Systematic Analysis of Clemastine, a Candidate Apicomplexan Parasite-Selective Tubulin-Targeting Agent

Apicomplexan parasites, such as Toxoplasma gondii, Plasmodium spp., Babesia spp., and Cryptosporidium spp., cause significant morbidity and mortality. Existing treatments are problematic due to toxicity and the emergence of drug-resistant parasites. Because protozoan tubulin can be selectively disrupted by small molecules to inhibit parasite growth, we assembled an in vitro testing cascade to fully delineate effects of candidate tubulin-targeting drugs on Toxoplasma gondii and vertebrate host cells. Using this analysis, we evaluated clemastine, an antihistamine that has been previously shown to inhibit Plasmodium growth by competitively binding to the CCT/TRiC tubulin chaperone as a proof-of-concept. We concurrently analyzed astemizole, a distinct antihistamine that blocks heme detoxification in Plasmodium. Both drugs have EC50 values of ~2 µM and do not demonstrate cytotoxicity or vertebrate microtubule disruption at this concentration. Parasite subpellicular microtubules are shortened by treatment with either clemastine or astemizole but not after treatment with pyrimethamine, indicating that this effect is not a general response to antiparasitic drugs. Immunoblot quantification indicates that the total α-tubulin concentration of 0.02 pg/tachyzoite does not change with clemastine treatment. In conclusion, the testing cascade allows profiling of small-molecule effects on both parasite and vertebrate cell viability and microtubule integrity.

4-O-Glucosylation of Trichothecenes by Fusarium Species: A Phase II Xenobiotic Metabolism for t-Type Trichothecene Producers

The t-type trichothecene producers Fusarium sporotrichioides and Fusarium graminearum protect themselves against their own mycotoxins by acetylating the C-3 hydroxy group with Tri101p acetylase. To understand the mechanism by which they deal with exogenously added d-type trichothecenes, the Δtri5 mutants expressing all but the first trichothecene pathway enzymes were fed with trichodermol (TDmol), trichothecolone (TCC), 8-deoxytrichothecin, and trichothecin. LC-MS/MS and NMR analyses showed that these C-3 unoxygenated trichothecenes were conjugated with glucose at C-4 by α-glucosidic linkage. As t-type trichothecenes are readily incorporated into the biosynthetic pathway following the C-3 acetylation, the mycotoxins were fed to the ΔFgtri5ΔFgtri101 mutant to examine their fate. LC-MS/MS and NMR analyses demonstrated that the mutant conjugated glucose at C-4 of HT-2 toxin (HT-2) by α-glucosidic linkage, while the ΔFgtri5 mutant metabolized HT-2 to 3-acetyl HT-2 toxin and T-2 toxin. The 4-O-glucosylation of exogenously added t-type trichothecenes appears to be a general response of the ΔFgtri5ΔFgtri101 mutant, as nivalenol and its acetylated derivatives appeared to be conjugated with hexose to some extent. The toxicities of 4-O-glucosides of TDmol, TCC, and HT-2 were much weaker than their corresponding aglycons, suggesting that 4-O-glucosylation serves as a phase II xenobiotic metabolism for t-type trichothecene producers.

Effects of Different Grazing Disturbances on the Plant Diversity and Ecological Functions of Alpine Grassland Ecosystem on the Qinghai-Tibetan Plateau

Grazing is one of the main human disturbance factors in alpine grassland on the Qinghai-Tibet Plateau (QTP), which can directly or indirectly influence the community structures and ecological functions of grassland ecosystems. However, despite extensive field grazing experiments, there is currently no consensus on how different grazing management approaches affect alpine grassland diversity, soil carbon (C), and nitrogen (N). Here, we conducted a meta-analysis of 70 peer-reviewed publications to evaluate the general response of 11 variables related to alpine grassland ecosystems plant diversity and ecological functions to grazing. Overall, the results showed that grazing significantly increased the species richness, Shannon–Wiener index, and Pielou evenness index values by 9.89% (95% CI: 2.75–17.09%), 7.28% (95% CI: 1.68–13.62%), and 3.74% (95% CI: 1.40–6.52%), respectively. Aboveground biomass (AGB) and belowground biomass (BGB) decreased, respectively, by 41.91% (95% CI: −50.91 to −32.88%) and 17.68% (95% CI: −26.94 to −8.52%). Soil organic carbon (SOC), soil total nitrogen (TN), soil C:N ratio, and soil moisture decreased by 13.06% (95% CI: −15.88 to −10.15%), 12.62% (95% CI: −13.35 to −8.61%), 3.27% (95% CI: −4.25 to −2.09%), and 20.75% (95% CI: −27.89 to −13.61%), respectively, whereas, soil bulk density and soil pH increased by 17.46% (95% CI: 11.88–24.53%) and 2.24% (95% CI: 1.01–3.64%), respectively. Specifically, moderate grazing, long-durations (>5 years), and winter grazing contributed to increases in the species richness, Shannon–Wiener index, and Pielou evenness index. However, AGB, BGB, SOC, TN, and soil C:N ratios showed a decrease with enhanced grazing intensity. The response ratio of SOC was positively associated with AGB and BGB but was negatively related to the Shannon–Wiener index and Pielou evenness index. Furthermore, the effects of grazing on plant diversity, AGB, BGB, SOC, and TN in alpine grassland varied with grazing duration, grazing season, livestock type, and grassland type. The findings suggest that grazing should synthesize other appropriate grazing patterns, such as seasonal and rotation grazing, and, furthermore, additional research on grazing management of alpine grassland on the QTP is needed in the future.

