Future Scenarios as a Research Tool: Investigating Climate Change Impacts, Adaptation Options and Outcomes for the Great Barrier Reef, Australia

Climate change is a significant future driver of change in coastal social-ecological systems. Our knowledge of impacts, adaptation options, and possible outcomes for marine environments and coastal industries is expanding, but remains limited and uncertain. Alternative scenarios are a way to explore potential futures under a range of conditions. We developed four alternative future scenarios for the Great Barrier Reef and its fishing and tourism industries positing moderate and more extreme (2–3 °C above pre-industrial temperatures) warming for 2050 and contrasting ‘limited’ and ‘ideal’ ecological and social adaptation. We presented these scenarios to representatives of key stakeholder groups to assess the perceived viability of different social adaptation options to deliver desirable outcomes under varied contexts.     

Differential localizations of and requirements for the two Drosophila ninaC kinase/myosins in photoreceptor cells

The ninaC gene encodes two retinal specific proteins (p132 and p174) consisting of a protein kinase domain joined to a domain homologous to the head region of the myosin heavy chain. The putative myosin domain of p174 is linked at the COOH-terminus to a tail which has some similarities to myosin-I tails. In the current report, we demonstrate that the ninaC mutation results in light- and age-dependent retinal degeneration. We also show that ninaC flies display an electrophysiological phenotype before any discernible retinal degeneration indicating that the electrophysiological defect is the primary effect of the mutation. This suggests that ninaC has a role in phototransduction and that the retinal degeneration is a secondary effect resulting from the defect in phototransduction. To examine the requirements for the individual ninaC isoforms, mutant alleles were generated which express only p132 or p174. Elimination of p174 resulted in a ninaC phenotype as strong as the null allele; however, elimination of p132 had little if any effect. As a first step in investigating the basis for the difference in requirements for p174 and p132 we performed immuno-localization at the electron microscopic level and found that the two isoforms display different subcellular distributions in the photoreceptor cells. The p132 protein is restricted primarily to the cytoplasm and p174 to the rhabdomeres, the microvillar structure which is the site of action of many of the steps in phototransduction. This suggests that the p174 myosin-I type tail is the domain responsible for association with the rhabdomeres and that the substrate for the p174 putative kinase may be a rhabdomeric protein important in photo-transduction.     

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures,Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability ({"type":"clinical-trial","attrs":{"text":"NCT01151124","term_id":"NCT01151124"}}NCT01151124 and {"type":"clinical-trial","attrs":{"text":"NCT02117635","term_id":"NCT02117635"}}NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycER^TAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3’UTRs by dual reporter plasmid transfection and dual luciferase assay.     

The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization

Translocation to the nucleus of diacylglycerol kinase (DGK)– ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP₂) levels, consistent with prior evidence that MARCKS can regulate PIP₂ levels. We also found increased staining for PIP₂ in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP₂ co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP₂ levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression.     

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.     

Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1-EC5). Studies on cadherin specificity have implicated the NH(2)-terminal EC1 domain in the homophilic binding interaction, but the roles of the other extracellular cadherin (EC) domains have not been evaluated. We have undertaken a systematic analysis of the binding properties of the entire cadherin extracellular domain and the contributions of the other EC domains to homophilic binding. Lateral (cis) dimerization of the extracellular domain is thought to be required for adhesive function. Sedimentation analysis of the soluble extracellular segment of C-cadherin revealed that it exists in a monomer-dimer equilibrium with an affinity constant of approximately 64 microm. No higher order oligomers were detected, indicating that homophilic binding between cis-dimers is of significantly lower affinity. The homophilic binding properties of a series of deletion constructs, lacking successive or individual EC domains fused at the COOH terminus to an Fc domain, were analyzed using a bead aggregation assay and a cell attachment-based adhesion assay. A protein with only the first two NH(2)-terminal EC domains (CEC1-2Fc) exhibited very low activity compared with the entire extracellular domain (CEC1-5Fc), demonstrating that EC1 alone is not sufficient for effective homophilic binding. CEC1-3Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5Fc. EC3 is not required for homophilic binding, however, since CEC1-2-4Fc and CEC1-2-4-5Fc exhibited high activity in both assays. These and experiments using additional EC combinations show that many, if not all, the EC domains contribute to the formation of the cadherin homophilic bond, and specific one-to-one interaction between particular EC domains may not be required. These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W. Brieher, N. Lavrik, B. Gumbiner, and D. Leckband. 1999. PROC: Natl. Acad. Sci. USA. 96:11820-11824; Sivasankar, S., B. Gumbiner, and D. Leckband. 2001. Biophys J. 80:1758-68). We propose new models for how the cadherin extracellular repeats may contribute to adhesive specificity and function.     

