, suggests that the length of the G2/M phase is regulated by nutrient levels whereas the beginning of S phase is clock controlled. In addition we found that up to 7% of individual cells achieved a DNA content higher than 2C, indicative of either zygote formation and replication (homothallism), or of double-haploid cells able to divide (polyploid forms). Cells belonging to different cell cycle phases (G1-S-G2/M) could be readily discriminated based on nuclear size. Our study provides evidence of cell-cycle plasticity during clonal growth and unambiguously characterizes the cell-cycle phases of this dinoflagellate species., SI

11 pages, 6 figures, supporting information http://onlinelibrary.wiley.com/doi/10.1002/ece3.1314/suppinfo, Ecology and Evolution published by John Wiley & Sons Ltd. The dynamics and consequences of host-parasite coevolution depend on the nature of host genotype-by-parasite genotype interactions (G × G) for host and parasite fitness. G × G with crossing reaction norms can yield cyclic dynamics of allele frequencies ("Red Queen" dynamics) while G × G where the variance among host genotypes differs between parasite genotypes results in selective sweeps ("arms race" dynamics). Here, we investigate the relative potential for arms race and Red Queen coevolution in a protist host-parasite system, the dinoflagellate Alexandrium minutum and its parasite Parvilucifera sinerae. We challenged nine different clones of A. minutum with 10 clones of P. sinerae in a fully factorial design and measured infection success and host and parasite fitness. Each host genotype was successfully infected by four to ten of the parasite genotypes. There were strong G × Gs for infection success, as well as both host and parasite fitness. About three quarters of the G × G variance components for host and parasite fitness were due to crossing reaction norms. There were no general costs of resistance or infectivity. We conclude that there is high potential for Red Queen dynamics in this host-parasite system. We investigate the relative potential for arms race and Red Queen coevolution in a protist host-parasite system by dissecting the nature of host geontype-by-parasite genotype interactions (G × G). G × Gs were mainly a result of crossing reaction norms, indicating high potential for Red Queen dynamics. © 2014 The Authors, This research was funded by the Crafoord Foundation (contract 2011:0882 to RF) and Spanish Ministry of Science and Innovation (project PARAL CTM2009-08399 to EG). L. Råberg was supported by a fellowship from the Swedish Research Council

16 pages, 7 figures, 4 tables, This study begins with a description of the infective process in the dinoflagellate type host Alexandrium minutum by a strain of the parasitoid, Parvilucifera sinerae, including the morphologies of the various dinoflagellate and parasitoid stages during the infection. Then, the susceptibility of 433 microalgal strains to P. sinerae infection was studied. The parasitoid was found to be capable of infecting several dinoflagellate species of the genera Alexandrium, Coolia, Dinophysis, Fragilidium, Gambierdiscus, Gymnodinium, Gyrodinium, Heterocapsa, Kryptoperidinium, Lepidodinium, Ostreopsis, Pentapharsodinium, Protoceratium, Scrippsiella, and Woloszynskia. Intra-strain variability was observed as well, such that within the same dinoflagellate species some strains were infected whereas others were not. Likewise, species of other dinoflagellate genera were not infected, such as Akashiwo, Amphidinium, Barrufeta, Bysmatrum, Karenia, Karlodinium, Prorocentrum, and Takayama. Moreover, P. sinerae was not able to infect any of the tested haptophyte, diatom, and chlorophyte species. In natural samples screened for P. sinerae infectivity, several dinoflagellate species of the genera Alexandrium, Coolia, Gonyaulax, Gymnodinium, Phalacroma, Protoperidinium, and Scrippsiella were identified as susceptible. Sporangia size was found to be proportional to the size of the host, and variations in the sporangia size were observed to influence their maturation time, This study was supported by the Spanish funded project PARAL (CTM2009-08399) and project CCVIEO. We thank N. Sampedro, A. Re˜né, S. Anglès, F. Rodriguez, and C. Satta for providing several strains used in this study. We also acknowledge the technical assistance of I. Ramilo, A. Fernández, and P. Rial in culturing the strains. W. Coats provided very useful comments on an earlier version of the manuscript

