An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.     

Use of Upland and Riparian Areas by Wintering Bald Eagles and Implications for Wind Energy

Weather can shape movements of animals and alter their exposure to anthropogenic threats. Bald eagles (Haliaeetus leucocephalus) are increasingly at risk from collision with turbines used in onshore wind energy generation. In the midwestern United States, development of this energy source typically occurs in upland areas that bald eagles use only intermittently. Our objective was to determine the factors that cause wintering bald eagles to occupy riparian areas and riskier, upland areas. We tracked 20 bald eagles using telemetry in the Upper Midwest (MN, IA, MO, WI, IL, USA) during winter 2014–2015 and 2015–2016 and evaluated habitat use by eagles in response to variation in weather and time of year. Eagles used riparian areas more when wind speed and atmospheric pressure were low. Exclusive use of uplands was more frequent during weather systems with low pressure and high humidity and after long periods of cold weather. There was a non-linear response to time of year (measured by days before migration) in the frequency of exclusive use of uplands or riparian areas. Probability of exclusive use of either landscape was generally constant within 95 days prior to migration. The probability of use of riparian areas, however, was markedly less during dates >100 days before migration. Our results suggest that eagles are most likely to be exposed to wind energy developments located in upland areas during low pressure systems, after long periods of cold weather, and several months before the onset of spring migration. This information helps to better understand the factors influencing bald eagle habitat use in winter and will be useful to managers and developers wishing to establish effective strategies to avoid, minimize, and mitigate take, and to survey for mortalities at wind energy developments. © 2020 The Wildlife Society.     

Polymorphisms and genetic linkage of histamine receptors

Histamine is a biogenic amine that plays an essential role in controlling many physiological functions, both in the central nervous system (CNS) and the peripheral nervous system (PNS). Most of these physiological effects are mediated through interactions with four histamine receptor subtypes, all of which are members of the larger family of rhodopsin-like class A G-protein coupled receptors (GPCRs) (Leurs et al., 2011; Lim et al., 2009). Here, we focus on the genetic variations and polymorphisms localized on the genes encoding for human histamine receptors where it provides an up to date collection of all polymorphisms found on genes encoding the histamine receptor subtypes and their association to diseases.     

Notes on the Anacroneuria (Plecoptera: Perlidae) of Guyana with the Description of a New Species

A new species of perlid, Anacroneuria takutu sp. n., from Guyana is described and a new record of A. arawak Stark is given. Two additional species of Anacroneuria, represented only by female specimens, are described under informal designations.     

Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol

Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP^−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG.     

Receptor Tyrosine Kinase-like Orphan Receptor 2 (Ror2) Expression Creates a Poised State of Wnt Signaling in Renal Cancer

Expression of the receptor tyrosine kinase-like orphan receptor 2 (Ror2) has been identified in an increasing array of tumor types and is known to play a role as an important mediator of Wnt signaling cascades. In this study, we aimed to clarify Ror2 interactions with the Wnt pathways within the context of renal cell carcinoma (RCC). An examination of Ror2 expression in primary human RCC tumors showed a significant correlation with several Wnt signaling genes, including the classical feedback target gene Axin2. We provide evidence that Ror2 expression results in a partially activated state for canonical Wnt signaling through an increased signaling pool of β-catenin, leading to an enhancement of downstream target genes following Wnt3a stimulation in both renal and renal carcinoma-derived cells. Additionally, inhibition of low-density lipoprotein receptor-related protein 6 (LRP6) with either siRNA or dickkopf decreased the response to Wnt3a stimulation, but no change was seen in the increased β-catenin pool associated with Ror2 expression, suggesting that LRP6 cofactor recruitment is necessary for a Wnt3a-induced signal but that it does not participate in the Ror2 effect on β-catenin signaling. These results highlight a new role for Ror2 in conveying a tonic signal to stabilize soluble β-catenin and create a poised state of enhanced responsiveness to Wnt3a exogenous signals in RCC.     