Motor plans under uncertainty reflect a trade-off between maximizing reward and success

When faced with multiple potential movement options, individuals either reach directly to one of the options, or initiate a reach intermediate between the options. It remains unclear why people generate these two types of behaviors. Using the go-before-you-know task (commonly used to study behavior under choice uncertainty), we examined two key questions. First, do these two types of responses reflect distinct movement strategies, or are they simply examples of a more general response to choice uncertainty? If the former, the relative desirability (i.e., weighing the likelihood of successfully hitting the target versus the attainable reward) of the two target options might be computed differently for direct versus intermediate reaches. We showed that indeed, when exogenous reward and success likelihood (i.e., endogenous reward) differ between the two options, direct reaches were more strongly biased by likelihood whereas intermediate movements were more strongly biased by reward. Second, what drives individual differences in how people respond under uncertainty? We found that risk/reward-seeking individuals generated a larger proportion of intermediate reaches and were more sensitive to trial-to-trial changes in reward, suggesting these movements reflect a strategy to maximize reward. In contrast, risk-adverse individuals tended to generate more direct reaches in an attempt to maximize success. Together, these findings suggest that when faced with choice uncertainty, individuals adopt movement strategies consistent with their risk/reward-seeking tendency, preferentially biasing behavior toward exogenous rewards or endogenous success and consequently modulating the relative desirability of the available options.

Signaling pathways of heat- and hypersalinity-induced polyp bailout in Pocillopora acuta

Polyp bailout is a drastic response to acute stress where coral coloniality breaks down and polyps detach. We induced polyp bailout in Pocillopora acuta with heat stress and tested for differential gene expression using RNAseq and a qPCR assay. Furthermore, we induced polyp bailout with hypersalinity and compared the results to identify stressor-independent signals and pathways active during polyp bailout. Both stressors led to the onset of polyp bailout and the detachment of vital polyps. We observed activation of microbe-associated molecular pattern receptors and downstream signaling pathways of the innate immune system. Further, we detected growth factors and genes active during Wnt-signaling potentially contributing to wound healing, regeneration, and proliferation. Upregulation of several genes encoding for matrix metalloproteinases and the fibroblast growth factor signaling pathway are the most likely involved in the remodeling of the extracellular matrix, as well as in the detachment of polyps from the calcareous skeleton during polyp bailout. Expression of genes of interest in our qPCR assay of vital polyps from our heat-stress experiment, showed a trend for a normalization of gene expression after polyp bailout. Our results provide new insights into the signaling cascades leading to the observed physiological responses during polyp bailout. Comparison between the two stressors showed that certain signaling pathways are independent of the stressor and suggested that polyp bailout is a general response of corals to acute stress. Furthermore, immune system responses during polyp bailout indicate that microbe-associated partners of corals may lead to the polyp bailout response.

Application of crude pectolytic enzymes from Saccaharomyces cerevisiae (ATCC 52,712) to starch extraction in Ghana: effects of enzyme technology on pasting characteristics in different cassava varieties

Previous work on enzyme application to starch extraction enhanced yield and starch recovery rates as well as modified some physicochemical properties of starches for potential alternative application to industry. The response of the technology, however, showed some sensitivity to variety. The knowledge gap therefore was to establish whether such physicochemical responses (by the technology) to variety affects the pasting parameters of the starches extracted. The pasting parameters of starches extracted from four different cassava varieties (‘Nkabom’, ‘Afisiafi’, ‘Bankyehemaa’ and ‘Esambankye’), with the aid of crude pectolytic enzymes from the Saccharomyces cerevisiae (ATCC 52,712), were investigated. Although a general response pattern was observed for most of the pasting parameters measured, which includes general enhancements (P < 0.05) in starch gelatinization viscosity, with improvements in gelatinization time and temperature and peak viscosities in most of the varieties, there were significant differences (P < 0.05) in their respective peak time and temperature requirements for the attainment of peak viscosity. Values for the breakdown viscosity were also generally increased (P < 0.05). The technology also increased values for setback viscosity in both the ‘Nkabom’ and ‘Bankyehemaa’ varieties but reduced setback values in the ‘Afisiafi’ and ‘Esambankye’ varieties. As pasting properties are one of the most important characteristics of starch that determine its overall utility, knowledge from this study should inform how adoption of the technology would help diversify the various cassava varieties for appropriate domestic and industrial applications while harnessing its benefits of improved starch yield.

