Protein extraction from Mycobacterium avium subsp. paratuberculosis: Comparison of methods for analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis, native PAGE and surface enhanced laser desorption/ionization time of flight mass spectrometry

Mycobacterium paratuberculosis causes Johne's disease, a chronic bowel disease in ruminants worldwide and is currently incurable. This study was conducted to compare methods for examining the proteome of M. paratuberculosis. SDS-PAGE, native PAGE and SELDI-TOF-MS were compared and the efficacy of various lysis buffers was assessed. Chaotropic agents (Urea CHAPS and potassium thiocyanate) and non-ionic detergent (Tween20 and Triton X-100) extracts were compared on three different ProteinChip surfaces along with two energy absorbing molecules (EAM): EAM-1 proprietary formulation and sinapinic acid (Ciphergen). Urea CHAPS was efficient for extraction of proteins and their detection on all the ProteinChip surfaces. However, potassium thiocyanate was the most effective buffer, leading to detection of the greatest number of protein peaks on the immobilized metal affinity chromatography (IMAC) surface. Sinapinic acid was more efficient than the EAM-1 proprietary formulation and resulted in additional peaks with higher intensity for both the low and the medium molecular weight range proteins. Intra-chip and inter-chip coefficient of variation for mass/charge varied from 0.01% to 0.07% and 0.00% to 0.08%, respectively. SELDI-TOF-MS was an efficient tool for the protein profiling of M. paratuberculosis and will be useful for investigation of novel proteins, although SDS-PAGE/2D gel electrophoresis is recommended for study of high molecular weight species. All buffers were suitable for protein extraction for SDS-PAGE, while Tween20 was best for native PAGE.     

Spatiotemporal railway use by grizzly bears in Canada's Rocky Mountains

Railway networks contribute to the direct mortality of wildlife through collisions with trains, which can threaten vulnerable wildlife populations even in protected areas, including grizzly bears (Ursus arctos) in Banff and Yoho National Parks, Canada. Mitigation to reduce bear-train collisions requires information about how grizzly bears use the railway spatially and temporally and how particular types of use might increase collision vulnerability. We used data from 27 grizzly bears fitted with global positioning system (GPS) collars between 2000 and 2016 to relate railway use by bears via resource selection functions to variables that described land cover, human use, and topography. We used the same suite of explanatory variables to distinguish pairs of 4 types of steps, in which 3 successive GPS points (with 2-hr fix rates) included ≥1 within 30 m of the rail (hereafter on) and 2 others that defined locations where bears effectively entered the railway (first fix off rail, next 2 on), crossed it (only the middle fix on the rail), continued along the railway (all 3 fixes on), or exited the railway corridor (first 2 on, last off). We compared both sites of higher use and each of these 4 step types to the relative frequency of bear-train collisions, predicting a positive correlation for continue step types. Relative to available locations, bears were more likely to use the railway close to railroad sidings (sections of twinned track where trains sometimes stop), at intermediate distances from human-use features (e.g., town sites, highways, trails), in areas with lower values of the compound topographic index (a proxy for wetness; within 500 m), and within 90 m of rugged terrain. Seasonally, bears made greater use of the railway in spring and fall. Among 1,515 sequences of 3 steps, crossing locations comprised >50% and were most distinct from continue locations (about 20%), which occurred in areas with more rugged terrain (within 300 m), closer to railway sidings, in spring and fall, and with steps that were 60% shorter. Contrary to our prediction, past reports of bear-train collisions were negatively correlated with continue locations and unrelated to overall use or any other movement type. Our results suggest that railway use by bears increased where it provided increased forage or easier travel, particularly in spring and fall, but more work will be needed to determine the mechanistic basis of bear-train collisions. Meanwhile, mitigation efforts such as habitat alteration or warning systems might target locations where past strikes are concentrated for grizzly bears or other sensitive populations. © 2019 The Wildlife Society.     

