Plasma and tissue transferrin and ferritin, and gene expression of ferritin, transferrin, and transferrin receptors I and II in channel catfish Ictalurus punctatus fed diets with different concentrations of inorganic or organic iron.

Ferritin, transferrin, and transferrin receptors I and II play a vital role in iron metabolism, health, and indication of iron deficiency anaemia in fish. To evaluate the use of high-iron diets to prevent or reverse channel catfish (Ictalurus punctatus) anaemia of unknown causes, we investigated the expression of these iron-regulatory genes and proteins in channel catfish fed plant-based diets. Catfish fingerlings were fed five diets supplemented with 0 (basal), 125, and 250 mg/kg of either inorganic iron or organic iron for 2 weeks. Ferritin, transferrin, and transferrin receptor I and II mRNA and protein expression levels in fish tissues (liver, intestine, trunk kidney, and head kidney) and plasma were determined. Transferrin (iron transporter) and TfR (I and II) genes were generally highly expressed in fish fed the basal diet compared to those fed the iron-supplemented diets. In contrast, ferritin (iron storage) genes were more expressed in the trunk kidney of fish fed the iron-supplemented diets than in those fed the basal diet. Our results demonstrate that supplementing channel catfish plant-based diets with iron from either organic or inorganic iron sources affected the expression of the iron-regulatory genes and increased body iron status in the fish.

Distinct regions of the kinesin-5 C-terminal tail are essential for mitotic spindle midzone localization and sliding force.

Kinesin-5 motor proteins play essential roles during mitosis in most organisms. Their tetrameric structure and plus-end-directed motility allow them to bind to and move along antiparallel microtubules, thereby pushing spindle poles apart to assemble a bipolar spindle. Recent work has shown that the C-terminal tail is particularly important to kinesin-5 function: The tail affects motor domain structure, ATP hydrolysis, motility, clustering, and sliding force measured for purified motors, as well as motility, clustering, and spindle assembly in cells. Because previous work has focused on presence or absence of the entire tail, the functionally important regions of the tail remain to be identified. We have therefore characterized a series of kinesin-5/Cut7 tail truncation alleles in fission yeast. Partial truncation causes mitotic defects and temperature-sensitive growth, while further truncation that removes the conserved BimC motif is lethal. We compared the sliding force generated by cut7 mutants using a kinesin-14 mutant background in which some microtubules detach from the spindle poles and are pushed into the nuclear envelope. These Cut7-driven protrusions decreased as more of the tail was truncated, and the most severe truncations produced no observable protrusions. Our observations suggest that the C-terminal tail of Cut7p contributes to both sliding force and midzone localization. In the context of sequential tail truncation, the BimC motif and adjacent C-terminal amino acids are particularly important for sliding force. In addition, moderate tail truncation increases midzone localization, but further truncation of residues N-terminal to the BimC motif decreases midzone localization.

Distinct regions of the kinesin-5 C-terminal tail are essential for mitotic spindle midzone localization and sliding force.

Kinesin-5 motor proteins play essential roles during mitosis in most organisms. Their tetrameric structure and plus-end-directed motility allow them to bind to and move along antiparallel microtubules, thereby pushing spindle poles apart to assemble a bipolar spindle. Recent work has shown that the C-terminal tail is particularly important to kinesin-5 function: the tail affects motor domain structure, ATP hydrolysis, motility, clustering, and sliding force measured for purified motors, as well as motility, clustering, and spindle assembly in cells. Because previous work has focused on presence or absence of the entire tail, the functionally important regions of the tail remain to be identified. We have therefore characterized a series of kinesin-5/Cut7 tail truncation alleles in fission yeast. Partial truncation causes mitotic defects and temperature-sensitive growth, while further truncation that removes the conserved BimC motif is lethal. We compared the sliding force generated by cut7 mutants using a kinesin-14 mutant background in which some microtubules detach from the spindle poles and are pushed into the nuclear envelope. These Cut7-driven protrusions decreased as more of the tail was truncated, and the most severe truncations produced no observable protrusions. Our observations suggest that the C-terminal tail of Cut7p contributes to both sliding force and midzone localization. In the context of sequential tail truncation, the BimC motif and adjacent C-terminal amino acids are particularly important for sliding force. In addition, moderate tail truncation increases midzone localization, but further truncation of residues N terminal to the BimC motif decreases midzone localization.

