Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson''s and Huntington''s disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 μm) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.     

Spatial heterogeneity in ecosystem structure and productivity in a moist Kenyan savanna

Topographic variations and disturbances are key sources of spatial heterogeneity in the ecosystem and may influence its functioning, productivity, and carbon (C) storage. In water controlled ecosystems, structural and functional heterogeneity become distinct during drought when the ecosystem processes are operating at their limits. We examined spatial heterogeneity arising from grazing, abandoned cropland, presence of Acacia trees, and termite mounds (termitaria). Soil water content (SWC) was significantly (P < 0.05) higher in termitaria and fenced (un-grazed) plots. Higher soil nitrogen (N) content occurred in the Acacia, termitaria, and fenced plots while total biomass was highest in the fenced plot. The termitaria plots showed the highest net ecosystem CO₂ exchange (NEE), ecosystem respiration (R _eco), and gross primary production (GPP) and were the only plots that were net CO₂ sinks. Except in fenced plots, maximum GPP was positively correlated with SWC and green biomass in all the other plots. Green biomass and R _eco were positively correlated with SWC. Shifting cultivation (abandoned farmlands) negatively affected soil quality, ecosystem CO₂ assimilation, and productivity. Removal of grazing (cattle) from the ecosystem negatively influenced GPP, while the presence of termitaria and Acacia trees facilitated soil water and N availability and ecosystem productivity. We concluded that soil water availability was responsible for most of the localized differences in the savanna and has a strong influence on ecosystem C capture and storage. We recommend that future studies on savanna productivity and ecosystem CO₂ fluxes should consider heterogeneity in the ecosystem in order to avoid bias and increase the accuracy of any estimates made.     

Structural analysis of enzyme-resistant isomaltooligosaccharides reveals the elongation of α-(1→3)-linked branches in Weissella confusa dextran

Weissella confusa VTT E-90392 is an efficient producer of a dextran that is mainly composed of α-(1→6)-linked D-glucosyl units and very few α-(1→3) branch linkages. A mixture of the Chaetomium erraticum endodextranase and the Aspergillus niger α-glucosidase was used to hydrolyze W. confusa dextran to glucose and a set of enzyme-resistant isomaltooligosaccharides. Two of the oligosaccharides (tetra- and hexasaccharide) were isolated in pure form and their structures elucidated. The tetrasaccharide had a nonreducing end terminal α-(1→3)-linked glucosyl unit (α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc), whereas the hexasaccharide had an α-(1→3)-linked isomaltosyl side group (α-D-Glcp-(1→6)[α-D-Glcp-(1→6)-α-D-Glcp-(1→3)]-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc). A mixture of two isomeric oligosaccharides was also obtained in the pentasaccharide fraction, which were identified as (α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc) and (α-D-Glcp-(1→6)[α-D-Glcp-(1→3)]-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc). The structures of the oligosaccharides indicated that W. confusa dextran contains both terminal and elongated α-(1→3)-branches. This is the first report evidencing the presence of elongated branches in W. confusa dextran. The (1)H and (13)C NMR spectroscopic data on the enzyme-resistant isomaltooligosaccharides with α-(1→3)-linked glucosyl and isomaltosyl groups are published here for the first time.     

Potent host-directed small-molecule inhibitors of myxovirus RNA-dependent RNA-polymerases

Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp) activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid adaptation to a pathogen-directed inhibitor of RdRp activity.     

Evaluation of three aqueous plant extracts and Lamdacot in controlling eggplant fruit borer (Daraba laisalis Wlk.) (Lepidoptera: Pyralidae) in north-eastern Nigeria

Field experiments carried out in Dadinkowa, Gombe State, Nigeria, during 2007 and 2008 cropping seasons evaluated the efficacy of three aqueous plant extracts namely neem (Azadirachta indica A.H.L. Juss.), bitter melon (Momordica balsamina Linn.) and siam weed (Chromolaena odorota (L.) King and Robinson) compared to a conventional insecticide, Lamdacot 500EC, in managing fruit borer (Daraba laisalis Wlk.) attack of eggplants in north-eastern Nigeria. The experiments were laid out in a randomised complete block design replicated four times including controls. The results showed that the application of aqueous extracts (5% w/v concentration) of A. indica and M. balsamina at one-week interval during the fruiting stage was effective and at par with the conventional insecticide, Lamdacot, in reducing eggplant fruit borer infestation and fruit damage, as well as the resulting increase in fruit yield. Farmers can therefore adopt the practice of applying 5% w/v concentration of the above aqueous plant extracts at weekly intervals to control fruit borer attack of eggplants in the north-eastern region of Nigeria.     

K-Cl cotransporter gene expression during human and murine erythroid differentiation

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.     

Intramolecular azo-bridge as a cystine disulfide bond surrogate: Somatostatin-14 and brain natriuretic peptide (BNP) analogs

Cystine disulfide bond is a common feature in numerous biologically active peptides and proteins and accordingly its replacement by various surrogates presents a potential route to obtain analogs with improved pharmacokinetic characteristics. The purpose of the present study was to assess whether an azo-bridge can serve as such a surrogate. In view of the marked clinical significance of somatostatin and the brain natriuretic peptide (BNP) we choose these peptides as a model. Three cyclic-azo somatostatin analogs and three cyclic-azo BNP analogs were effectively prepared in solution through azo bond formation between p-amino phenylalanine and His or Tyr residues that were positioned in the peptide sequences in place of the native Cys residues. The peptides binding affinities to the sst? and ANP-receptor (NPR-A) expressed on rat acinar pancreating carcinoma AR4-2J cell membranes and HeLa cells, respectively, were examined. The somatostatin analogs displayed good to moderate affinities to the rat sst? in the nM range with best results obtained with peptide 2, that is, IC?? = 8.1 nM. Molecular dynamics simulations on these peptides suggests on a correlation between the observed binding potencies and the degree of conformational space overlapping with that of somatostatin. The BNP analogs exhibited binding affinities to the NPR-A in the nM range with best results obtained with BNP-1, that is, IC?? = 60 nM.     

