Roadmap for the expression of canonical and extended endocannabinoid system receptors and metabolic enzymes in peripheral organs of preclinical animal models.

The endocannabinoid system is widely expressed throughout the body and is comprised of receptors, ligands, and enzymes that maintain metabolic, immune, and reproductive homeostasis. Increasing interest in the endocannabinoid system has arisen due to these physiologic roles, policy changes leading to more widespread recreational use, and the therapeutic potential of Cannabis and phytocannabinoids. Rodents have been the primary preclinical model of focus due to their relative low cost, short gestational period, genetic manipulation strategies, and gold-standard behavioral tests. However, the potential for lack of clinical translation to non-human primates and humans is high as cross-species comparisons of the endocannabinoid system have not been evaluated. To bridge this gap in knowledge, we evaluate the relative gene expression of 14 canonical and extended endocannabinoid receptors in seven peripheral organs of C57/BL6 mice, Sprague-Dawley rats, and non-human primate rhesus macaques. Notably, we identify species- and organ-specific heterogeneity in endocannabinoid receptor distribution where there is surprisingly limited overlap among the preclinical models. Importantly, we determined there were no receptors with identical expression patterns among mice (three males and two females), rats (six females), and rhesus macaques (four males). Our findings demonstrate a critical, yet previously unappreciated, contributor to challenges of rigor and reproducibility in the cannabinoid field, which has implications in hampering progress in understanding the complexity of the endocannabinoid system and development of cannabinoid-based therapies.

Roadmap For The Expression Of Canonical and Extended Endocannabinoid System Receptors and Proteins in Peripheral Organs of Preclinical Animal Models.

The endocannabinoid system is widely expressed throughout the body and is comprised of receptors, ligands, and enzymes that maintain metabolic, immune, and reproductive homeostasis. Increasing interest in the endocannabinoid system has arisen due to these physiologic roles, policy changes leading to more widespread recreational use, and the therapeutic potential of Cannabis and phytocannabinoids. Rodents have been the primary preclinical model of focus due to their relative low cost, short gestational period, genetic manipulation strategies, and gold-standard behavioral tests. However, the potential for lack of clinical translation to non-human primates and humans is high as cross-species comparisons of the endocannabinoid system has not been evaluated. To bridge this gap in knowledge, we evaluate the relative gene expression of 14 canonical and extended endocannabinoid receptors in seven peripheral organs of C57/BL6 mice, Sprague-Dawley rats, and non-human primate rhesus macaques. Notably, we identify species- and organ-specific heterogeneity in endocannabinoid receptor distribution where there is surprisingly limited overlap among the preclinical models. Importantly, we determined there were only five receptors (CB2, GPR18, GPR55, TRPV2, and FAAH) that had identical expression patterns in mice, rats, and rhesus macaques. Our findings demonstrate a critical, yet previously unappreciated, contributor to challenges of rigor and reproducibility in the cannabinoid field, which has profound implications in hampering progress in understanding the complexity of the endocannabinoid system and development of cannabinoid-based therapies.

Daily patterns in parasite processes: diel variation in fish louse transcriptomes.

Parasites, similar to all other organisms, time themselves to environmental cues using a molecular clock to generate and maintain rhythms. Chronotherapeutic (timed treatment) techniques based on such rhythms offer great potential for improving control of chronic, problematic parasites. Fish lice are a key disease threat in aquaculture, with current control insufficient. Assessing the rhythmicity of fish lice transcriptomes offers not only insight into the viability of chronotherapy, but the opportunity to identify new drug targets. Here, for the first known time in any crustacean parasite, diel changes in gene transcription are examined, revealing that approximately half of the Argulus foliaceus annotated transcriptome displays significant daily rhythmicity. We identified rhythmically transcribed putative clock genes including core clock/cycle and period/timeless pairs, alongside rhythms in feeding-associated genes and processes involving immune response, as well as fish louse drug targets. A substantial number of gene pathways showed peak transcription in hours immediately preceding onset of light, potentially in anticipation of peak host anti-parasite responses or in preparation for increased feeding activity. Genes related to immune haemocyte activity and chitin development were more highly transcribed 4 h post light onset, although inflammatory gene transcription was highest during dark periods. Our study provides an important resource for application of chronotherapy in fish lice; timed application could increase efficacy and/or reduce dose requirement, improving the current landscape of drug resistance and fish health while reducing the economic cost of infection.