A cis-regulatory element promoting increased transcription at low temperature in cultured ectothermic Drosophila cells

Background Temperature change affects the myriad of concurrent cellular processes in a non-uniform, disruptive manner. While endothermic organisms minimize the challenge of ambient temperature variation by keeping the core body temperature constant, cells of many ectothermic species maintain homeostatic function within a considerable temperature range. The cellular mechanisms enabling temperature acclimation in ectotherms are still poorly understood. At the transcriptional level, the heat shock response has been analyzed extensively. The opposite, the response to sub-optimal temperature, has received lesser attention in particular in animal species. The tissue specificity of transcriptional responses to cool temperature has not been addressed and it is not clear whether a prominent general response occurs. Cis-regulatory elements (CREs), which mediate increased transcription at cool temperature, and responsible transcription factors are largely unknown. Results The ectotherm Drosophila melanogaster with a presumed temperature optimum around 25 °C was used for transcriptomic analyses of effects of temperatures at the lower end of the readily tolerated range (14–29 °C). Comparative analyses with adult flies and cell culture lines indicated a striking degree of cell-type specificity in the transcriptional response to cool. To identify potential cis-regulatory elements (CREs) for transcriptional upregulation at cool temperature, we analyzed temperature effects on DNA accessibility in chromatin of S2R+ cells. Candidate cis-regulatory elements (CREs) were evaluated with a novel reporter assay for accurate assessment of their temperature-dependency. Robust transcriptional upregulation at low temperature could be demonstrated for a fragment from the pastrel gene, which expresses more transcript and protein at reduced temperatures. This CRE is controlled by the JAK/STAT signaling pathway and antagonizing activities of the transcription factors Pointed and Ets97D. Conclusion Beyond a rich data resource for future analyses of transcriptional control within the readily tolerated range of an ectothermic animal, a novel reporter assay permitting quantitative characterization of CRE temperature dependence was developed. Our identification and functional dissection of the pst_E1 enhancer demonstrate the utility of resources and assay. The functional characterization of this CoolUp enhancer provides initial mechanistic insights into transcriptional upregulation induced by a shift to temperatures at the lower end of the readily tolerated range.

Altered collective mitochondrial dynamics in an Arabidopsis msh1 mutant compromising organelle DNA maintenance

Mitochondria form highly dynamic populations in the cells of plants (and all eukaryotes). The characteristics of this collective behaviour, and how it is influenced by nuclear features, remain to be fully elucidated. Here, we use a recently-developed quantitative approach to reveal and analyse the physical and collective "social" dynamics of mitochondria in an Arabidopsis msh1 mutant where organelle DNA maintenance machinery is compromised. We use a newly-created line combining the msh1 mutant with mitochondrially-targeted GFP, and characterise mitochondrial dynamics with a combination of single-cell timelapse microscopy, computational tracking and network analysis. The collective physical behaviour of msh1 mitochondria is altered from wildtype in several ways: mitochondria become less evenly spread, and networks of inter-mitochondrial encounters become more connected with greater potential efficiency for inter-organelle exchange. We find that these changes are similar to those observed in friendly, where mitochondrial dynamics are altered by a physical perturbation, suggesting that this shift to higher connectivity may reflect a general response to mitochondrial challenges.

Design and Development of Customer Relationship Management Recommendations by Clustering and Profiling of Customers Using RFM

RFM (recency, frequency, monetary) examination is a method to perceive high-response customers in promoting progressions and to improve general response rates, which is remarkable and is comprehensively associated today. Less extensively understood is the estimation of applying RFM scoring to a customer database and evaluating customer advantage. A customer who has passed by an e-keeping cash site recently (R) and frequently (F) and influenced a huge amount of monetary to esteem (M) through portion and standing solicitations is presumably going to visit and make portions yet again. After appraisal of the customer's lead using specific RFM criteria, the RFM score is related to the bank excitement, with a high RFM score being more important to the bank by and by and later on.

Computational mechanics of the paddlefish rostrum

Purpose – The rostrum of a paddlefish provides hydrodynamic stability during feeding process in addition to detect the food using receptors that are randomly distributed in the rostrum. The exterior tissue of the rostrum covers the cartilage that surrounds the bones forming interlocking star shaped bones. Design/methodology/approach – The aim of this work is to assess the mechanical behavior of four finite element models varying the type of formulation as follows: linear-reduced integration, linear-full integration, quadratic-reduced integration and quadratic-full integration. Also presented is the load transfer mechanisms of the bone structure of the rostrum. Findings – Conclusions are based on comparison among the four models. There is no significant difference between integration orders for similar type of elements. Quadratic-reduced integration formulation resulted in lower structural stiffness compared with linear formulation as seen by higher displacements and stresses than using linearly formulated elements. It is concluded that second-order elements with reduced integration and can model accurately stress concentrations and distributions without over stiffening their general response. Originality/value – The use of advanced computational mechanics techniques to analyze the complex geometry and components of the paddlefish rostrum provides a viable avenue to gain fundamental understanding of the proper finite element formulation needed to successfully obtain the system behavior and hot spot locations.