Bacteriophages Isolated in China for the Control of Pectobacterium carotovorum Causing Potato Soft Rot in Kenya

Soft rot is an economically significant disease in potato and one of the major threats to sustainable potato production. This study aimed at isolating lytic bacteriophages and evaluating methods for and the efficacy of applying phages to control potato soft rot caused by Pectobacterium carotovorum. Eleven bacteriophages isolated from soil and water samples collected in Wuhan, China, were used to infect P. carotovorum host strains isolated from potato tubers showing soft rot symptoms in Nakuru county, Kenya. The efficacy of the phages in controlling soft rot disease was evaluated by applying individual phage strains or a phage cocktail on potato slices and tubers at different time points before or after inoculation with a P. carotovorum strain. The phages could lyse 20 strains of P. carotovorum, but not Pseudomonas fluorescens control strains. Among the 11 phages, Pectobacterium phage Wc5 r, interestingly showed cross-activity against Pectobacterium atrosepticum and two phage-resistant P. carotovorum strains. Potato slice assays showed that the phage concentration and timing of application are crucial factors for effective soft rot control. Phage cocktail applied at a concentration of 1 9 109 plaque-forming units per milliliter before or within an hour after bacterial inoculation on potato slices, resulted in C 90%reduction of soft rot symptoms. This study provides a basis for the development and application of phages to reduce the impact of potato soft rot disease.     

Loading into Nanoparticles Improves Quercetin's Efficacy in Preventing Neuroinflammation Induced by Oxysterols

Chronic inflammatory events appear to play a fundamental role in Alzheimer''s disease (AD)-related neuropathological changes, and to result in neuronal dysfunction and death. The inflammatory responses observed in the AD brain include activation and proliferation of glial cells, together with up-regulation of inflammatory mediators and of free radicals. Along with glial cells, neurons themselves can also react and contribute to neuroinflammatory changes in the AD brain, by serving as sources of inflammatory mediators. Because excess cholesterol cannot be degraded in the brain, it must be excreted from that organ as cholesterol oxidation products (oxysterols), in order to prevent its accumulation. Among risk factors for this neurodegenerative disease, a mechanistic link between altered cholesterol metabolism and AD has been suggested; oxysterols appear to be the missing linkers between the two, because of their neurotoxic effects. This study shows that 24-hydroxycholesterol, 27-hydroxycholesterol, and 7β-hydroxycholesterol, the three oxysterols potentially implicated in AD pathogenesis, induce some pro-inflammatory mediator expression in human neuroblastoma SH-SY5Y cells, via Toll-like receptor-4/cyclooxygenase-2/membrane bound prostaglandin E synthase (TLR4/COX-2/mPGES-1); this clearly indicates that oxysterols may promote neuroinflammatory changes in AD. To confirm this evidence, cells were incubated with the anti-inflammatory flavonoid quercetin; remarkably, its anti-inflammatory effects in SH-SY5Y cells were enhanced when it was loaded into β-cyclodextrin-dodecylcarbonate nanoparticles, versus cells pretreated with free quercetin. The goal of loading quercetin into nanoparticles was to improve its permeation across the blood-brain barrier into the brain, and its bioavailability to reach target cells. The findings show that this drug delivery system might be a new therapeutic strategy for preventing or reducing AD progression.     

Prioritizing key resilience indicators to support coral reef management in a changing climate

Managing coral reefs for resilience to climate change is a popular concept but has been difficult to implement because the empirical scientific evidence has either not been evaluated or is sometimes unsupportive of theory, which leads to uncertainty when considering methods and identifying priority reefs. We asked experts and reviewed the scientific literature for guidance on the multiple physical and biological factors that affect the ability of coral reefs to resist and recover from climate disturbance. Eleven key factors to inform decisions based on scaling scientific evidence and the achievability of quantifying the factors were identified. Factors important to resistance and recovery, which are important components of resilience, were not strongly related, and should be assessed independently. The abundance of resistant (heat-tolerant) coral species and past temperature variability were perceived to provide the greatest resistance to climate change, while coral recruitment rates, and macroalgae abundance were most influential in the recovery process. Based on the 11 key factors, we tested an evidence-based framework for climate change resilience in an Indonesian marine protected area. The results suggest our evidence-weighted framework improved upon existing un-weighted methods in terms of characterizing resilience and distinguishing priority sites. The evaluation supports the concept that, despite high ecological complexity, relatively few strong variables can be important in influencing ecosystem dynamics. This is the first rigorous assessment of factors promoting coral reef resilience based on their perceived importance, empirical evidence, and feasibility of measurement. There were few differences between scientists' perceptions of factor importance and the scientific evidence found in journal publications but more before and after impact studies will be required to fully test the validity of all the factors. The methods here will increase the feasibility and defensibility of including key resilience metrics in evaluations of coral reefs, as well as reduce costs. Adaptation, marine protected areas, priority setting, resistance, recovery.