Mapping Conformational Changes in the Saliva Proteome Potentially Associated with Oral Cancer Aggressiveness.

Diverse proteomics-based strategies have been applied to saliva to quantitatively identify diagnostic and prognostic targets for oral cancer. Considering that these targets may be regulated by events that do not imply variation in protein abundance levels, we hypothesized that changes in protein conformation can be associated with diagnosis and prognosis, revealing biological processes and novel targets of clinical relevance. For this, we employed limited proteolysis-mass spectrometry in saliva samples to explore structural alterations, comparing the proteome of healthy control and oral squamous cell carcinoma (OSCC) patients with and without lymph node metastasis. Thirty-six proteins with potential structural rearrangements were associated with clinical patient features including transketolase and its interacting partners. Moreover, N-glycosylated peptides contribute to structural rearrangements of potential diagnostic and prognostic markers. Altogether, this approach utilizes saliva proteins to search for targets for diagnosing and prognosing oral cancer and can guide the discovery of potential regulated sites beyond protein-level abundance.

Exploring the Potential of Structure-Based Deep Learning Approaches for T cell Receptor Design.

Deep learning methods, trained on the increasing set of available protein 3D structures and sequences, have substantially impacted the protein modeling and design field. These advancements have facilitated the creation of novel proteins, or the optimization of existing ones designed for specific functions, such as binding a target protein. Despite the demonstrated potential of such approaches in designing general protein binders, their application in designing immunotherapeutics remains relatively unexplored. A relevant application is the design of T cell receptors (TCRs). Given the crucial role of T cells in mediating immune responses, redirecting these cells to tumor or infected target cells through the engineering of TCRs has shown promising results in treating diseases, especially cancer. However, the computational design of TCR interactions presents challenges for current physics-based methods, particularly due to the unique natural characteristics of these interfaces, such as low affinity and cross-reactivity. For this reason, in this study, we explored the potential of two structure-based deep learning protein design methods, ProteinMPNN and ESM-IF, in designing fixed-backbone TCRs for binding target antigenic peptides presented by the MHC through different design scenarios. To evaluate TCR designs, we employed a comprehensive set of sequence- and structure-based metrics, highlighting the benefits of these methods in comparison to classical physics-based design methods and identifying deficiencies for improvement.

Cavity Characterization in Supramolecular Cages.

Confining molecular guests within artificial hosts has provided a major driving force in the rational design of supramolecular cages with tailored properties. Over the last 30 years, a set of design strategies have been developed that enabled the controlled synthesis of a myriad of cages. Recently, there has been a growing interest in involving in silico methods in this toolbox. Cavity shape and size are important parameters that can be easily accessed by inexpensive geometric algorithms. Although these algorithms are well developed for the detection of nonartificial cavities (e.g., enzymes), they are not routinely used for the rational design of supramolecular cages. In order to test the capabilities of this tool, we have evaluated the performance and characteristics of seven different cavity characterization software in the context of 22 analogues of well-known supramolecular cages. Among the tested software, KVFinder project and Fpocket proved to be the most software to characterize supramolecular cavities. With the results of this work, we aim to popularize this underused technique within the supramolecular community.

KVFinder-web: a web-based application for detecting and characterizing biomolecular cavities.

Molecular interactions that modulate catalytic processes occur mainly in cavities throughout the molecular surface. Such interactions occur with specific small molecules due to geometric and physicochemical complementarity with the receptor. In this scenario, we present KVFinder-web, an open-source web-based application of parKVFinder software for cavity detection and characterization of biomolecular structures. The KVFinder-web has two independent components: a RESTful web service and a web graphical portal. Our web service, KVFinder-web service, handles client requests, manages accepted jobs, and performs cavity detection and characterization on accepted jobs. Our graphical web portal, KVFinder-web portal, provides a simple and straightforward page for cavity analysis, which customizes detection parameters, submits jobs to the web service component, and displays cavities and characterizations. We provide a publicly available KVFinder-web at https://kvfinder-web.cnpem.br, running in a cloud environment as docker containers. Further, this deployment type allows KVFinder-web components to be configured locally and customized according to user demand. Hence, users may run jobs on a locally configured service or our public KVFinder-web.

Are There Edge Effects on the Genetic Diversity of the Trap-Jaw Ant Odontomachus chelifer (Formicidae: Ponerinae) in a Neotropical Savanna Fragment? A First Assessment.

