Geographic Tools for Eradication Programs of Insular Non-native Mammals

Non-native mammals are major drivers of ecosystem change and biodiversity loss; this is especially apparent on islands. However, techniques exist to remove non-native mammals, providing a powerful conservation tool. Conservation practitioners are now targeting larger islands for restoration. Leveraging existing and developing new techniques and technologies will prove critical to successful eradications on large islands. Using the removal of introduced goats (Capra hircus) from Santiago Island, Galápagos as a case study, we present a suite of Geographic Information System (GIS) tools that aid island conservation actions. GIS tools were incorporated into the three phases of the eradication campaign: planning, hunting, and monitoring. Further, these tools were adopted for three eradication techniques: ground-based hunting, aerial hunting by helicopter, and Judas goats. These geographic approaches provide a foundation for statistical, spatial, and economic analyses that should increase the capability and efficiency of removal campaigns. Given limited conservation funds and the dire status of many insular species, efficiently removing non-native mammals from islands is of paramount global conservation importance.     

Quality control of human tissues-experience from the Indiana University Cancer Center-Lilly Research Labs human tissue bank

The success of molecular research and its applications in both the clinical and basic research arenas is strongly dependent on the collection, handling, storage, and quality control of fresh human tissue samples. This tissue bank was set up to bank fresh surgically obtained human tissue using a Clinical Annotated Tissue Database (CATD) in order to capture the associated patient clinical data and demographics using a one way patient encryption scheme to protect patient identification. In this study, we determined that high quality of tissue samples is imperative for both genomic and proteomic molecular research. This paper also contains a brief compilation of the literature involved in the patient ethics, patient informed consent, patient de-identification, tissue collection, processing, and storage as well as basic molecular research generated from the tissue bank using good clinical practices. The current applicable rules, regulations, and guidelines for handling human tissues are briefly discussed. More than 6,610 cancer patients have been consented (97% of those that were contacted by the consenter) and 16,800 tissue specimens have been banked from these patients in 9 years. All samples collected in the bank were QC’d by a pathologist. Approximately 1,550 tissue samples have been requested for use in basic, clinical, and/or biomarker cancer research studies. Each tissue aliquot removed from the bank for a research study were evaluated by a second H&E, if the samples passed the QC, they were submitted for genomic and proteomic molecular analysis/study. Approximately 75% of samples evaluated were of high histologic quality and used for research studies. Since 2003, we changed the patient informed consent to allow the tissue bank to gather more patient clinical follow-up information. Ninety two percent of the patients (1,865 patients) signed the new informed consent form and agreed to be re-contacted for follow-up information on their disease state. In addition, eighty five percent of patients (1,584) agreed to be re-contacted to provide a biological fluid sample to be used for biomarker research.     

Identification of proteins involved in microglial endocytosis of alpha-synuclein

Aggregated alpha-synuclein, a protein playing pivotal roles in the pathogenesis of Parkinson disease (PD) and related synucleinopathy, has been shown to activate microglia, the key cells in neuroinflammation. However, the mechanisms by which aggregated alpha-synuclein enters microglia remain uncharacterized. In this study, we first replicated our previous results with a modified protocol that generated aggregated alpha-synuclein more efficiently. Next, using two recently developed proteomic techniques, SILAC (Stable Isotope Labeling of Amino acid in Cell cultures) and PROCEED (PROteome of Cell Exposed Extracellular Domains), we studied the plasma membrane proteins of primary cultured microglia that might be interacting with aggregated alpha-synuclein and mediating its internalization. The results demonstrated that 250 nM alpha-synuclein, aged for 6 h with a magnetic stir bar, was just as potent in activating microglia as the aggregated alpha-synuclein produced by aging without constant agitation for 7 days. The proteomic analysis identified 111 membrane proteins; of these, 46 proteins were altered in relative abundance in the membrane compartment after treatment with aggregated alpha-synuclein for 3 h. Two of these proteins, clathrin and calnexin, were further evaluated with Western blotting, demonstrating good agreement with quantitative proteomics. Finally, immunocytochemical as well as co-immunoprecipitation studies indicated that clathrin was indeed co-localized with internalized alpha-synuclein in microglia. These results suggest for the first time that microglial activation secondary to internalization of aggregated alpha-synuclein likely requires participation of clathrin, which is an essential protein of the polyhedral coat of coated pits and vesicles that play major roles in endocytosis and vesicular trafficking.     

High Fat Diet Increases [3H] Flunitrazepam Binding in the Mouse Brain that is Dependent on the Expression of the Dopamine D2 Gene

Neurochemical Research - Dopamine is an important neuromodulator in the brain that binds to dopamine D1-like receptors (D1, D5) as well as dopamine D2-like receptors (D2, D3, D4). The D2 receptor...     

CHARMM-GUI HMMM Builder for Membrane Simulations with the Highly Mobile Membrane-Mimetic Model

Slow diffusion of the lipids in conventional all-atom simulations of membrane systems makes it difficult to sample large rearrangements of lipids and protein-lipid interactions. Recently, Tajkhorshid and co-workers developed the highly mobile membrane-mimetic (HMMM) model with accelerated lipid motion by replacing the lipid tails with small organic molecules. The HMMM model provides accelerated lipid diffusion by one to two orders of magnitude, and is particularly useful in studying membrane-protein associations. However, building an HMMM simulation system is not easy, as it requires sophisticated treatment of the lipid tails. In this study, we have developed CHARMM-GUI HMMM Builder (http://www.charmm-gui.org/input/hmmm) to provide users with ready-to-go input files for simulating HMMM membrane systems with/without proteins. Various lipid-only and protein-lipid systems are simulated to validate the qualities of the systems generated by HMMM Builder with focus on the basic properties and advantages of the HMMM model. HMMM Builder supports all lipid types available in CHARMM-GUI and also provides a module to convert back and forth between an HMMM membrane and a full-length membrane. We expect HMMM Builder to be a useful tool in studying membrane systems with enhanced lipid diffusion.     

Stigmatellin Probes the Electrostatic Potential in the QB Site of the Photosynthetic Reaction Center

The electrostatic potential in the secondary quinone (Q_B) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to Q_B. It is controlled by the ionization states of residues in a strongly interacting cluster around the Q_B site. Reduction of the Q_B induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the Q_B binding pocket. It binds to the Q_B site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the Q_B pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the Q_B site was modulated by a single mutation, L213Asp→Ala, or double mutations, L213Asp-L212Glu→Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (ΔpK > 3.5) decreased dramatically to (ΔpK > 0.75) in the RC of the compensatory mutant (AA+M44Asn→AA+M44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the Q_B site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay.     

cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca²⁺ channel inositol 1,4,5-trisphosphate receptor 1 (IP₃R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP₃R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3^−/−/Nrl^−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP₃R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca²⁺ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.     

DNA loops generate intracentromere tension in mitosis

The centromere is the DNA locus that dictates kinetochore formation and is visibly apparent as heterochromatin that bridges sister kinetochores in metaphase. Sister centromeres are compacted and held together by cohesin, condensin, and topoisomerase-mediated entanglements until all sister chromosomes bi-orient along the spindle apparatus. The establishment of tension between sister chromatids is essential for quenching a checkpoint kinase signal generated from kinetochores lacking microtubule attachment or tension. How the centromere chromatin spring is organized and functions as a tensiometer is largely unexplored. We have discovered that centromere chromatin loops generate an extensional/poleward force sufficient to release nucleosomes proximal to the spindle axis. This study describes how the physical consequences of DNA looping directly underlie the biological mechanism for sister centromere separation and the spring-like properties of the centromere in mitosis.