We isolated eleven strains of the harmful algal bloom (HAB)-forming dinoflagellate Karlodinium veneficum during a bloom event in the NW Mediterranean coastal waters and we studied the inter-strain variability in several of their physiological and biochemical traits. These included autotrophic growth parameters, feeding capabilities (mixotrophy), lipid composition, and, in some cases, their responses to biotic and abiotic factors. The strains were found to differ in their growth rates (0.27–0.53 d−1) and in the maximum cell concentrations achieved during stationary phase (6.1 × 104–8.6 × 104 cells mL−1). Their ingestion performance, when offered Rhodomonas salina as prey, was also diverse (0.22–1.3 cells per K. veneficum per day; 8–52% of their daily ration). At least two strains survived for several months under strict heterotrophic conditions (no light, low inorganic nutrients availability, and R. salina as food source). These strains also showed very distinct fatty acid compositions, with very low contents of monounsaturated and polyunsaturated fatty acids. According to a Bray Curtis similarity analysis, three or four strain groups able to perform different roles in bloom development were identified. We further analyzed one strain from each of the two most distinct groups with respect to prey concentration, light intensity, nutrient availability, and we determined the functional responses (growth and feeding rates) to food concentration. Taken together, the results served to highlight the role of mixotrophy and clone variability in the formation of HABs, This work was funded through projects CTM2004-02575/MAR and CTM2009-08783 from the Ministry of Science and Innovation to A.C. E.G. was supported by a Ramon y Cajal contract of the Spanish Ministry of Science and Innovation (MICINN), and C.F-G. by Comisión Nacional de Investigación Ciencia y Tecnología (CONICYT) Chile

8 pages, 5 figures, 3 tables, A parasite threat stimulates adaptive shifts in the life-history strategy (sexual recombination rate) of the toxic bloom-forming dinoflagellate Alexandrium minutum Halim. This microalgae divides asexually when clonal but can also form mobile zygotes (planozygotes) when compatible clones are crossed. Planozygotes usually form resistant dormant stages (resting cysts) although they can also divide. In this study, asexual and sexual cultures were infected with the parasite Parvilucifera sinerae (Perkinsozoa) and the resulting clones classified as susceptible (S), low susceptible (LS), or resistant (R) to the infection. R and LS clones were never of Mediterranean origin, pointing to local adaptation of the parasite. (S × S) crosses were infected faster than either of the parental clones growing asexually. By contrast, (S × R) crosses were resistant to the parasite and produced no resting cysts, even when planozygotes were formed. Therefore, in infected cultures, the planozygotes mainly divided instead of encysting, thus increasing the rate at which recombinant progeny formed. This strategy against infection seems to combine the benefits of quickly producing asexual offspring and increasing recombination. As the susceptibility of the crosses was dependent on parental sexual compatibility, and cultures established by the division of (R × S) planozygotes (F1 offspring) also formed R or LS cultures, resistance may be regulated by several genes or through maternal effects, This study was supported by the Spanish funded project PARAL (CTM2009-08399). The work of R.I. Figueroa and E. Garcés was supported by a postdoctoral grant I3P and a Ramon y Cajal award respectively, from the CSIC and the Spanish Ministry of Science and Innovation

Slow granular flows play an important role in industries ranging from food to pharmaceuticals to ceramics. This subject has received much attention in recent literature; however, heat transfer in even the simplest particle flows is poorly understood. Heat transfer depends on the flow of granular materials: the bed conductivity is a function of the (changing) micro-structure; heat redistribution also depends on particle mixing/segregation. In this work, a multi-scale, multi-physics modeling technique, thermal particle dynamics (TPD), is used to examine the interplay between transient heat transfer and particle mixing in rotating tumblers. We study the effect of the mixing rate on the heating rate of the granular material by changing the tumbler cross-sectional shape and operation parameters—rotation rate and tumbler filling level. We use the Peclet number in the granular bed to determine the dominant heating mechanism—conduction or convection—and to predict conditions that will favor more rapid flow of thermal energy in the granular bed. The rate of heat transport is characterized using the Nusselt number and a fluids-inspired relationship correlating this quantity with the Peclet values is attempted. Finally, we use a continuum model to study the relationship between the mixing patterns and temperature contours.