Habitat fragmentation is considered an important threat to biodiversity, increasing species exposure to edge effects. The Brazilian Cerrado savanna is considered a biodiversity hotspot and has been converted to small, isolated fragments due to human activities. Ant communities and colony survivorship are known to be affected by edge effects in Cerrado, but to date there is no information on the genetic diversity of ant colonies at the edge of fragmented areas. Here, we investigate if colony genetic diversity and structure of Odontomachus chelifer (Latreille) ants (Hymenoptera: Formicidae) are subject to edge effects in a Cerrado reserve in southeast Brazil. Using microsatellites, we evaluated the number of breeders (queens and males) and the genetic diversity in O. chelifer colonies located in the interior versus edge of a Cerrado fragment. All O. chelifer nests had multiple queens, which presented a low mating frequency. The number of breeders and most estimates of genetic diversity did not differ between colonies at the edge versus interior of the fragment. Genetic structure was not influenced by nest location as well. However, we detected a small and positive increase in the observed heterozygosity in colonies located at fragment edges. High heterozygosity is thought to be particularly important in fast-changing environments, such as edges, providing an advantage for genetic diversity. Further investigation is needed to assess in greater detail how habitat loss affects O. chelifer biology. Our study is a first step toward elucidating edge effects on genetic diversity of ant colonies, a topic still poorly explored in tropical environments.

Habitat and Host Species Drive the Structure of Bacterial Communities of Two Neotropical Trap-Jaw Odontomachus Ants : Habitat and Host Species Drive the Structure of Bacterial Communities of Two Neotropical Trap-Jaw Odontomachus Ants.

Ants have long been known for their associations with other taxa, including macroscopic fungi and symbiotic bacteria. Recently, many ant species have had the composition and function of their bacterial communities investigated. Due to its behavioral and ecological diversity, the subfamily Ponerinae deserves more attention regarding its associated microbiota. Here, we used the V4 region of the 16S rRNA gene to characterize the bacterial communities of Odontomachus chelifer (ground-nesting) and Odontomachus hastatus (arboreal), two ponerine trap-jaw species commonly found in the Brazilian savanna ("Cerrado") and Atlantic rainforest. We investigated habitat effects (O. chelifer in the Cerrado and the Atlantic rainforest) and species-specific effects (both species in the Atlantic rainforest) on the bacterial communities' structure (composition and abundance) in two different body parts: cuticle and gaster. Bacterial communities differed in all populations studied. Cuticular communities were more diverse, while gaster communities presented variants common to other ants, including Wolbachia and Candidatus Tokpelaia hoelldoblerii. Odontomachus chelifer populations presented different communities in both body parts, highlighting the influence of habitat type. In the Atlantic rainforest, the outcome depended on the body part targeted. Cuticular communities were similar between species, reinforcing the habitat effect on bacterial communities, which are mainly composed of environmentally acquired taxa. Gaster communities, however, differed between the two Odontomachus species, suggesting species-specific effects and selective filters. Unclassified Firmicutes and uncultured Rhizobiales variants are the main components accounting for the observed differences. Our study indicates that both host species and habitat act synergistically, but to different degrees, to shape the bacterial communities in these Odontomachus species.

Identification and characterization of the anti-SARS-CoV-2 activity of cationic amphiphilic steroidal compounds.

The ongoing COVID-19 pandemic caused a significant loss of human lives and a worldwide decline in quality of life. Treatment of COVID-19 patients is challenging, and specific treatments to reduce COVID-19 aggravation and mortality are still necessary. Here, we describe the discovery of a novel class of epiandrosterone steroidal compounds with cationic amphiphilic properties that present antiviral activity against SARS-CoV-2 in the low micromolar range. Compounds were identified in screening campaigns using a cytopathic effect-based assay in Vero CCL81 cells, followed by hit compound validation and characterization. Compounds LNB167 and LNB169 were selected due to their ability to reduce the levels of infectious viral progeny and viral RNA levels in Vero CCL81, HEK293, and HuH7.5 cell lines. Mechanistic studies in Vero CCL81 cells indicated that LNB167 and LNB169 inhibited the initial phase of viral replication through mechanisms involving modulation of membrane lipids and cholesterol in host cells. Selection of viral variants resistant to steroidal compound treatment revealed single mutations on transmembrane, lipid membrane-interacting Spike and Envelope proteins. Finally, in vivo testing using the hACE2 transgenic mouse model indicated that SARS-CoV-2 infection could not be ameliorated by LNB167 treatment. We conclude that anti-SARS-CoV-2 activities of steroidal compounds LNB167 and LNB169 are likely host-targeted, consistent with the properties of cationic amphiphilic compounds that modulate host cell lipid biology. Although effective in vitro, protective effects were cell-type specific and did not translate to protection in vivo, indicating that subversion of lipid membrane physiology is an important, yet complex mechanism involved in SARS-CoV-2 replication and pathogenesis.

