Fauna Associated with American Alligator (Alligator mississippiensis) Nests in Coastal South Carolina, USA.

Crocodilians are considered to be "ecosystem engineers" because their modification of habitats provides opportunities for feeding, drinking, breeding, and other vital life activities to a wide variety of other animals. One such habitat modification is the construction of nest mounds during the breeding season by most crocodilian species, including American alligators (Alligator mississippiensis). While many reports exist describing wildlife associated with alligator nests, no studies have quantified faunal associates and their corresponding behaviors while visiting nests. To address this data gap, we used automated game cameras to monitor wildlife and their behaviors at alligator nests during the egg incubation period (June-September) in coastal South Carolina, USA (2016-2021). We documented a total of 81 species (79 vertebrates and 2 invertebrates) at 78 alligator nests representing six taxonomic groups, including 48 birds (59.2%), 9 mammals (11.1%), 19 reptiles (23.4%), 3 amphibians (3.7%), 1 malacostracan (1.2%), and 1 insect (1.2%). Collectively, faunal associates primarily used alligator nests for feeding/foraging (51.8%), traveling (29.3%), and loafing (19.9%) and to a much lesser extent basking, burrowing/shelter, breeding, and nesting. However, trends in alligator nest use varied among faunal associate groups (birds, mammals, reptiles, amphibians, etc.), subgroups (e.g., passerines, raptors, wading birds, and waterfowl), and species. Several novel behaviors by some nest associates were also noted during the study, including the first observations of Virginia oppossum (Didelphis virginiana) opening and predating nests, bobcat (Lynx rufus) consuming alligator hatchlings, and Carolina wren (Thryothorus ludovicianus) feeding on the contents of a recently predated alligator egg. The results of this study indicate that a diverse assemblage of vertebrates (and some invertebrates) use alligator nest sites in coastal South Carolina for a variety of life activities during the egg incubation period, and the proportion of the behaviors exhibited varies among animal groups and species. This study provides a first step for investigations regarding the net impacts of alligator nest-faunal associate interactions and ultimately the greater ecological role of alligators and other crocodilians.

Knockdown of the Non-canonical Wnt Gene Prickle2 Leads to Cerebellar Purkinje Cell Abnormalities While Cerebellar-Mediated Behaviors Remain Intact.

Autism spectrum disorders (ASD) involve brain wide abnormalities that contribute to a constellation of symptoms including behavioral inflexibility, cognitive dysfunction, learning impairments, altered social interactions, and perceptive time difficulties. Although a single genetic variation does not cause ASD, genetic variations such as one involving a non-canonical Wnt signaling gene, Prickle2, has been found in individuals with ASD. Previous work looking into phenotypes of Prickle2 knock-out (Prickle2-/-) and heterozygous mice (Prickle2-/+) suggest patterns of behavior similar to individuals with ASD including altered social interaction and behavioral inflexibility. Growing evidence implicates the cerebellum in ASD. As Prickle2 is expressed in the cerebellum, this animal model presents a unique opportunity to investigate the cerebellar contribution to autism-like phenotypes. Here, we explore cerebellar structural and physiological abnormalities in animals with Prickle2 knockdown using immunohistochemistry, whole-cell patch clamp electrophysiology, and several cerebellar-associated motor and timing tasks, including interval timing and eyeblink conditioning. Histologically, Prickle2-/- mice have significantly more empty spaces or gaps between Purkinje cells in the posterior lobules and a decreased propensity for Purkinje cells to fire action potentials. These structural cerebellar abnormalities did not impair cerebellar-associated behaviors as eyeblink conditioning and interval timing remained intact. Therefore, although Prickle-/- mice show classic phenotypes of ASD, they do not recapitulate the involvement of the adult cerebellum and may not represent the pathophysiological heterogeneity of the disorder.

Senescence-induced immune remodeling facilitates metastatic adrenal cancer in a sex-dimorphic manner.

Aging markedly increases cancer risk, yet our mechanistic understanding of how aging influences cancer initiation is limited. Here we demonstrate that the loss of ZNRF3, an inhibitor of Wnt signaling that is frequently mutated in adrenocortical carcinoma, leads to the induction of cellular senescence that remodels the tissue microenvironment and ultimately permits metastatic adrenal cancer in old animals. The effects are sexually dimorphic, with males exhibiting earlier senescence activation and a greater innate immune response, driven in part by androgens, resulting in high myeloid cell accumulation and lower incidence of malignancy. Conversely, females present a dampened immune response and increased susceptibility to metastatic cancer. Senescence-recruited myeloid cells become depleted as tumors progress, which is recapitulated in patients in whom a low myeloid signature is associated with worse outcomes. Our study uncovers a role for myeloid cells in restraining adrenal cancer with substantial prognostic value and provides a model for interrogating pleiotropic effects of cellular senescence in cancer.