Twenty year contrast of non-native parrotfeather distribution and abundance in an unregulated river

Hydrobiologia - Efficient management of invasive species benefits from understanding patterns of persistence and change over time. In this study, we compare distribution and abundance of the...     

Toxic introns and parasitic intein in Coxiella burnetii: legacies of a promiscuous past

The genome of the obligate intracellular pathogen Coxiella burnetii contains a large number of selfish genetic elements, including two group I introns (Cbu.L1917 and Cbu.L1951) and an intervening sequence that interrupts the 23S rRNA gene, an intein (Cbu.DnaB) within dnaB and 29 insertion sequences. Here, we describe the ability of the intron-encoded RNAs (ribozymes) to retard bacterial growth rate (toxicity) and examine the functionality and phylogenetic history of Cbu.DnaB. When expressed in Escherichia coli, both introns repressed growth, with Cbu.L1917 being more inhibitory. Both ribozymes were found to associate with ribosomes of Coxiella and E. coli. In addition, ribozymes significantly reduced in vitro luciferase translation, again with Cbu.L1917 being more inhibitory. We analyzed the relative quantities of ribozymes and genomes throughout a 14-day growth cycle of C. burnetii and found that they were inversely correlated, suggesting that the ribozymes have a negative effect on Coxiella's growth. We determined possible sites for ribozyme associations with 23S rRNA that could explain the observed toxicities. Further research is needed to determine whether the introns are being positively selected because they promote bacterial persistence or whether they were fixed in the population due to genetic drift. The intein, Cbu.DnaB, is able to self-splice, leaving the host protein intact and presumably functional. Similar inteins have been found in two extremophilic bacteria (Alkalilimnicola ehrlichei and Halorhodospira halophila) that are distantly related to Coxiella, making it difficult to determine whether the intein was acquired by horizontal gene transfer or was vertically inherited from a common ancestor.     

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth.

spo0H encodes a sigma factor, sigma-H, of RNA polymerase that is required for sporulation in Bacillus subtilis. Null mutations in spo0H block the initiation of sporulation but have no obvious effect on vegetative growth. We have characterized an insertion mutation, csh203::Tn917lac, that makes spo0H essential for normal growth. In otherwise wild-type cells, the csh203::Tn917lac insertion mutation has no obvious effect on cell growth, viability, or sporulation. However, in combination with a mutation in spo0H, the csh203 mutation causes a defect in vegetative growth. The csh203::Tn917lac insertion mutation was found to be located within orf23, the first gene of the rpoD (sigma-A) operon. The transposon insertion separates the major vegetative promoters P1 and P2 from the coding regions of two essential genes, dnaG (encoding DNA primase) and rpoD (encoding the major sigma factor, sigma-A) and leaves these genes under the control of minor promoters, including P4, a promoter controlled by sigma-H. The chs203 insertion mutation caused a 2- to 10-fold increase in expression of promoters recognized by RNA polymerase containing sigma-H. The increased expression of genes controlled by sigma-H in the csh203 single mutant, as well as the growth defect of the csh203 spo0H double mutant, was due to effects on rpoD and not to a defect in orf23 or dnaG.     

Recovery from an activity-induced metabolic acidosis in the American alligator, Alligator mississippiensis

The metabolic acidosis resulting from an intense exercise bout is large in crocodilians. Here we studied recovery from this pH perturbation in the American alligator. Metabolic rate, minute ventilation, arterial pH and gases, and strong ion concentration were measured for 10 h after exhaustion to elucidate the mechanisms and time course of recovery. Exhaustion resulted in a significant increase in lactate, metabolic rate, and ventilation, and a decrease in arterial PCO2), pH and bicarbonate. By 15 min after exhaustion, oxygen consumption returned to rest though carbon dioxide excretion remained elevated for 30 min. Arterial PO2), [Na+], and [K+], increased following exhaustion and recovered by 30 min post-exercise. Minute ventilation, tidal volume, [Cl-], and respiratory exchange ratio returned to resting values by 1 h. The air convection requirement for oxygen was elevated between 15 and 60 min of recovery. Breathing frequency and pH returned to resting values by 2 h of recovery. Lactate levels remained elevated until 6 h post-exercise. Arterial PCO2) and [HCO3-] were depressed until 8 h post-exercise. Compensation during recovery of acid-base balance was achieved by altering ventilation: following the initial metabolic acidosis and titration of bicarbonate, a relative hyperventilation prevented a further decrease in pH.