Cloning and characterization of SK2 channel from chicken short hair cells

In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74±0.17 M. The expressed channels were blocked by apamin (IC₅₀=73.3±5.0 pM) and d-tubocurarine (IC₅₀=7.6±1.0 M), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates.     

ATP formation from deoxyadenosine in human erythrocytes: Evidence for a hitherto unidentified route involving adenine and S-adenosylhomocysteine hydrolase

A novel route of ATP formation has been identified using erythrocytes from patients deficient in four different enzymes associated with ATP formation. It entails prior adenine production from deoxyadenosine (or adenosine) in a reaction involving S-adenosylhomocysteine hydrolase. The postulated route has been demonstrated in human erythrocytes which, unlike other human cells, cannot form ATP from IMP. It is based on studies by others using purified S-adenosylhomocysteine hydrolase preparationsin vitro. The results provide the first confirmation that this reaction occurs in intact human cellsin vitro and thus most probablyin vivo. This adenine production is normally masked in intact cells by further metabolism to ATP. Clinical significance for such a route is suggested by the fact that some adenosine analogues with potent oncostatic and antiviral properties also release adenine (or analogues)in vitro.     

Do not feed the wildlife: associations between garbage use,aggression, and disease in banded mongooses (Mungos mungo)

Urbanization and other human modifications of the landscape may indirectly affect disease dynamics by altering host behavior in ways that influence pathogen transmission. Few opportunities arise to investigate behaviorally mediated effects of human habitat modification in natural host–pathogen systems, but we provide a potential example of this phenomenon in banded mongooses (Mungos mungo), a social mammal. Our banded mongoose study population in Botswana is endemically infected with a novel Mycobacterium tuberculosis complex pathogen, M. mungi, that primarily invades the mongoose host through the nasal planum and breaks in the skin. In this system, several study troops have access to human garbage sites and other modified landscapes for foraging. Banded mongooses in our study site (N = 4 troops, ~130 individuals) had significantly higher within‐troop aggression levels when foraging in garbage compared to other foraging habitats. Second, monthly rates of aggression were a significant predictor of monthly number of injuries in troops. Finally, injured individuals had a 75% incidence of clinical tuberculosis (TB) compared to a 0% incidence in visibly uninjured mongooses during the study period. Our data suggest that mongoose troops that forage in garbage may be at greater risk of acquiring TB by incurring injuries that may allow for pathogen invasion. Our study suggests the need to consider the indirect effects of garbage on behavior and wildlife health when developing waste management approaches in human‐modified areas.     

Physio-climatic classification of South Africa's woodland biome

In an effort to develop more holistic ecosystem approaches to resource assessment and management, landscapes need to be stratified into homogeneous geographic regions. These regions can then be used in a monitoring framework to develop reliable estimates of ecosystem productivity. A regional characterization of the woodland biome has been developed for South Africa, delineated by satellite imagery and using environmental data and a rigorous statistical methodology. Distribution maps of key environmental variables are analyzed by factor analysis, an iterative clustering technique and maximum likelihood classification to quantify and identify homogeneous physio-climatic units.A spatial clustering technique was used to identify regions, which are statistically different with regard to five physiographic, climatic and edaphic variables deemed important within southern African savanna woodlands. The woodland biome of South Africa at 1km resolution was successively divided. Thirty year mean monthly temperature, total plant-available water balance of soil, elevation, landscape topographic position, and landscape soil fertility were used as input classification variables.The map data were submitted to a factor analysis and varimax axis rotation. The factor analysis removes correlations from the input variables, reduces the dimensionality, and normalizes the axis measurements. A cluster analysis was performed on the three principal factor scores using a modified iterative optimization clustering procedure to determine the finest level of classes statistically permitable. Twenty-seven identified unimodal cluster signatures were then submitted to a maximum likelihood classification where the statistical probability of the GIS cell assignment is carried out to determine class membership. The final map of custom physio-climatic regions is described, and these custom regions are compared with a vegetation potential map of the woodland types identified in the South African summer rainfall zone.     