Processing of fine powders is a relevant operation in many industries, from pharmaceuticals to material synthesis. As the size of the particles decreases, van der Waals forces start to play an important role in the behavior of the granular material and can dramatically impact the degree of mixing. In this work, we study the mixing and segregation behavior of fine particle systems using models capable of solving both normal and tangential interactions of elastic-plastic particles in the presence of van der Waals forces. We use scaling arguments to analytically predict the asymptotic state of the system by comparing the relative magnitude of the adhesive force with the other relevant forces. These predictions are most easily summarized by phase-space diagrams which exhibit both mixed and segregated regions. We compare the result of our Particle Dynamics simulations for different particle systems with our predictions. Graphical abstract Here we develop a predictive model for assessing the impact of adhesive forces on the mixing and segregation of fine powders. Interestingly, these forces may both enhance mixing or cause segregation, depending on the properties of the differing particles.

The microalgal genus Alexandrium includes species known to produce paralytic shellfish poisoning (PSP). Due to the importance of discriminating between HAB-forming species, we compared the undescribed life-cycle pattern of Alexandrium tamutum Montresor, Beran et U. John and of its toxic relative Alexandrium minutum Halim. Sexual stages, asexual and sexual division, mating type, and nuclear morphology were studied in both species. Sexual cysts are known to be morphologically identical. However, the relative size of the U-shaped nucleus may be used to differentiate between the cysts of these species since DNA packaging in the resting cysts was lower in A. tamutum than in A. minutum, species in which the planozygote nucleus was reduced to half its volume prior to encystment. The dormancy period of the cysts was

12 pages, 7 figures, 4 tables, Alexandrium taylori Balech is a cyst‐forming dinoflagellate species responsible for recurrent blooms in Mediterranean coastal waters. The nuclear development of the cells during the sexual cycle and the effect of different external nitrate and phosphate levels were studied. Nuclear fusion of gametes occurred 6–12 h after the complete cytoplasmic fusion. The U‐shaped nuclei fused through the end of one nucleus and the mid‐area of the other. The mobile and biflagellated zygote had a large, U‐shaped nucleus and may follow three different fates: direct division, short‐term encystment (ecdysal), and long‐term encystment (resting). Ecdysal cysts may divide in >24–96 h into two, four, six, or eight cells before germinating. Meiosis presumably occurred in three locations: in the planozygote, within the ecdysal cyst, and in the planomeiocyte (germling) liberated either from ecdysal or resting cysts. The effects of nutrients on these routes were studied in individually isolated sexual stages. (1) Direct divisions occurred mainly under replete conditions (L1), whereas no direct planozygote divisions were recorded in media with no phosphate added (L‐P). (2) Short‐term encystment was larger in media lacking phosphate (L‐P and L/30) than in medium with no nitrate added (L‐N) or under replete conditions (L1). (3) Long‐term encystment was only observed in medium with no nitrate added (L‐N). The long‐lived resting cyst, not previously described for this species, had a clear double wall, an irregular shape, a flat morphology, and a middle orange spot. No cysts germinated in 1–2 months, whereas 86% of the cysts germinated 2–3 months after being formed. A flow cytometry analysis showed that sexual induction and zygote formation were very fast and highly common processes, zygotes being nearly half of the population at days 3 and 5 after the induction of sexuality in the cultures, This study was supported by the EC‐funded Research Project SEED (Life cycle transformations among HAB species, and the environmental and physiological factors that regulate them) GOCE‐CT‐2005‐003875. E. Garcés's work was supported by the Ramon y Cajal contract of the Spanish Ministry of Education and Science