The kinesin-5 protein Cut7 moves bidirectionally on fission yeast spindles with activity that increases in anaphase.

Kinesin-5 motors are essential to separate mitotic spindle poles and assemble a bipolar spindle in many organisms. These motors crosslink and slide apart antiparallel microtubules via microtubule plus-end-directed motility. However, kinesin-5 localization is enhanced away from antiparallel overlaps. Increasing evidence suggests this localization occurs due to bidirectional motility or trafficking. The purified fission-yeast kinesin-5 protein Cut7 moves bidirectionally, but bidirectionality has not been shown in cells, and the function of the minus-end-directed movement is unknown. Here, we characterized the motility of Cut7 on bipolar and monopolar spindles and observed movement toward both plus- and minus-ends of microtubules. Notably, the activity of the motor increased at anaphase B onset. Perturbations to microtubule dynamics only modestly changed Cut7 movement, whereas Cut7 mutation reduced movement. These results suggest that the directed motility of Cut7 contributes to the movement of the motor. Comparison of the Cut7 mutant and human Eg5 (also known as KIF11) localization suggest a new hypothesis for the function of minus-end-directed motility and spindle-pole localization of kinesin-5s.

Effects of Different Feeding Regimes on Growth Rates and Fatty Acid Composition of Largemouth Bass Micropterus nigricans at High Water Temperatures.

As the northern Largemouth bass (LMB) (Micropterus nigricans) industry shifts toward fingerling production, implementing practical feeding strategies to ensure efficient growth during high water temperatures is paramount. Twenty (12.7 ± 0.2 g) (Trial 1) and fifteen (7.2 ± 0.1 g) (Trial 2) LMB fingerlings were stocked in two recirculating systems (each containing nine tanks), acclimated to 30 °C, with one system fed daily rations of 3, 5 and 7% body weight (Trial 1), and the second system fed to satiation daily, every second day, or every third day (Trial 2), for 28 days each. All treatments were triplicated. Multiple growth metrics and lipid composition were analyzed. The 3% treatment yielded the lowest final average weight (36.05 g) and FCR (0.83), with no difference in final biomass in Trial 1 treatments. Fish fed to satiation daily and every second day produced FCRs and biomasses of 0.83 and 356.78 g, and 0.93 and 272.26 g, respectively. There were no differences in total lipid concentration, however, fatty acid profiles differed significantly between all treatments within their respective trials. Feeding LMB fingerlings 3% of total body weight or feeding daily to satiation allows for efficient growth at 30 °C and implements cost-effective feeding strategies.

Key phosphorylation events in Spc29 and Spc42 guide multiple steps of yeast centrosome duplication.

Phosphorylation modulates many cellular processes during cell cycle progression. The yeast centrosome (called the spindle pole body, SPB) is regulated by the protein kinases Mps1 and Cdc28/Cdk1 as it nucleates microtubules to separate chromosomes during mitosis. Previously we completed an SPB phosphoproteome, identifying 297 sites on 17 of the 18 SPB components. Here we describe mutagenic analysis of phosphorylation events on Spc29 and Spc42, two SPB core components that were shown in the phosphoproteome to be heavily phosphorylated. Mutagenesis at multiple sites in Spc29 and Spc42 suggests that much of the phosphorylation on these two proteins is not essential but enhances several steps of mitosis. Of the 65 sites examined on both proteins, phosphorylation of the Mps1 sites Spc29-T18 and Spc29-T240 was shown to be critical for function. Interestingly, these two sites primarily influence distinct successive steps; Spc29-T240 is important for the interaction of Spc29 with Spc42, likely during satellite formation, and Spc29-T18 facilitates insertion of the new SPB into the nuclear envelope and promotes anaphase spindle elongation. Phosphorylation sites within Cdk1 motifs affect function to varying degrees, but mutations only have significant effects in the presence of an MPS1 mutation, supporting a theme of coregulation by these two kinases.

Evaluations of Insecticides and Fungicides for Reducing Attack Rates of a new invasive ambrosia beetle (Euwallacea Sp., Coleoptera: Curculionidae: Scolytinae) in Infested Landscape Trees in California.