Structural mechanism of a drug-binding process involving a large conformational change of the protein target.

Proteins often undergo large conformational changes when binding small molecules, but atomic-level descriptions of such events have been elusive. Here, we report unguided molecular dynamics simulations of Abl kinase binding to the cancer drug imatinib. In the simulations, imatinib first selectively engages Abl kinase in its autoinhibitory conformation. Consistent with inferences drawn from previous experimental studies, imatinib then induces a large conformational change of the protein to reach a bound complex that closely resembles published crystal structures. Moreover, the simulations reveal a surprising local structural instability in the C-terminal lobe of Abl kinase during binding. The unstable region includes a number of residues that, when mutated, confer imatinib resistance by an unknown mechanism. Based on the simulations, NMR spectra, hydrogen-deuterium exchange measurements, and thermostability measurements and estimates, we suggest that these mutations confer imatinib resistance by exacerbating structural instability in the C-terminal lobe, rendering the imatinib-bound state energetically unfavorable.

Schizencephaly in Hereditary Hemorrhagic Telangiectasia.

BACKGROUND AND PURPOSE: The presence of malformations of cortical development in patients with hereditary hemorrhagic telangiectasia has been reported on previous occasions. We evaluated a sample of adults with hereditary hemorrhagic telangiectasia for the presence of malformations of cortical development, spatial coincidence of malformations of cortical development and AVMs, and the coincidence of brain and pulmonary AVMs. MATERIALS AND METHODS: A total of 141 patients 18 years of age or older who were referred to the Augusta University hereditary hemorrhagic telangiectasia clinic and underwent brain MR imaging between January 19, 2018, and December 3, 2020, were identified. MR imaging examinations were reviewed retrospectively by 2 experienced neuroradiologists, and the presence of malformations of cortical development and AVMs was confirmed by consensus. Demographic and clinical information was collected for each case, including age, sex, hereditary hemorrhagic telangiectasia status by the Curacao Criteria, mutation type, presence of malformations of cortical development, presence of brain AVMs, presence of pulmonary AVMs, and a history of seizures or learning disabilities. RESULTS: Five of 141 (3.5%) patients with hereditary hemorrhagic telangiectasia had malformations of cortical development. Two of the 5 patients with polymicrogyria also had closed-lip schizencephaly. One of the patients had a porencephalic cavity partially lined with heterotopic GM. The incidence of spatially coincident polymicrogyria and brain AVMs was 40% (2/5 cases). Of the patients with hereditary hemorrhagic telangiectasia and malformations of cortical development, 4/5 (80%) had pulmonary AVMs and 2/5 (40%) had brain AVMs. CONCLUSIONS: To our knowledge, we are the first group to report the presence of schizencephaly in patients with hereditary hemorrhagic telangiectasia. The presence of schizencephaly and porencephaly lends support to the hypothesis of regional in utero cerebral hypoxic events as the etiology of malformations of cortical development in hereditary hemorrhagic telangiectasia.

Evolution of Cortical Functional Networks in Healthy Infants.

During normal childhood development, functional brain networks evolve over time in parallel with changes in neuronal oscillations. Previous studies have demonstrated differences in network topology with age, particularly in neonates and in cohorts spanning from birth to early adulthood. Here, we evaluate the developmental changes in EEG functional connectivity with a specific focus on the first 2 years of life. Functional connectivity networks (FCNs) were calculated from the EEGs of 240 healthy infants aged 0-2 years during wakefulness and sleep using a cross-correlation-based measure and the weighted phase lag index. Topological features were assessed via network strength, global clustering coefficient, characteristic path length, and small world measures. We found that cross-correlation FCNs maintained a consistent small-world structure, and the connection strengths increased after the first 3 months of infancy. The strongest connections in these networks were consistently located in the frontal and occipital regions across age groups. In the delta and theta bands, weighted phase lag index networks decreased in strength after the first 3 months in both wakefulness and sleep, and a similar result was found in the alpha and beta bands during wakefulness. However, in the alpha band during sleep, FCNs exhibited a significant increase in strength with age, particularly in the 21-24 months age group. During this period, a majority of the strongest connections in the networks were located in frontocentral regions, and a qualitatively similar distribution was seen in the beta band during sleep for subjects older than 3 months. Graph theory analysis suggested a small world structure for weighted phase lag index networks, but to a lesser degree than those calculated using cross-correlation. In general, graph theory metrics showed little change over time, with no significant differences between age groups for the clustering coefficient (wakefulness and sleep), characteristics path length (sleep), and small world measure (sleep). These results suggest that infant FCNs evolve during the first 2 years with more significant changes to network strength than features of the network structure. This study quantifies normal brain networks during infant development and can serve as a baseline for future investigations in health and neurological disease.