Gymnodinium catenatum Graham is an unarmored, cyst-forming dinoflagellate species responsible for outbreaks of paralytic shellfish poisoning. The nuclear development of the cells during the sexual cycle and the effect of different nitrate and phosphate external levels on sexual stages were studied. Nuclear fusion of gametes occurred before or at the same time as cytoplasmic fusion. During this process, either both nuclei migrated to a central area in the sulcal region, or only one of them migrated to the other nucleus. The motile and longitudinally biflagellated zygote presented a large, pear-shaped nucleus, and either divided or encysted. Planozygotes and germlings underwent similar division processes, which suggested an uncoordinated meiosis in both encysting and non-encysting zygotes. Encystment in culture was greater under low nitrate and phosphate limitation (L/15) than when only one or neither of these nutrients were added (L-N, L-P, and -N-P). However, planozygotes individually monitored achieved the maximum encystment (40%) in a medium with no phosphate or nitrate added (-N-P), while most of them divided (70%-90%) in replete (L1) or half-replete (L-N and L-P) media. Low levels of nitrate in the medium of cyst formation promoted a deficient development of the cyst wall. On the other hand, low phosphate levels in the medium of germination prevented both planozygote and germling division and lowered the final germination frequencies of cysts. The minimum dormancy, with an average value of 13.7 ± 5.5 days, was not affected by any of the nutritional conditions studied.

Unreported aspects in the sexual cycle of the marine dinoflagellate Lingulodinium polyedrum (Stein) Dodge were described. Our observations included the description of two types of hypnozygote formation, because culture planozygotes were observed to encyst in two different ways: an ecdysal sexual stage or a spiny resting cyst. Phosphate deficiency was the main nutritional condition required for fusing gamete pairs to form resting cysts, whereas replete conditions prevented their appearance and favored the formation of ecdysal sexual forms. Mating experiments revealed the existence of two sexual types (+/-), which were enough to explain resting cyst appearance (simple heterothallism). Morphological aspects and timing of gamete mating, fusion, and the efficiency of encystment under different external levels of nitrate and phosphate were analyzed after isolating and monitoring individual pairs of fusing gametes. The staining of sexual stages showed that nuclear fusion was completed at the same time as the cytoplasmic fusion. After 1 to 2 h, the planozygotes presented one quadrolobulated nucleus. Germination of ecdysal sexual stages occurred after < 24-72 h, whereas excystment of resting cysts was dependent on the studied parental cross and took place after 2-4 months. Newly germinated cells from both types of cysts had a similar, big, U-shaped nucleus. Twenty-four to 48 h after excystment, the germlings divided by desmoschisis, a process before which enlargement of the nucleus was observed.

Cell abundances and distributions of Alexandrium catenella resting cysts in recent sediments were studied along time at two locations in the Chilean Inland Sea exposed to different oceanographic conditions: Low Bay, which is much more open to the ocean than the more interior and protected Ovalada Island. The bloom began in interior areas but maximum cyst concentrations were recorded in locations more open to the ocean, at the end of the Moraleda channel. Our results showed a time lapse of around 3 months from the bloom peak (planktonic population) until the number of resting cysts in the sediments reached a maximum. Three months later, less than 10% of the A. catenella cysts remained in the sediments. Maximum cyst numbers in the water column occurred one month after the planktonic peak, when no cells were present. The dinoflagellate assemblage at both study sites was dominated by heterotrophic cysts, except during the A. catenella bloom. CCA analyses of species composition and environmental factors indicated that the frequency of A. catenella blooms was associated with low temperatures, but not with salinity, chlorophyll a concentration, and predator presence (measured as clam biomass). However, resting cyst distribution was only related to cell abundance and location. The occurrence of A. catenella cysts was also associated with that of cysts from the toxic species Protoceratium reticulatum. By shedding light on the ecological requirements of A. catenella blooms, our observations support the relevance of encystment as a mechanism of bloom termination and show a very fast depletion of cysts from the sediments (, SI