Gene expression varies within and between enzootic and epizootic lineages of Batrachochytrium dendrobatidis (Bd) in the Americas.

While much research focus is paid to hypervirulent fungal lineages during emerging infectious disease outbreaks, examining enzootic pathogen isolates can be equally fruitful in delineating infection dynamics and determining pathogenesis. The fungal pathogen of amphibians, Batrachochytrium dendrobatidis (Bd), exhibits markedly different patterns of disease in natural populations, where it has caused massive amphibian declines in some regions, yet persists enzootically in others. Here we compare in vitro gene expression profiles of a panel of Bd isolates representing both the enzootic Bd-Brazil lineage, and the more recently diverged, panzootic lineage, Bd-GPL. We document significantly different lineage-specific and intralineage gene expression patterns, with Bd-Brazil upregulating genes with aspartic-type peptidase activity, and Bd-GPL upregulating CBM18 chitin-binding genes, among others. We also find pronounced intralineage variation in membrane integrity and transmembrane transport ability within our Bd-GPL isolates. Finally, we highlight unexpectedly divergent expression profiles in sympatric panzootic isolates, underscoring microgeographic functional variation in a largely clonal lineage. This variation in gene expression likely plays an important role in the relative pathogenesis and host range of Bd-Brazil and Bd-GPL isolates. Together, our results demonstrate that functional genomics approaches can provide information relevant to studies of virulence evolution within the Bd clade.

How do climate change experiments alter plot-scale climate?

To understand and forecast biological responses to climate change, scientists frequently use field experiments that alter temperature and precipitation. Climate manipulations can manifest in complex ways, however, challenging interpretations of biological responses. We reviewed publications to compile a database of daily plot-scale climate data from 15 active-warming experiments. We find that the common practices of analysing treatments as mean or categorical changes (e.g. warmed vs. unwarmed) masks important variation in treatment effects over space and time. Our synthesis showed that measured mean warming, in plots with the same target warming within a study, differed by up to 1.6  ∘ C (63% of target), on average, across six studies with blocked designs. Variation was high across sites and designs: for example, plots differed by 1.1  ∘ C (47% of target) on average, for infrared studies with feedback control (n = 3) vs. by 2.2  ∘ C (80% of target) on average for infrared with constant wattage designs (n = 2). Warming treatments produce non-temperature effects as well, such as soil drying. The combination of these direct and indirect effects is complex and can have important biological consequences. With a case study of plant phenology across five experiments in our database, we show how accounting for drier soils with warming tripled the estimated sensitivity of budburst to temperature. We provide recommendations for future analyses, experimental design, and data sharing to improve our mechanistic understanding from climate change experiments, and thus their utility to accurately forecast species' responses.

A neglected fish stressor: mechanical disturbance during transportation impacts susceptibility to disease in a globally important ornamental fish.

The transport of fish in aquaculture and the ornamental trade exposes fish to multiple stressors that can cause mass mortalities and economic loss. Previous research on fish transport has largely focussed on chemical stress related to deterioration in water quality. However, mechanical disturbance during routine fish transport is unpredictable and is a neglected potential stressor when studying fish welfare. Stress-induced immunosuppression caused by mechanical disturbance can increase the chances of contracting infections and can significantly increase infection burden. Here, using a model host-parasite system (guppy Poecilia reticulata and the monogenean ectoparasite Gyrodactylus turnbulli) and a new method of bagging fish (Breathing Bags™), which reduces mechanical disturbance during fish transport, we investigated how parasite infections contracted after simulated transport impact infection trajectories on a globally important ornamental freshwater species. Guppies exposed to mechanical transport disturbance suffered significantly higher parasite burden compared to fish that did not experience transport disturbance. Unfortunately, there was no significant reduction in parasite burden of fish transported in the Breathing Bags™ compared to standard polythene carrier bags. Thus, transport-induced mechanical disturbance, hitherto neglected as a stressor, can be detrimental to disease resistance and highlights the need for specific management procedures to reduce the impact of infectious diseases following routine fish transport.

Maintaining functional major histocompatibility complex diversity under inbreeding: the case of a selfing vertebrate.