Acoustic shock: an update review.

BACKGROUND: This paper discusses the concept of acoustic shock based on a literature review and the results of our own research into cases seen in both clinical and medicolegal practice. With the demise of traditional 'metal bashing' and 'smoke stack' heavy industries, there has been a decline in the incidence of noise-induced hearing loss and tinnitus in this form of employment. However, with the increasing establishment of call centre work, the emergence of acoustic shock has been noted. Acoustic shock is recognised as a clinical entity distinct from noise-induced hearing loss and acoustic trauma. OBJECTIVE: This article discusses clinical implications, medicolegal considerations in light of a recent high-profile court case and proposed criteria for diagnosis.

Inferring diet, feeding behaviour and causes of mortality from prey-induced injuries in a New Zealand fur seal.

New Zealand fur seals Arctocephalus forsteri are the most abundant of the 4 otariid (eared seal) species distributed across Australasia. Analyses of stomach contents, scats and regurgitates suggest a diet dominated by bony fish and squid, with cartilaginous species (e.g. sharks and rays) either absent or underrepresented because of a lack of preservable hard parts. Here we report on a subadult specimen from south-eastern Australia, which was found ashore emaciated and with numerous puncture wounds across its lips, cheeks, throat and the inside of its oral cavity. Fish spines embedded in the carcass revealed that these injuries were inflicted by chimaeras and myliobatiform rays (stingrays and relatives), which matches reports on the diet of A. forsteri from New Zealand, but not South Australia. Shaking and tearing of prey at the surface may help to avoid ingestion of the venomous spines, perhaps contributing to their absence from scats and regurgitates. Nevertheless, the number and severity of the facial stab wounds, some of which led to local necrosis, likely affected the animal's ability to feed, and may account for its death. Despite their detrimental effects, fish spine-related injuries are difficult to spot, and may be a common, albeit cryptic, type of trauma. We therefore recommend that stranded seals be systematically examined for this potentially life-threatening pathology.

Molecular mechanisms of fentanyl mediated ß-arrestin biased signaling.

The development of novel analgesics with improved safety profiles to combat the opioid epidemic represents a central question to G protein coupled receptor structural biology and pharmacology: What chemical features dictate G protein or ß-arrestin signaling? Here we use adaptively biased molecular dynamics simulations to determine how fentanyl, a potent ß-arrestin biased agonist, binds the µ-opioid receptor (µOR). The resulting fentanyl-bound pose provides rational insight into a wealth of historical structure-activity-relationship on its chemical scaffold. Following an in-silico derived hypothesis we found that fentanyl and the synthetic opioid peptide DAMGO require M153 to induce ß-arrestin coupling, while M153 was dispensable for G protein coupling. We propose and validate an activation mechanism where the n-aniline ring of fentanyl mediates µOR ß-arrestin through a novel M153 "microswitch" by synthesizing fentanyl-based derivatives that exhibit complete, clinically desirable, G protein biased coupling. Together, these results provide molecular insight into fentanyl mediated ß-arrestin biased signaling and a rational framework for further optimization of fentanyl-based analgesics with improved safety profiles.

Structure of formylpeptide receptor 2-Gi complex reveals insights into ligand recognition and signaling.

Formylpeptide receptors (FPRs) as G protein-coupled receptors (GPCRs) can recognize formylpeptides derived from pathogens or host cells to function in host defense and cell clearance. In addition, FPRs, especially FPR2, can also recognize other ligands with a large chemical diversity generated at different stages of inflammation to either promote or resolve inflammation in order to maintain a balanced inflammatory response. The mechanism underlying promiscuous ligand recognition and activation of FPRs is not clear. Here we report a cryo-EM structure of FPR2-Gi signaling complex with a peptide agonist. The structure reveals a widely open extracellular region with an amphiphilic environment for ligand binding. Together with computational docking and simulation, the structure suggests a molecular basis for the recognition of formylpeptides and a potential mechanism of receptor activation, and reveals conserved and divergent features in Gi coupling. Our results provide a basis for understanding the molecular mechanism of the functional promiscuity of FPRs.