Noncanonical Gβ Gib2 Is a Scaffolding Protein Promoting cAMP Signaling through Functions of Ras1 and Cac1 Proteins in Cryptococcus neoformans

Gβ-like/RACK1 functions as a key mediator of various pathways and contributes to numerous cellular functions in eukaryotic organisms. In the pathogenic fungus Cryptococcus neoformans, noncanonical Gβ Gib2 promotes cAMP signaling in cells lacking normal Gpa1 function while displaying versatility in interactions with Gα Gpa1, protein kinase Pkc1, and endocytic intersectin Cin1. To elucidate the Gib2 functional mechanism(s), we demonstrate that Gib2 is required for normal growth and virulence. We show that Gib2 directly binds to Gpa1 and Gγ Gpg1/Gpg2 and that it interacts with phosphodiesterase Pde2 and monomeric GTPase Ras1. Pde2 remains functionally dispensable, but Ras1 is found to associate with adenylyl cyclase Cac1 through the conserved Ras association domain. In addition, the ras1 mutant exhibits normal capsule formation, whereas the ras1 gpa1 mutant displays enhanced capsule formation, and the ras1 gpa1 cac1 mutant is acapsular. Collectively, these findings suggest that Gib2 promotes cAMP levels by relieving an inhibitory function of Ras1 on Cac1 in the absence of Gpa1. In addition, using GST affinity purification combined with mass spectrometry, we identified 47 additional proteins that interact with Gib2. These proteins have putative functions ranging from signal transduction, energy generation, metabolism, and stress response to ribosomal function. After establishing and validating a protein-protein interactive network, we believe Gib2 to be a key adaptor/scaffolding protein that drives the formation of various protein complexes required for growth and virulence. Our study reveals Gib2 as an essential component in deciphering the complexity of regulatory networks that control growth and virulence in C. neoformans.     

Sphingosine,a Modulator of Human Translesion DNA Polymerase Activity

Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼3000 small molecules, including one comprising ∼600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.     

Quantitative analysis of brain microstructure following mild blunt and blast trauma

We induced mild blunt and blast injuries in rats using a custom-built device and utilized in-house diffusion tensor imaging (DTI) software to reconstruct 3-D fiber tracts in brains before and after injury (1, 4, and 7 days). DTI measures such as fiber count, fiber length, and fractional anisotropy (FA) were selected to characterize axonal integrity. In-house image analysis software also showed changes in parameters including the area fraction (AF) and nearest neighbor distance (NND), which corresponded to variations in the microstructure of Hematoxylin and Eosin (H&E) brain sections. Both blunt and blast injuries produced lower fiber counts, but neither injury case significantly changed the fiber length. Compared to controls, blunt injury produced a lower FA, which may correspond to an early onset of diffuse axonal injury (DAI). However, blast injury generated a higher FA compared to controls. This increase in FA has been linked previously to various phenomena including edema, neuroplasticity, and even recovery. Subsequent image analysis revealed that both blunt and blast injuries produced a significantly higher AF and significantly lower NND, which correlated to voids formed by the reduced fluid retention within injured axons. In conclusion, DTI can detect subtle pathophysiological changes in axonal fiber structure after mild blunt and blast trauma. Our injury model and DTI method provide a practical basis for studying mild traumatic brain injury (mTBI) in a controllable manner and for tracking injury progression. Knowledge gained from our approach could lead to enhanced mTBI diagnoses, biofidelic constitutive brain models, and specialized pharmaceutical treatments.     

Growth responses, biomass partitioning, and nitrogen isotopes of prairie legumes in response to elevated temperature and varying nitrogen source in a growth chamber experiment

? Premise of the Study: Because legumes can add nitrogen (N) to ecosystems through symbiotic fixation, they play important roles in many plant communities, such as prairies and grasslands. However, very little research has examined the effect of projected climate change on legume growth and function. Our goal was to study the effects of temperature on growth, nodulation, and N chemistry of prairie legumes and determine whether these effects are mediated by source of N. ? Methods: We grew seedlings of Amorpha canescens, Dalea purpurea, Lespedeza capitata, and Lupinus perennis at 25/20°C (day/night) or 28/23°C with and without rhizobia and mineral N in controlled-environment growth chambers. Biomass, leaf area, nodule number and mass, and shoot N concentration and δ(15)N values were measured after 12 wk of growth. ? Key Results: Both temperature and N-source affected responses in a species-specific manner. Lespedeza showed increased growth and higher shoot N content at 28°C. Lupinus showed decreases in nodulation and lower shoot N concentration at 28°C. The effect of temperature on shoot N concentration occurred only in individuals whose sole N source was N(2)-fixation, but there was no effect of temperature on δ(15)N values in these plants. ? Conclusions: Elevated temperature enhanced seedling growth of some species, while inhibiting nodulation in another. Temperature-induced shifts in legume composition or nitrogen dynamics may be another potential mechanism through which climate change affects unmanaged ecosystems.     