A recently discovered ambrosia beetle with the proposed common name of polyphagous shot hole borer (Euwallacea sp., Coleoptera: Curculionidae: Scolytinae), is reported to attack >200 host tree species in southern California, including many important native and urban landscape trees. This invasive beetle, along with its associated fungi, causes branch dieback and tree mortality in a large variety of tree species including sycamore (Platanus racemosa Nutt.). Due to the severity of the impact of this Euwallacea sp., short-term management tools must include chemical control options for the arboriculture industry and private landowners to protect trees. We examined the effectiveness of insecticides, fungicides, and insecticide-fungicide combinations for controlling continued Euwallacea sp. attacks on previously infested sycamore trees which were monitored for 6 mo after treatment. Pesticide combinations were generally more effective than single pesticide treatments. The combination of a systemic insecticide (emamectin benzoate), a contact insecticide (bifenthrin), and a fungicide (metconazole) provided some level of control when applied on moderate and heavily infested trees. The biological fungicide Bacillus subtilis provided short-term control. There was no difference in the performance of the three triazole fungicides (propiconazole, tebuconazole, and metconazole) included in this study. Although no pesticide combination provided substantial control over time, pesticide treatments may be more effective when trees are treated during early stages of attack by this ambrosia beetle.

The molecular architecture of the yeast spindle pole body core determined by Bayesian integrative modeling.

Microtubule-organizing centers (MTOCs) form, anchor, and stabilize the polarized network of microtubules in a cell. The central MTOC is the centrosome that duplicates during the cell cycle and assembles a bipolar spindle during mitosis to capture and segregate sister chromatids. Yet, despite their importance in cell biology, the physical structure of MTOCs is poorly understood. Here we determine the molecular architecture of the core of the yeast spindle pole body (SPB) by Bayesian integrative structure modeling based on in vivo fluorescence resonance energy transfer (FRET), small-angle x-ray scattering (SAXS), x-ray crystallography, electron microscopy, and two-hybrid analysis. The model is validated by several methods that include a genetic analysis of the conserved PACT domain that recruits Spc110, a protein related to pericentrin, to the SPB. The model suggests that calmodulin can act as a protein cross-linker and Spc29 is an extended, flexible protein. The model led to the identification of a single, essential heptad in the coiled-coil of Spc110 and a minimal PACT domain. It also led to a proposed pathway for the integration of Spc110 into the SPB.

Long-term T-DNA insert stability and transgene expression consistency in field propagated sugarcane.

KEY MESSAGE: This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. T-DNA inserts are stable; no transgene rearrangements were observed. AmCYAN1 and PMI protein accumulation levels were maintained. There was no evidence that production of either protein declined across generations and no transgene silencing was observed in three commercial sugarcane varieties through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes over 4 years of field testing. Long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized. This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. These data are critical supporting information needed for successful commercialization of GM sugarcane. Here seventeen transgenic events, containing the AmCYAN1 gene driven by a CMP promoter and the E. coli PMI gene driven by either a CMP or Ubi promoter, were used to monitor T-DNA insert stability and consistency of transgene encoded protein accumulation through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes. The experiments were conducted in three commercial sugarcane varieties over 4 years of field testing. DNA gel blot analysis showed that the T-DNA inserts are stable; no transgene rearrangements were observed. Quantitative ELISA showed no evidence of decreasing AmCYAN1 and PMI protein levels across generations and no transgene silencing was observed. These results indicate that long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized.

Effect of Chipping and Solarization on Emergence and Boring Activity of a Recently Introduced Ambrosia Beetle (Euwallacea sp., Coleoptera: Curculionidae: Scolytinae) in Southern California.