Major histocompatibility complex (MHC) genes encode proteins that present pathogen-derived antigens to T-cells, initiating the adaptive immune response in vertebrates. Although populations with low MHC diversity tend to be more susceptible to pathogens, some bottlenecked populations persist and even increase in numbers despite low MHC diversity. Thus, the relative importance of MHC diversity versus genome-wide variability for the long-term viability of populations after bottlenecks and/or under high inbreeding is controversial. We tested the hypothesis that genome-wide inbreeding (estimated using microsatellites) should be more critical than MHC diversity alone in determining pathogen resistance in the self-fertilizing fish Kryptolebias marmoratus by analysing MHC diversity and parasite loads in natural and laboratory populations with different degrees of inbreeding. Both MHC and neutral diversities were lost after several generations of selfing, but we also found evidence of parasite selection acting on MHC diversity and of non-random loss of alleles, suggesting a possible selective advantage of those individuals with functionally divergent MHC, in accordance with the hypothesis of divergent allele advantage. Moreover, we found that parasite loads were better explained by including MHC diversity in the model than by genome-wide (microsatellites) heterozygosity alone. Our results suggest that immune-related overdominance could be the key in maintaining variables rates of selfing and outcrossing in K. marmoratus and other mixed-mating species.

Decoding emotional prosody: resolving differences in functional neuroanatomy from fMRI and lesion studies using TMS.

BACKGROUND: Prosody conveys information about the emotional state and intention of others. Lesion studies have shown that damage to the right posterior temporal region is associated with prosody decoding deficits. Dissimilarly to findings from lesion studies, neuroimaging data show substantial bilateral peri-Sylvian activation. OBJECTIVE: This study aimed to investigate the involvement of the left and right superior temporal gyrus (STG) in prosodic and semantic processing using transcranial magnetic stimulation (TMS). These two regions of interest were chosen for their correspondence to Wernicke's area in the left hemisphere and its analog in the right. METHODS: Offline TMS with a stimulation frequency of 1 Hz and intensity of 60% of stimulator output (approximately 1.1 Tesla) with one pulse applied per second for 10 minutes (600 pulses) was performed. Directly after TMS on the right STG, the left STG or sham-stimulation, participants completed a prosody decoding or a semantic judgment task (whether the tone/meaning was happy or sad). RESULTS: Reaction times (RT) for the prosodic task were significantly slower when TMS was applied in the right STG in comparison to left STG and sham conditions. TMS over both right and left STG delayed RT in the semantic task, significantly when the tone of voice was incongruent with the meaning. CONCLUSIONS: Our data strongly suggests that left temporal regions are not crucial to the basic task of prosody decoding per se; however, the analogous region on the right is. Hence, involvement of the left STG in prosodic decoding revealed in previous imaging data is incidental.

Contralateral visual search deficits following TMS.

Transcranial magnetic stimulation of posterior parietal vs. superior temporal sites cause differential effects on conventional conjunction vs. feature search tasks, respectively. We now report that when a decision has to be made on the target's left/right location, different lateralized deficits emerge in these two cases. With full-field arrays, we found a specific PPC search deficit for contralateral space. With smaller, structured arrays presented in left or right hemispace, we found a specific STG deficit for contralateral parts of the array.

Full recovery of electron damage in glass at ambient temperatures.

An unusually complete recovery of extensive electron-beam-induced damage in a thin film of a CaO-Al2O3-SiO2 glass was discovered. Nanoscale measurements show that the Ca ions migrate about 10 nm away during irradiation and return during recovery. Oxygen atoms are trapped largely as molecular oxygen and do not migrate. Electron energy loss measurements demonstrate that the glass returns completely to the original compositional and structural state thus indicating that the glass is in a deep thermodynamic energy minimum.

Nutrient limitation and stoichiometry of carnivorous plants.

The cost-benefit model for the evolution of carnivorous plants posits a trade-off between photosynthetic costs associated with carnivorous structures and photosynthetic benefits accrued through additional nutrient acquisition. The model predicts that carnivory is expected to evolve if its marginal benefits exceed its marginal costs. Further, the model predicts that when nutrients are scarce but neither light nor water is limiting, carnivorous plants should have an energetic advantage in competition with non-carnivorous plants. Since the publication of the cost-benefit model over 20 years ago, marginal photosynthetic costs of carnivory have been demonstrated but marginal photosynthetic benefits have not. A review of published data and results of ongoing research show that nitrogen, phosphorus, and potassium often (co-)limit growth of carnivorous plants and that photosynthetic nutrient use efficiency is 20 - 50 % of that of non-carnivorous plants. Assessments of stoichiometric relationships among limiting nutrients, scaling of leaf mass with photosynthesis and nutrient content, and photosynthetic nutrient use efficiency all suggest that carnivorous plants are at an energetic disadvantage relative to non-carnivorous plants in similar habitats. Overall, current data support some of the predictions of the cost-benefit model, fail to support others, and still others remain untested and merit future research. Rather than being an optimal solution to an adaptive problem, botanical carnivory may represent a set of limited responses constrained by both phylogenetic history and environmental stress.