N-acetylmannosamine-6-phosphate 2-epimerase uses a novel substrate-assisted mechanism to catalyze amino sugar epimerization

There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation–reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the β-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.     

The phylogenetic potential of entire 26S rDNA sequences in plants

18S ribosomal RNA genes are the most widely used nuclear sequences for phylogeny reconstruction at higher taxonomic levels in plants. However, due to a conservative rate of evolution, 18S rDNA alone sometimes provides too few phylogenetically informative characters to resolve relationships adequately. Previous studies using partial sequences have suggested the potential of 26S or large-subunit (LSU) rDNA for phylogeny retrieval at taxonomic levels comparable to those investigated with 18S rDNA. Here we explore the patterns of molecular evolution of entire 26S rDNA sequences and their impact on phylogeny retrieval. We present a protocol for PCR amplification and sequencing of entire (approximately 3.4 kb) 26S rDNA sequences as single amplicons, as well as primers that can be used for amplification and sequencing. These primers proved useful in angiosperms and Gnetales and likely have broader applicability. With these protocols and primers, entire 26S rDNA sequences were generated for a diverse array of 15 seed plants, including basal eudicots, monocots, and higher eudicots, plus two representatives of Gnetales. Comparisons of sequence dissimilarity indicate that expansion segments (or divergence domains) evolve 6.4 to 10.2 times as fast as conserved core regions of 26S rDNA sequences in plants. Additional comparisons indicate that 26S rDNA evolves 1.6 to 2.2 times as fast as and provides 3.3 times as many phylogenetically informative characters as 18S rDNA; compared to the chloroplast gene rbcL, 26S rDNA evolves at 0.44 to 1.0 times its rate and provides 2.0 times as many phylogenetically informative characters. Expansion segment sequences analyzed here evolve 1.2 to 3.0 times faster than rbcL, providing 1.5 times the number of informative characters. Plant expansion segments have a pattern of evolution distinct from that found in animals, exhibiting less cryptic sequence simplicity, a lower frequency of insertion and deletion, and greater phylogenetic potential.      

Feasibility of 3D UV-C treatment to reduce fungal growth and mycotoxin loads on maize and wheat kernels

Fungal disease of grain crops is a concern for the agricultural industry, resulting in economic losses. Aside from severe yield losses, mycotoxigenic fungi such as Penicillium and Fusarium can produce harmful mycotoxins, including deoxynivalenol (DON), zearalenone (ZEN), and ochratoxin A (OTA). This proof-of-concept study explored the feasibility and effects of ultraviolet (UV) C light at 253.7 nm to reduce fungal and mycotoxin loads on model surfaces as well as on maize and wheat kernels using benchtop 2D and 3D illumination strategies. Reduction of Penicillium verrucosum (98.6%) and Fusarium graminearum (88.8%) on agar was achieved using a UV-C dose of 100 mJ cm^?2. Naturally occurring fungal growth resembling P. verrucosum on maize was reduced by 79% after exposure to 5000 mJ cm^?2. Similarly, fungal growth resembling F. graminearum on maize was reduced by 60% with 1000 mJ cm^?2. On wheat, significant reduction of fungal growth was not observed. Maximal reduction of DON (97.3%), ZEN (75.4%), and OTA (91.2%) on filter paper was obtained using 15,000 mJ cm^?2. The overall reduction of DON (30%; 14%), ZEN (52%; 42%), and OTA (17%; 6%) on maize and wheat, respectively, was lower than on filter paper. Moisture and crude protein content as well as percent germination of maize kernels were not affected by UV-C treatment up to 5000 mJ cm^?2. This study has shown that 3D UV-C treatment is a feasible option for reducing Fusarium and Penicillium growth on maize kernels and, at higher doses, decreasing ZEN by ~?50%.     

Demographic costs of inbreeding revealed by sex-specific genetic rescue effects

Background  

Inbreeding can slow population growth and elevate extinction risk. A small number of unrelated immigrants to an inbred population can substantially reduce inbreeding and improve fitness, but little attention has been paid to the sex-specific effects of immigrants on such "genetic rescue". We conducted two subsequent experiments to investigate demographic consequences of inbreeding and genetic rescue in guppies.