Polyphagous shot hole borer (Euwallacea sp., Coleoptera: Curculionidae: Scolytinae) has recently invaded southern California. The beetle, along with its associated fungi, Fusarium euwallaceae, Graphium sp., and Acremonium sp., causes branch dieback and tree mortality in a large variety of tree species including avocado (Persea americana Mill.) and box elder (Acer negundo L.). With the spread of the beetle through Los Angeles, Orange, and San Diego Counties in California, there is increasing concern that felled trees and pruned branches infested with polyphagous shot hole borer should receive sanitation treatment to reduce the potential spread of the beetle from the movement of untreated wood. We tested two sanitation methods to reduce beetle populations, chipping with a commercial chipper and solarization by covering logs with clear or black plastic in full sun. Both chipping and solarization decreased beetle emergence and boring activity compared to untreated control logs. Chipping was most effective for chip sizes <5 cm. Solarization was most effective using clear polyethylene sheeting during hot summer months, particularly August, when daily maximum temperatures were ≥35°C. Beetles persisted for 2 mo or more when solarization was applied during the spring or fall.

Activation of the yeast Hippo pathway by phosphorylation-dependent assembly of signaling complexes.

Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.

Cell cycle phosphorylation of mitotic exit network (MEN) proteins.

Phosphorylation of proteins is an important mechanism used to regulate most cellular processes. Recently, we completed an extensive phosphoproteomic analysis of the core proteins that constitute the Saccharomyces cerevisiae centrosome. Here, we present a study of phosphorylation sites found on the mitotic exit network (MEN) proteins, most of which are associated with the cytoplasmic face of the centrosome. We identified 55 sites on Bfa1, Cdc5, Cdc14 and Cdc15. Eight sites lie in cyclin-dependent kinase motifs (Cdk, S/T-P), and 22 sites are completely conserved within fungi. More than half of the sites were found in centrosomes from mitotic cells, possibly in preparation for their roles in mitotic exit. Finally, we report phosphorylation site information for other important cell cycle and regulatory proteins.

A cell cycle phosphoproteome of the yeast centrosome.

Centrosomes organize the bipolar mitotic spindle, and centrosomal defects cause chromosome instability. Protein phosphorylation modulates centrosome function, and we provide a comprehensive map of phosphorylation on intact yeast centrosomes (18 proteins). Mass spectrometry was used to identify 297 phosphorylation sites on centrosomes from different cell cycle stages. We observed different modes of phosphoregulation via specific protein kinases, phosphorylation site clustering, and conserved phosphorylated residues. Mutating all eight cyclin-dependent kinase (Cdk)-directed sites within the core component, Spc42, resulted in lethality and reduced centrosomal assembly. Alternatively, mutation of one conserved Cdk site within γ-tubulin (Tub4-S360D) caused mitotic delay and aberrant anaphase spindle elongation. Our work establishes the extent and complexity of this prominent posttranslational modification in centrosome biology and provides specific examples of phosphorylation control in centrosome function.

Structure-function analysis of the C-terminal domain of CNM67, a core component of the Saccharomyces cerevisiae spindle pole body.

The spindle pole body of the budding yeast Saccharomyces cerevisiae has served as a model system for understanding microtubule organizing centers, yet very little is known about the molecular structure of its components. We report here the structure of the C-terminal domain of the core component Cnm67 at 2.3 Å resolution. The structure determination was aided by a novel approach to crystallization of proteins containing coiled-coils that utilizes globular domains to stabilize the coiled-coils. This enhances their solubility in Escherichia coli and improves their crystallization. The Cnm67 C-terminal domain (residues Asn-429-Lys-581) exhibits a previously unseen dimeric, interdigitated, all α-helical fold. In vivo studies demonstrate that this domain alone is able to localize to the spindle pole body. In addition, the structure reveals a large functionally indispensable positively charged surface patch that is implicated in spindle pole body localization. Finally, the C-terminal eight residues are disordered but are critical for protein folding and structural stability.

Sex differences in the haptic change task.

Three experiments examined gender differences in picture location memory in the sense of touch involving the change task. In Experiments 1 and 2, blindfolded sighted participants felt 15 tangible raised-line pictures and memorized their locations for 8 min. Subsequently, they felt another set of raised-line pictures that were identical to the original set, but locations were exchanged for 6 of them. The change task required participants to indicate which raised-line pictures were moved to new locations and which were not moved. It was expected that females would show superior picture location memory compared with males. The results showed that females had a significantly higher number of correct picture location judgments than males in Experiment 1. No differences appeared between males and females on the change task when the task was much more difficult and the raised-line pictures were irregularly arrayed in the second experiment. Task difficulty was too high in Experiment 2, and a third experiment reduced the number of pictures in the irregular array to 12. Females performed better than males in Experiment 3. Gender differences in the change task are small in magnitude in touch as well as in vision. The results of the present experiments using touch are consistent with the larger literature in vision.