Structural analysis of TrkA mutations in patients with congenital insensitivity to pain reveals PLCγ as an analgesic drug target.

Chronic pain is a major health issue, and the search for new analgesics has become increasingly important because of the addictive properties and unwanted side effects of opioids. To explore potentially new drug targets, we investigated mutations in the NTRK1 gene found in individuals with congenital insensitivity to pain with anhidrosis (CIPA). NTRK1 encodes tropomyosin receptor kinase A (TrkA), the receptor for nerve growth factor (NGF) and that contributes to nociception. Molecular modeling and biochemical analysis identified mutations that decreased the interaction between TrkA and one of its substrates and signaling effectors, phospholipase Cγ (PLCγ). We developed a cell-permeable phosphopeptide derived from TrkA (TAT-pQYP) that bound the Src homology domain 2 (SH2) of PLCγ. In HEK-293T cells, TAT-pQYP inhibited the binding of heterologously expressed TrkA to PLCγ and decreased NGF-induced, TrkA-mediated PLCγ activation and signaling. In mice, intraplantar administration of TAT-pQYP decreased mechanical sensitivity in an inflammatory pain model, suggesting that targeting this interaction may be analgesic. The findings demonstrate a strategy to identify new targets for pain relief by analyzing the signaling pathways that are perturbed in CIPA.

Boosting antitumor response with PSMA-targeted immunomodulatory VLPs, harboring costimulatory TNFSF ligands and GM-CSF cytokine.

Therapeutic strategies based on immunomodulation have improved cancer therapy. Most approaches target co-stimulatory pathways or the inhibition of immunosuppressive mechanisms, to enhance immune response and overcome the immune tolerance of tumors. Here, we propose a novel platform to deliver targeted immunomodulatory signaling, enhancing antitumor response. The platform is based on virus-like particles derived from lentiviral capsids. These particles may be engineered to harbor multifunctional ligands on the surface that drive tropism to the tumor site and deliver immunomodulatory signaling, boosting the antitumor response. We generated virus-like particles harboring a PSMA-ligand, TNFSF co-stimulatory ligands 4-1BBL or OX40L, and a membrane-anchored GM-CSF cytokine. The virus-like particles are driven to PSMA-expressing tumors and deliver immunomodulatory signaling from the TNFSF surface ligands and the anchored GM-CSF, inducing T cell proliferation, inhibition of regulatory T cells, and potentiating elimination of tumor cells. The PSMA-targeted particles harboring immunomodulators enhanced antitumor activity in immunocompetent challenged mice and may be explored as a potential tool for cancer immunotherapy.

Foraging and Spatial Ecology of a Polydomous Carpenter Ant, Camponotus leydigi (Hymenoptera: Formicidae), in Tropical Cerrado Savanna: A Natural History Account.

Carpenter ants (genus Camponotus) are considered to be predominantly omnivorous, mixing several feeding habits that include predation, scavenging of animal matter, and plant-derived resources. Nitrogen acquisition is crucial for the nutritional ecology of ant colonies because growing larvae require sustainable protein provisioning. Here, we investigate the foraging ecology and the spatial nesting structure of the carpenter ant, Camponotus leydigi Forel, in Brazilian cerrado savanna. By marking workers from different nests with distinct colors, we revealed that C. leydigi occupies physically separated but socially connected nests (up to 30 m apart), a phenomenon known as polydomy. Observational data on aboveground internest movements in C. leydigi corroborate cooperative exchanges between nest units and confirm several types of social connections, including internest transfer of liquid and solid food, transport of colony members (brood, workers), movement of solitary workers, and internest recruitment. Polydomous C. leydigi allocate foragers throughout 1,700 m2, feeding mostly on termites and plant-derived exudates. Influx of exudates is threefold higher compared with solid food. Uric acid pellets excreted by lizards comprise 20% of the solid diet in C. leydigi, a rare quantitative assessment of this peculiar type of nitrogen complementation in ants. Based on video recordings, we hypothesize that nest decentralization in C. leydigi may reduce foraging constraints caused by overt interference by the aggressive ant, Ectatomma brunneum Smith, F. (Hymenoptera: Formicidae), which regularly blocks nest entrances. Our field study enhances the importance of natural history data to clarify selective pressures underlying the evolution of particular behavioral patterns (nutritional and nesting habits) in ants.