Molecular Basis for Hormone Recognition and Activation of Corticotropin-Releasing Factor Receptors.

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.

Intermittent gastric feeds lower insulin requirements without worsening dysglycemia: A pilot randomized crossover trial.

INTRODUCTION: We hypothesized that critically ill medical patients would require less insulin when fed intermittently. METHODS: First, 26 patients were randomized to receive intermittent or continuous gastric feeds. Once at goal nutrition, data were collected for the first 4-hr data collection period. Next, the enteral feed type was switched, goal nutrition was repeated, and a second 4-h data collection period was completed. The primary endpoint was the total amount of insulin infused; secondary endpoints were glucose concentration mean, maximum, minimum, and standard deviation, as well as episodes of hypoglycemia. RESULTS: Sixteen of the 26 patients successfully completed the protocol. One patient experienced a large, rapid, and sustained decline in insulin requirement from liver failure, creating a bias of lesser insulin in the intermittent arm; this patient was removed from the analysis. For the remaining 15 patients, the average total amount of insulin infused was 1.4 U/patient/h less following intermittent feeds: P =0.027, 95% confidence interval (0.02, 11.17), and effect size 0.6. Secondary endpoints were statistically similar. CONCLUSIONS: Critically ill medical patients who require an insulin infusion have a reduced insulin requirement when fed intermittently, whereas dysglycemia metrics are not adversely affected. A larger clinical study is required to confirm these findings.

Development of "Plug and Play" Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM.

"Universal" synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric Gi and Gs, as well as mini-Gs, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αßγ) protein. Several Gßγ-specific sABs, cross-reactive between trimeric Gi and Gs, were identified suggesting they could be used across all subclasses in a "plug and play" fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes.

A complex structure of arrestin-2 bound to a G protein-coupled receptor.

Arrestins comprise a family of signal regulators of G-protein-coupled receptors (GPCRs), which include arrestins 1 to 4. While arrestins 1 and 4 are visual arrestins dedicated to rhodopsin, arrestins 2 and 3 (Arr2 and Arr3) are ß-arrestins known to regulate many nonvisual GPCRs. The dynamic and promiscuous coupling of Arr2 to nonvisual GPCRs has posed technical challenges to tackle the basis of arrestin binding to GPCRs. Here we report the structure of Arr2 in complex with neurotensin receptor 1 (NTSR1), which reveals an overall assembly that is strikingly different from the visual arrestin-rhodopsin complex by a 90° rotation of Arr2 relative to the receptor. In this new configuration, intracellular loop 3 (ICL3) and transmembrane helix 6 (TM6) of the receptor are oriented toward the N-terminal domain of the arrestin, making it possible for GPCRs that lack the C-terminal tail to couple Arr2 through their ICL3. Molecular dynamics simulation and crosslinking data further support the assembly of the Arr2‒NTSR1 complex. Sequence analysis and homology modeling suggest that the Arr2‒NTSR1 complex structure may provide an alternative template for modeling arrestin-GPCR interactions.

Gravity Compensation of an Exoskeleton Joint Using Constant-Force Springs.

Stroke is one of the leading causes of impairment in the world. Many of those who have suffered a stroke experience long-term loss of upper-limb function as a result. BLUE SABINO is an exoskeleton device being developed at the University of Idaho to help assess these patients and aid in their rehabilitation. One of the central design challenges with exoskeletons is limiting the overall weight of the device. Motors used in actuation of these devices are often oversized to allow gravity balancing of the device and user and the creation of torques to facilitate patient movements. If the torques required for gravity balancing are achieved through elastic elements, the motor and other upstream components can be lighter, potentially greatly reducing the overall weight of the device. In this paper, constant-force springs may provide an effective method of generating a constant offsetting torque to compensate for gravity. In experimental testing of multiple mounting configurations of C-shaped constant-force springs (single, back-to-back, double-wrapped), the force output fluctuated less than 8.6% over 180° of wrapping, with friction values below 2.6%, validating the viability of constant-force springs for this application. The results suggest the back-to-back configuration provides a simpler implementation with better force consistency while the double-wrapped configuration adds less friction to the system.

Chest wall pain following lung stereotactic body radiation therapy using 48Gy in three fractions: A search for predictors.