Rhythm generation through period concatenation in rat somatosensory cortex

Rhythmic voltage oscillations resulting from the summed activity of neuronal populations occur in many nervous systems. Contemporary observations suggest that coexistent oscillations interact and, in time, may switch in dominance. We recently reported an example of these interactions recorded from in vitro preparations of rat somatosensory cortex. We found that following an initial interval of coexistent gamma ( approximately 25 ms period) and beta2 ( approximately 40 ms period) rhythms in the superficial and deep cortical layers, respectively, a transition to a synchronous beta1 ( approximately 65 ms period) rhythm in all cortical layers occurred. We proposed that the switch to beta1 activity resulted from the novel mechanism of period concatenation of the faster rhythms: gamma period (25 ms)+beta2 period (40 ms) = beta1 period (65 ms). In this article, we investigate in greater detail the fundamental mechanisms of the beta1 rhythm. To do so we describe additional in vitro experiments that constrain a biologically realistic, yet simplified, computational model of the activity. We use the model to suggest that the dynamic building blocks (or motifs) of the gamma and beta2 rhythms combine to produce a beta1 oscillation that exhibits cross-frequency interactions. Through the combined approach of in vitro experiments and mathematical modeling we isolate the specific components that promote or destroy each rhythm. We propose that mechanisms vital to establishing the beta1 oscillation include strengthened connections between a population of deep layer intrinsically bursting cells and a transition from antidromic to orthodromic spike generation in these cells. We conclude that neural activity in the superficial and deep cortical layers may temporally combine to generate a slower oscillation.     

Quantifying how acquired interactions with native and invasive insects influence population growth rates of a non-indigenous plant

Non-native species often acquire novel interspecific interactions, which are central to several hypotheses of invasion success, including biotic resistance and invasional meltdown. However, the outcome of these interactions is not often linked with the demographic evidence based on the full life cycle of the species. The Philippine Ground Orchid (Spathoglottis plicata) has invaded Puerto Rico and has acquired both negative and positive interspecific interactions involving the native weevil Stethobaris polita and the invasive red fire ant Solenopsis invicta, respectively. We studied a population in the Rio Abajo Forest, and asked how these interactions affect population demography by using a combination of field, experimental and modelling approaches. Stage-structured matrix population models based on four years of field observations showed that the population of S. plicata is growing at a rate (λ) of 1.05 under natural conditions. When we modified fecundity values based on experimental exclusion of weevils and ants, the control treatment showed a similar λ. Excluding weevils increased λ to 1.20, whereas the exclusion of ants decreased λ to 1.03. When we incorporate demographic and environmental stochasticity in our models, exclusion of invasive red fire ants significantly reduces the orchid abundance over time. Although weevils offer some biotic resistance to S. plicata, these effects do not prevent orchid population growth and expansion. On the other hand, invasive red fire ants have a positive effect on the invasive orchid’s λ, partially supporting the invasional meltdown hypothesis. This study presents a method that allows one to combine opposing mechanisms of species interactions within the same quantitative framework, and the results highlight the importance of considering acquired plant–animal interactions and stochastic processes when evaluating the population growth rates and dynamics of invasive plants.     

The influence of vertical mixing on the photoinhibition of variable chlorophyll a fluorescence and its inclusion in a model of phytoplankton photosynthesis

The maximum effective quantum yield of photosystem II was estimatedfrom measurements of variable chlorophyll a fluorescence [(F'_m- F'_o)/F'_m = F'_v/F'_m] in samples of phytoplankton collectedfrom various depths in Chaffey Reservoir, Australia. Duringstratified conditions, F'_v/F'_m showed depth-dependent decreasesas irradiance increased during the morning, and increases asirradiancereduced in the afternoon. Wind-induced mixing disrupted thediel pattern, but even under well-mixed conditions a verticalgradient in F'_v/F'_m remained. Differences in F'_v/F'_m valuesbetween samples incubated at fixed depths and unconstrainedlake samples enabled identification of the phytoplankton mixingdepth. Recovery of F'_v/F'_m was modelled as a function of timeand the degree of F'_v/F'_m inhibition, while damage was considereda function of photon dose. A combined, numerical model was fittedto diel sequences of F'_v/F'_m to estimate rate constants fordamage and repair. Recovery rate constants ranged from r = 0.7x 10^-4 to 9.1 x 10^-4 s^-1, while damage rate constants rangedfrom k = 0.03 to 0.22 m² mol photon^-1. A fluorescence-basedmodel of photosynthesis was used to investigate the effectsof wind speed, euphotic depth and mixed layer depth on photoinhibition.At different mixing rates, depth-integrated photosynthesis wasenhanced by up to 16% under the conditions tested, while increasesof 9% occurred between phytoplankton with different measureddamage and repair characteristics.