Microsatellites for the Neotropical Ant, Odontomachus chelifer (Hymenoptera: Formicidae).

Odontomachus chelifer (Latreille) (Ponerinae) is a ground-dwelling, predominantly carnivorous ant whose colonies may contain multiple egg-laying queens and are potentially susceptible to border effects in the Brazilian savanna known as Cerrado. The ecology and natural history of O. chelifer is well studied, but very little is known about the genetic diversity of O. chelifer colonies. In this study, we developed microsatellite markers for the study of genetic variation in O. chelifer. We created a microsatellite-enriched library that resulted in the development and characterization of 22 markers, of which 18 were found to be polymorphic in the population studied. The mean expected heterozygosity was 0.59, whereas the mean rarified allelic richness was determined as 4.27 alleles per locus. The polymorphism level detected was similar to genetic diversity estimates found in other poneromorph ant species. The microsatellites developed here are likely to be useful for the investigation of colony structure, functional polygyny, breeding system, and population genetics in O. chelifer. Moreover, the description of O. chelifer's genetic diversity is crucial for its conservation and maintenance of its ecological role in the Cerrado savanna.

Symbiotic bacterial communities in rainforest fungus-farming ants: evidence for species and colony specificity.

Animals may host diverse bacterial communities that can markedly affect their behavioral physiology, ecology, and vulnerability to disease. Fungus-farming ants represent a classical example of mutualism that depends on symbiotic microorganisms. Unraveling the bacterial communities associated with fungus-farming ants is essential to understand the role of these microorganisms in the ant-fungus symbiosis. The bacterial community structure of five species of fungus-farmers (non-leaf-cutters; genera Mycocepurus, Mycetarotes, Mycetophylax, and Sericomyrmex) from three different environments in the Brazilian Atlantic rainforest (lowland forest, restinga forest, and sand dunes) was characterized with amplicon-based Illumina sequencing of 16 S ribosomal RNA gene. Possible differences in bacterial communities between ants internal to the nest (on the fungus garden) and external foragers were also investigated. Our results on the richness and diversity of associated bacteria provide novel evidence that these communities are host- and colony-specific in fungus-farming ants. Indeed, the bacterial communities associated with external foragers differ among the five species, and among colonies of the same species. Furthermore, bacterial communities from internal ants vs. foragers do not differ or differ only slightly within each ant species. This study highlights the importance of describing ant-associated bacterial communities to better understand this host-bacterial interaction in the social environment of insect colonies and provides the foundation for future studies on the ecological and evolutionary processes that drive the success of fungus-farming ants.

Foraging Ecology of the Leaf-Cutter Ant, Acromyrmex subterraneus (Hymenoptera: Formicidae), in a Neotropical Cerrado Savanna.

Fungus-farming ants cultivate a fungal symbiont inside the nest that serves as a food source. Leaf-cutter ants are distinctive among fungus-farmers because they forage for fresh plant material to nurture the fungus. Here we investigate the foraging ecology of Acromyrmex subterraneus (Forel) in the Brazilian cerrado savanna. We examined the species activity pattern, forage material collected, and the relationship between load mass and forager size. Ant activity peaked at night and was negatively related to temperature but positively related to relative air humidity. The majority of the items collected by ants was plant material: dry and fresh leaves, flowers, and fruits. Trunk trails ranged from 0.7 to 13 m and colony home ranged from 2 to 28 m2, indicating that ants collect material nearby the nest. Total load mass was positively associated with forager size, especially in the case of leaves. The negative relationship between ant size and burden suggests that ants might optimize their delivery rate by collecting lighter substrates more frequently. Given their pest status, most studies on leaf-cutters are undertaken in human-altered environments. Information on A. subterraneus in native cerrado is imperative given the threatened status of this vegetation. Leaf-cutters thrive in disturbed cerrado and severe seedling herbivory may hinder vegetation recovery. Our fieldwork may provide insights for management techniques of Acromyrmex colonies in agroecosystems, as well as for restoration programs of degraded cerrado areas.