PURPOSE: Chest wall pain is an uncommon but bothersome late complication following lung stereotactic body radiation therapy. Despite numerous studies investigating predictors of chest wall pain, no clear consensus has been established for a chest wall constraint. The aim of our study was to investigate factors related to chest wall pain in a homogeneous group of patients treated at our institution. PATIENTS AND METHODS: All 122 patients were treated with the same stereotactic body radiation therapy regimen of 48Gy in three fractions, seen for at least 6 months of follow-up, and planned with heterogeneity correction. Chest wall pain was scored according to the Common Terminology Criteria for Adverse Events classification v3.0. Patient (age, sex, diabetes, osteoporosis), tumour (planning target volume, volume of the overlapping region between planning target volume and chest wall) and chest wall dosimetric parameters (volumes receiving at least 30, 40, and 50Gy, the minimal doses received by the highest irradiated 1, 2, and 5cm3, and maximum dose) were collected. The correlation between chest wall pain (grade 2 or higher) and the different parameters was evaluated using univariate and multivariate logistic regression. RESULTS: Median follow-up was 18 months (range: 6-56 months). Twelve patients out of 122 developed chest wall pain of any grade (seven with grade 1, three with grade 2 and two with grade 3 pain). In univariate analysis, only the volume receiving 30Gy or more (P=0.034) and the volume of the overlapping region between the planning target volume and chest wall (P=0.038) significantly predicted chest wall pain, but these variables were later proved non-significant in multivariate regression. CONCLUSION: Our analysis could not find any correlation between the studied parameters and chest wall pain. Considering our present study and the wide range of differing results from the literature, a reasonable conclusion is that a constraint for chest wall pain is yet to be defined.

Structure and dynamics of the active human parathyroid hormone receptor-1.

The parathyroid hormone receptor-1 (PTH1R) is a class B G protein-coupled receptor central to calcium homeostasis and a therapeutic target for osteoporosis and hypoparathyroidism. Here we report the cryo-electron microscopy structure of human PTH1R bound to a long-acting PTH analog and the stimulatory G protein. The bound peptide adopts an extended helix with its amino terminus inserted deeply into the receptor transmembrane domain (TMD), which leads to partial unwinding of the carboxyl terminus of transmembrane helix 6 and induces a sharp kink at the middle of this helix to allow the receptor to couple with G protein. In contrast to a single TMD structure state, the extracellular domain adopts multiple conformations. These results provide insights into the structural basis and dynamics of PTH binding and receptor activation.

Freeze-Drying To Produce Efficacious CPMV Virus-like Particles.

In situ cancer vaccination that uses immune stimulating agents is revolutionizing the way that cancer is treated. In this realm, viruses and noninfectious virus-like particles have gained significant traction in reprogramming the immune system to recognize and eliminate malignancies. Recently, cowpea mosaic virus-like particles (VLPs) have shown exceptional promise in their ability to fight a variety of cancers. However, the current methods used to produce CPMV VLPs rely on agroinfiltration in plants. These protocols remain complicated and labor intensive and have the potential to introduce unwanted immunostimulatory agents, like lipopolysaccharides. This Letter describes a simple "post-processing" method to remove RNA from wild-type CPMV, while retaining the structure and function of the capsid. Lyophilization was able to eject encapsulated RNA to form lyo-eCPMV and, when purified, eliminated nearly all traces of encapsulated RNA. Lyo-eCPMV was characterized by cryo-electron microscopy single particle reconstruction to confirm the structural integrity of the viral capsid. Finally, lyo-eCPMV showed  equivalent anticancer efficacy as eCPMV, produced by agroinfiltration, when using an invasive melanoma model. These results describe a straightforward method to prepare CPMV VLPs from infectious virions.

Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK).

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and a promising drug target for managing metabolic diseases such as type 2 diabetes. Many pharmacological AMPK activators, and possibly unidentified physiological metabolites, bind to the allosteric drug and metabolite (ADaM) site at the interface between the kinase domain (KD) in the α-subunit and the carbohydrate-binding module (CBM) in the ß-subunit. Here, using double electron-electron resonance (DEER) spectroscopy, we demonstrate that the CBM-KD interaction is partially dissociated and the interface highly disordered in the absence of pharmacological ADaM site activators as inferred from a low depth of modulation and broad DEER distance distributions. ADaM site ligands such as 991, and to a lesser degree phosphorylation, stabilize the KD-CBM association and strikingly reduce conformational heterogeneity in the ADaM site. Our findings that the ADaM site, formed by the KD-CBM interaction, can be modulated by diverse ligands and by phosphorylation suggest that it may function as a hub for integrating regulatory signals.