Functional analysis of human MLH1 and MSH2 missense variants and hybrid human-yeast MLH1 proteins in Saccharomyces cerevisiae.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant inherited disease caused by defects in the process of DNA mismatch repair (MMR), and mutations in the hMLH1 or hMSH2 genes are responsible for the majority of HNPCC. In addition to clear loss-of-function mutations conferred by nonsense or frameshift alterations in the coding sequence or by splice variants, genetic screening has revealed a large number of missense codons with less obvious functional consequences. The ability to discriminate between a loss-of-function mutation and a silent polymorphism is important for genetic testing for inherited diseases like HNPCC where the opportunity exists for early diagnosis and preventive intervention. In this study, quantitative in vivo DNA MMR assays in the yeast Saccharomyces cerevisiae were performed to determine the functional significance of amino acid replacements observed in the human population. Missense codons previously observed in human genes were introduced at the homologous residue in the yeast MLH1 or MSH2 genes. This study also demonstrated feasibility of constructing genes that encode functional hybrid human-yeast MLH1 proteins. Three classes of missense codons were found: (i) complete loss of function, i.e. mutations; (ii) variants indistinguishable from wild-type protein, i.e. silent polymorphisms; and (iii) functional variants which support MMR at reduced efficiency, i.e. efficiency polymorphisms. There was a good correlation between the functional results in yeast and available human clinical data regarding penetrance of the missense codon. The results reported here raise the intriguing possibility that differences in the efficiency of DNA MMR exist between individuals in the human population due to common polymorphisms.

The role of transcranial magnetic stimulation (TMS) in studies of vision, attention and cognition.

Transcranial magnetic stimulation (TMS) can be conceptualized as a virtual lesion technique, capable of disrupting organized cortical activity, transiently and reversibly. The technique combines good spatial and temporal resolution and, moreover, because it represents an interference technique, can be said to have excellent functional resolution. The following is a review and discussion of the contribution which TMS has made to the study of vision, attention, development and plasticity and speech and language.

Complementary localization and lateralization of orienting and motor attention.

It is widely agreed that the right posterior parietal cortex has a preeminent role in visuospatial and orienting attention. A number of lines of evidence suggest that although orienting and the preparation of oculomotor responses are dissociable from each other, the two are intimately related. If this is true, then it should be possible to identify other attentional mechanisms tied to other response modalities. We used repetitive transcranial magnetic stimulation (rTMS) to demonstrate the existence of a distinct anterior parietal mechanism of motor attention. The critical area for motor attention is anterior to the one concerned with orienting, and it is lateralized to the left hemisphere in humans.

Interspecific and intraspecific variation in seed size and germination requirements of Sarracenia (Sarraceniaceae).

Seed size and germination requirements of eight (of nine) Sarracenia species, and 13 populations of S. purpurea were studied. All species except for S. purpurea are restricted to the southeastern United States, whereas S. purpurea ranges across Canada, southward along the eastern United States into Maryland and Virginia (S. purpurea ssp. purpurea), and from New Jersey southward into northern Florida and the coast of the Gulf of Mexico (S. purpurea ssp. venosa). I tested the hypotheses that dormancy-breaking requirements vary predictably among species across a latitudinal gradient. I also sought to determine whether seed size and germination requirements were useful characters for resolving systematic and phylogenetic questions within this genus. Seed size varied significantly among species, but variability in seed size within S. purpurea exceeded the variability in seed size observed across all eight species studied. Seeds of all species are morphophysiologically dormant upon dispersal. Length of required cool, moist pretreatment varied among species, and germination in higher latitude populations is enhanced with longer pretreatment. In contrast, variability in germination requirements of subspecies, varieties, and populations of the geographically wide-ranging S. purpurea was not related clearly to geographic location (latitude or elevation). Germination requirements do not map onto a proposed phylogeny of Sarracenia, but observed differences in germination requirements of S. purpurea ssp. venosa var. burkii relative to other populations of S. purpurea support the recent proposal to elevate this variety to species status.