Natural History of a Sit-and-Wait Dipteran Predator That Uses Extrafloral Nectar as Prey Attractant.

Sit-and-wait predators use different strategies to encounter potential prey. Rhinoleucophenga myrmecophaga Vidal et (Vidal et Vilela; Diptera: Drosophilidae) larvae build sticky shelters on top of extrafloral nectaries (EFNs) of Qualea grandiflora Mart (Vochysiaceae), a common plant in the Brazilian cerrado savanna. Although larval shelters block the EFNs, nectar production is not obstructed and is used by the larvae to attract and trap nectar-gathering ants that are eventually eaten by the dipteran. Here we describe the natural history of R. myrmecophaga, its infestation pattern in Q. grandiflora, the ant assemblage at EFNs, and the insects used as prey. We use stable isotope composition (δ13C and δ15N) of R. myrmecophaga and potential food sources to infer its diet, and perform chemical analyses of the droplets found at shelter openings to determine whether nectar is used as a prey attractant. We found that Rhinoleucophenga larvae occur on the majority of Qualea plants and occupy active EFNs mainly in the rainy season. The two most frequent visiting species were also the most common insects found trapped at larval shelters. The stable isotope analyses confirmed that ants are the main food sources of R. myrmecophaga. Chemical analyses and field observations revealed that Rhinoleucophenga larvae use extrafloral nectar to attract prey to their shelters by pushing this liquid to the shelter opening where it forms a droplet. This is a rare case of sit-and-wait predator exploiting an ant-plant mutualism through the use of the very food reward produced by the plant to attract and capture potential ant mutualists.

First Nonphosphorylated Inhibitors of Phosphoglucose Isomerase Identified by Chemical Library Screening.

Human African trypanosomiasis, Chagas disease, and leishmaniasis are human infections caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania. Besides their severity and global impact, treatments are still challenging. Currently available drugs have important limitations, highlighting the urgent need to develop new drugs. Phosphoglucose isomerase (PGI) is considered a promising target for the development of antiparasitic drugs, as it acts on two essential metabolic pathways, glycolysis and gluconeogenesis. Herein, we describe the identification of new nonphosphorylated inhibitors of Leishmania mexicana PGI ( LmPGI), with the potential for the development of antiparasitic drugs. A fluorescence-based high-throughput screening (HTS) assay was developed by coupling the activities of recombinant LmPGI with glucose-6-phosphate dehydrogenase and diaphorase. This coupled assay was used to screen 42,720 compounds from ChemBridge and TimTec commercial libraries. After confirmatory assays, selected LmPGI inhibitors were tested against homologous Trypanosoma cruzi and humans. The PGI hits are effective against trypanosomatid PGIs, with IC50 values in the micromolar range, and also against the human homologous enzyme. A computational analysis of cavities present on PGI's crystallographic structure suggests a potential binding site for the proposed mixed-type inhibition mechanism.

Generating Conformation-Specific Polyclonal and Monoclonal Anti-Protein Kinase C Antibodies and Anti-Active State Specific PKC Antibodies.

The protein kinase C (PKC) family of serine/ threonine kinases has been shown to play active roles as either suppressors or promoters of carcinogenesis in different types of tumors. Using antibodies that preferentially recognize the active conformation of classical PKCs (cPKCs), we have previously shown that in breast cancer samples the expression levels of cPKCs were similar in estrogen receptor-positive (ER+ ) as compared to triple-negative tumors; however, the levels of active cPKCs were different. Determining the activation status of PKCs and other kinases in tumors may thus aid therapeutic decisions. Further, in basic science these tools may be used to understand the spatial and temporal dynamics of PKC signaling under different stimuli and for co-immunoprecipitation studies to detect binding partners and substrates of active cPKCs. In this article, we describe how monoclonal and polyclonal anti-active state PKC antibodies can be obtained using rational approaches to select bona fide epitopes through inspection of the crystal structure of classical PKCs coupled to molecular modeling studies. We believe that this methodology can be used for other kinases and multi-domain enzymes that undergo changes in their conformation upon activation. © 2018 by John Wiley & Sons, Inc.

Thioredoxin-1 Negatively Modulates ADAM17 Activity Through Direct Binding and Indirect Reductive Activity.