The Leishmania major BBSome subunit BBS1 is essential for parasite virulence in the mammalian host

Bardet–Biedl syndrome (BBS) is a human genetic disorder with a spectrum of symptoms caused by primary cilium dysfunction. The disease is caused by mutations in one of at least 17 identified genes, of which seven encode subunits of the BBSome, a protein complex required for specific trafficking events to and from the primary cilium. The molecular mechanisms associated with BBSome function remain to be fully elucidated. Here, we generated null and complemented mutants of the BBSome subunit BBS1 in the protozoan parasite, Leishmania. In the absence of BBS1, extracellular parasites have no apparent defects in growth, flagellum assembly, motility or differentiation in vitro but there is accumulation of vacuole‐like structures close to the flagellar pocket. Infectivity of these parasites for macrophages in vitro is reduced compared with wild‐type controls but the null parasites retain the ability to differentiate to the intracellular amastigote stage. However, infectivity of BBS1 null parasites is severely compromised in a BALB/c mouse footpad model. We hypothesize that the absence of BBS1 in Leishmania leads to defects in specific trafficking events that affect parasite persistence in the host. This is the first report of an association between the BBSome complex and pathogen infectivity.     

Differences in motivations and weight loss behaviors in young adults and older adults in the national weight control registry

Objective:

The goal of this study was to compare young adults (YA) and older adults (OA) in the National Weight Control Registry on motivations for weight loss and weight‐loss behaviors.

Design and Methods:

Participants (n = 2,964, 82% female, 94% White, BMI = 24.8 ± 4.4) were divided into two age groups (18‐35 vs. 36‐50) and compared on motivations, strategies for weight loss, diet, physical activity (PA), and the three‐factor eating questionnaire.

Results:

YA were 28.6% of the sample (n = 848). YA and OA achieved similar weight losses (P = 0.38), but duration of maintenance was less in YA (43 vs. 58 months, P < 0.001). YA were more likely to cite appearance and social motivations for weight loss, were less motivated by health, and were less likely to report a medical trigger for weight loss (P's < 0.001). YA were more likely to use exercise classes and to lose weight on their own, and less likely to use a commercial program (P's < 0.001). YA reported engaging in more high‐intensity PA (P = 0.001). There were no group differences in total calories consumed (P = 0.47), or percent calories from fat (P = 0.97), alcohol (P = 0.52), or sugar‐sweetened beverages (P = 0.26).

Conclusions:

YA successful weight losers (SWL) are motivated more by appearance and social influences than OA, and physical activity appears to play an important role in their weight‐loss efforts. The differences reported by YA and OA SWL should be considered when developing weight‐loss programs for YA.     

Clinical Profiles Associated with Influenza Disease in the Ferret Model

Influenza A viruses continue to pose a threat to human health; thus, various vaccines and prophylaxis continue to be developed. Testing of these products requires various animal models including mice, guinea pigs, and ferrets. However, because ferrets are naturally susceptible to infection with human influenza viruses and because the disease state resembles that of human influenza, these animals have been widely used as a model to study influenza virus pathogenesis. In this report, a statistical analysis was performed to evaluate data involving 269 ferrets infected with seasonal influenza, swine influenza, and highly pathogenic avian influenza (HPAI) from 16 different studies over a five year period. The aim of the analyses was to better qualify the ferret model by identifying relationships among important animal model parameters (endpoints) and variables of interest, which include survival, time-to-death, changes in body temperature and weight, and nasal wash samples containing virus, in addition to significant changes from baseline in selected hematology and clinical chemistry parameters. The results demonstrate that a disease clinical profile, consisting of various changes in the biological parameters tested, is associated with various influenza A infections in ferrets. Additionally, the analysis yielded correlates of protection associated with HPAI disease in ferrets. In all, the results from this study further validate the use of the ferret as a model to study influenza A pathology and to evaluate product efficacy.