The effect of nitrogen additions on oak foliage and herbivore communities at sites with high and low atmospheric pollution.

To evaluate plant and herbivore responses to nitrogen we conducted a fertilization study at a low and high pollution site in the mixed conifer forests surrounding Los Angeles, California. Contrary to expectations, discriminant function analysis of oak herbivore communities showed significant response to N fertilization when atmospheric deposition was high, but not when atmospheric deposition was low. We hypothesize that longer-term fertilization treatments are needed at the low pollution site before foliar N nutrition increases sufficiently to affect herbivore communities. At the high pollution site, fertilization was also associated with increased catkin production and higher densities of a byturid beetle that feeds on the catkins of oak. Leaf nitrogen and nitrate were significantly higher at the high pollution site compared to the low pollution site. Foliar nitrate concentrations were positively correlated with abundance of sucking insects, leafrollers and plutellids in all three years of the study.

Centrosome duplication: is asymmetry the clue?

The structure of the yeast Sfi1-centrin complex, and its asymmetric position within the yeast centrosome, suggest a model for the initiation of centrosome duplication and provides a target for licensing this event.

Reducing cancer disparities for minorities: a multidisciplinary research agenda to improve patient access to health systems, clinical trials, and effective cancer therapy.

Ethnic diversity in recruitment is a vital prerequisite to eliminating health disparities in cancer treatment, control and prevention programs. Much anecdotal reporting, but little scientific rigor, has been applied to the study of methods to improve the recruitment of minorities into cancer control or treatment trials. Even the most innovative research is stymied by the lack of representative samples of the populations that the research is designed to serve. The goals of this article are to describe a theory-driven framework for improving minority recruitment to clinical and cancer control trials, to explain organizational prerequisites to improving minority recruitment, and to provide empirical evidence of success in initial efforts to recruit to cancer control studies. These programs are offered as models for improving minority recruitment to cancer-control and -treatment trials, and minority access to cancer treatment in general.

Detecting changes in insect herbivore communities along a pollution gradient.

The forests surrounding the urban areas of the Los Angeles basin are impacted by ozone and nitrogen pollutants arising from urban areas. We examined changes in the herbivore communities of three prominent plant species (ponderosa pine, California black oak and bracken fern) at six sites along an air pollution gradient. Insects were extracted from foliage samples collected in spring, as foliage reached full expansion. Community differences were evaluated using total herbivore abundance, richness, Shannon-Weiner diversity, and discriminant function analysis. Even without conspicuous changes in total numbers, diversity or richness of herbivores, herbivore groups showed patterns of change that followed the air pollution gradient that were apparent through discriminant function analysis. For bracken fern and oak, chewing insects were more dominant at high pollution sites. Oak herbivore communities showed the strongest effect. These changes in herbivore communities may affect nutrient cycling in forest systems.

Chemical genetics reveals a role for Mps1 kinase in kinetochore attachment during mitosis.

Accurate chromosome segregation depends on proper assembly and function of the kinetochore and the mitotic spindle. In the budding yeast, Saccharomyces cerevisiae, the highly conserved protein kinase Mps1 has well-characterized roles in spindle pole body (SPB, yeast centrosome equivalent) duplication and the mitotic checkpoint. However, an additional role for Mps1 is suggested by phenotypes of MPS1 mutations that include genetic interactions with kinetochore mutations and meiotic chromosome segregation defects and also by the localization of Mps1 at the kinetochore, the latter being independent of checkpoint activation. We have developed a new MPS1 allele, mps1-as1, that renders the kinase specifically sensitive to a cell-permeable ATP analog inhibitor, allowing us to perform high-resolution execution point experiments that identify a novel role for Mps1 subsequent to SPB duplication. We demonstrate, by using both fixed- and live-cell fluoresence techniques, that cells lacking Mps1 function show severe defects in mitotic spindle formation, sister kinetochore positioning at metaphase, and chromosome segregation during anaphase. Taken together, our experiments are consistent with an important role for Mps1 at the kinetochore in mitotic spindle assembly and function.