AIMS: A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17. RESULTS: Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1K72A and catalytic site mutant Trx-1C32/35S rescued ADAM17 activity, although the interaction with Trx-1C32/35S was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1C32/35S mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1K72A mutant showed similar oxidant levels to Trx-1C32/35S, even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity. INNOVATION: We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases. CONCLUSION: This unexpected Trx-1K72A behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass spectrometry analysis. Antioxid. Redox Signal. 29, 717-734.

Indirect effects of mutualism: ant-treehopper associations deter pollinators and reduce reproduction in a tropical shrub.

Animal-pollinated plants can be susceptible to changes in pollinator availability. Honeydew-producing treehoppers frequently occur on inflorescences, potentially enhancing ant-mediated negative effects on pollination services. However, the effect of ant-attended, honeydew-producing insects on plant reproduction remains uncertain. We recorded the abundance of treehoppers and ants on Byrsonima intermedia (Malpighiaceae), and monitored floral visitors in a Brazilian cerrado savanna. We manipulated the presence of ants and ant-treehopper associations on inflorescences to assess their effect on pollination and fruit formation. We used dried ants pinned to inflorescences to evaluate the effect of ant presence and ant identity on potential pollinators. Results show that the presence of treehoppers increases ant abundance on flowers and disrupts pollination by oil-collecting bees, decreasing the frequency and duration of floral visits and reducing fruit and seed set. Treehopper herbivory has no direct effect on fruit or seed production, which are independent of treehopper density. Pinned ants promote avoidance by floral visitors, reducing the number of visits. Ant identity mediates visitation decisions, with Ectatomma brunneum causing greater avoidance by floral visitors than Camponotus rufipes. Field videos show that pollinating bees are harassed by ants near flowers, prompting avoidance behavior by the bees. This is the first demonstration of indirect effects by honeydew-gathering ants, via disrupted pollination, on plant reproduction in tropical cerrado savanna. Our results highlight the importance of studying other interactions near flowers, in addition to just observing pollinators, for a proper understanding of plant reproduction.

An activating mutation in the CRHR1 gene is rarely associated with pituitary-dependent hyperadrenocorticism in poodles.

OBJECTIVES: Pituitary-dependent hyperadrenocorticism is the most common cause of naturally occurring hypercortisolism in dogs. CRHR1 expression in human and dog corticotrophinomas suggested that this gene affects pituitary tumorigenesis. The present study aimed to investigate mutations in the CRHR1 coding region in poodles with pituitary-dependent hyperadrenocorticism. METHODS: Fifty poodles with pituitary-dependent hyperadrenocorticism and 50 healthy poodles were studied. Genomic DNA was amplified by PCR and analyzed by Sanger sequencing. RESULTS: The novel CRHR1 p.V97M mutation was identified in one dog. This valine residue, located in the amino-terminal extracellular domain, exhibits high affinity for its corticotropin-releasing hormone (CRH) ligand. Bioinformatic analysis revealed structural rearrangements in the mutant protein, with a 17% increase in the surface binding affinity between CRHR1 and CRH. In vitro functional studies showed that mutant CRHR1 induced higher ACTH secretion than the wild type after stimulation with human CRH. CONCLUSION: These results suggest that germline activating mutations in CRHR1 may be a rare cause of pituitary hyperadrenocorticism in poodles.

Fear Mediates Trophic Cascades: Nonconsumptive Effects of Predators Drive Aquatic Ecosystem Function.

Predators control prey populations and influence communities and the functioning of ecosystems through a combination of consumptive and nonconsumptive effects. These effects can be locally confined to one ecosystem but can also be extended to neighboring ecosystems. In this study, we investigated the nonconsumptive effects of terrestrial avian predators on the communities of aquatic invertebrates inhabiting bromeliads and on the functioning of these natural ecosystems. Bromeliads with stuffed birds placed nearby showed a decrease in aquatic damselfly larvae abundance and biomass, and we can infer that these changes were caused by antipredator responses. These larvae, which are top predators in bromeliad ecosystems, changed the composition of the entire aquatic invertebrate community. While total species richness, mesopredator richness, and shredder abundance increased in the presence of birds, scraper biomass decreased, possibly as a consequence of the increase in mesopredator richness. High scraper biomass in the absence of birds may have accelerated detrital decomposition, making more nutrients available for bromeliads, which grew more. These results show that nonconsumptive effects triggered by terrestrial predators can cascade down to lower trophic levels and dramatically affect the functioning of aquatic ecosystems, which can in turn alter nutrient provision to terrestrial ecosystems.