Chronic pain is a debilitating condition leading to impaired quality of life but has few efficacious therapeutic options. The use of opioids to treat chronic pain is associated with well‐known ...drawbacks including drug dependence and addiction. The development of opioid alternatives to treat chronic pain is an urgent, yet unmet, need. Here we show the therapeutic potential of GPR171, a G‐Protein Coupled Receptor that was recently identified to regulate morphine antinociception. Previously shown that systemic administration of the GPR171 agonist, MS15203, enhances morphine antinociception in acute pain tests in mice. Here, we show that once‐daily systemic administration of MS15203 (10 mg/kg, i.p.) over the course of 1 week improved chronic pain outcomes in two different mouse pain models. Chemotherapy‐induced peripheral neuropathy (CIPN) was induced by injecting Paclitaxel (16 mg/kg cumulative) and inflammatory pain was induced by injecting Complete Freund’s Adjuvant (CFA) under the epidermis into the plantar surface of both hind paws (20 µl/paw). MS15203 improved mechanical allodynia caused by CIPN and reduced thermal hypersensitivity caused by CFA‐induced inflammatory pain. The reduction of chronic pain duration was observed only in male mice, but not in female mice, indicating a sex‐dependent effect at this particular dose of GPR171 agonist. Analysis of GPR171 protein levels in the midbrain Periaqueductal Gray showed that neuropathic pain decreased GPR171 receptor levels in males, but treatment by MS15203 rescued this decrease. Taken together, our findings show that GPR171 activity exhibits sexual dimorphism and can regulate multiple modalities of chronic pain.
Morphine is a potent opioid analgesic with high propensity for the development of antinociceptive tolerance. Morphine antinociception and tolerance are partially regulated by the midbrain ...ventrolateral periaqueductal gray (vlPAG). However, the majority of research evaluating mu-opioid receptor signaling has focused on males. Here, we investigate kinase activation and localization patterns in the vlPAG following acute and chronic morphine treatment in both sexes. Male and female mice developed rapid antinociceptive tolerance to morphine (10 mg/kg i.p.) on the hot plate assay, but tolerance did not develop in males on the tail flick assay. Quantitative fluorescence immunohistochemistry was used to map and evaluate the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2), protein kinase-C (PKC), and protein kinase-A (PKA). We observed significantly greater phosphorylated ERK 1/2 in the vlPAG of chronic morphine-treated animals which co-localized with the endosomal marker, Eea1. We note that pPKC is significantly elevated in the vlPAG of both sexes following chronic morphine treatment. We also observed that although PKA activity is elevated following chronic morphine treatment in both sexes, there is a significant reduction in the nuclear translocation of its phosphorylated substrate. Taken together, this study demonstrates increased activation of ERK 1/2, PKC, and PKA in response to repeated morphine treatment. The study opens avenues to explore the impact of chronic morphine treatment on G-protein signaling and kinase nuclear transport.
Glucagon-like peptide-2 (GLP-2) increases small intestinal mass and blood flow in nonruminants but its effect in ruminants is unknown. Eight Holstein calves with an ultrasonic flow probe around the ...superior mesenteric artery and catheters in the carotid artery and mesenteric vein were paired by age and randomly assigned to treatment of a control (0.5% of BSA in saline; n=4) or GLP-2 (50 μg/kg of body weight of bovine GLP-2 in BSA; n=4) given subcutaneously every 12h for 10 d. Blood flow was measured on d 0 (acute) and d 10 (chronic) and included 3 periods: baseline (saline infusion), treatment (infusion of BSA or 1,000 pmol of GLP-2/kg of body weight per h), and recovery (saline infusion). On d 11, calves were killed 2h after injection of 5-bromo-2′-deoxyuridine (BrdU). Gastrointestinal tissues were weighed and epithelial samples were obtained to determine villus height, crypt depth, and BrdU staining. Infusion of GLP-2 increased superior mesenteric artery blood flow to 175% of baseline on d 0 but to only 137% of baseline after chronic treatment. Compared with that of the control, GLP-2 increased small intestinal mass by 24% by increasing epithelial mass in the jejunum and ileum. Additionally, GLP-2 increased villus height, crypt depth, and BrdU-labeling in small intestinal segments. These results demonstrate that GLP-2 induces similar increases in small intestinal blood flow and growth in ruminants to those observed in nonruminants. Furthermore, GLP-2 increases small intestinal blood flow in ruminants but this response is attenuated after 10 d of GLP-2 administration. In cattle, GLP-2 may be an important hormone in the regulation of intestinal blood flow and epithelial growth.
IgG oligoclonal bands (OCBs) are present in the cerebrospinal fluid (CSF) of more than 95% of patients with multiple sclerosis (MS), and are considered to be the immunological hallmark of disease. ...However, the target specificities of the IgG in MS OCBs have remained undiscovered. Nevertheless, evidence that OCBs are associated with increased levels of disease activity and disability support their probable pathological role in MS. We investigated the antigen specificity of individual MS CSF IgG from 20 OCB-positive patients and identified 40 unique peptides by panning phage-displayed random peptide libraries. Utilizing our unique techniques of phage-mediated real-time Immuno-PCR and phage-probed isoelectric focusing immunoblots, we demonstrated that these peptides were targeted by intrathecal oligoclonal IgG antibodies of IgG1 and IgG3 subclasses. In addition, we showed that these peptides represent epitopes sharing sequence homologies with proteins of viral origin, and proteins involved in cell stress, apoptosis, and inflammatory processes. Although homologous peptides were found within individual patients, no shared peptide sequences were found among any of the 42 MS and 13 inflammatory CSF control specimens. The distinct sets of oligoclonal IgG-reactive peptides identified by individual MS CSF suggest that the elevated intrathecal antibodies may target patient-specific antigens.
Burton-Edwards asserts that as a denominational worship consultant for nearly thirteen years, and a freelance consultant since 2018, I've witnessed and been part of many conversations (and debates) ...about online worship. Some of those, including offering the sacraments to viewers watching online rather than gathered physically with their authorized presider, remained--or became more hotly debated--during the pandemic. Other worship questions, such as whether worship should be made available to people online at all, however, became no longer debatable, but simply necessary while it was physically unsafe to gather in person. The pastor side of me has lived with a very different set of realities. I had no production team to assist me in creating online worship services while we could not meet in person. I had to do them entirely myself. Pastors want to say yes to many things we've had to say no to for a long time. Just remember to do so with the reality of your situation--both your personal and congregational capacity and the degree of healing you require--firmly in heart and mind.
Aging is associated with a reduced magnitude of primary immune responses to vaccination. mRNA-based SARS-CoV-2 vaccines have shown efficacy in older adults but virus variant escape is still unclear. ...Here we analyze humoral and cellular immunity against an early-pandemic viral isolate and compare that to the P.1 (Gamma) and B.1.617.2 (Delta) variants in two cohorts (<50 and >55 age) of mRNA vaccine recipients. We further measure neutralizing antibody titers for B.1.617.1 (Kappa) and B.1.595, with the latter SARS-CoV-2 isolate bearing the spike mutation E484Q. Robust humoral immunity is measured following second vaccination, and older vaccinees manifest cellular immunity comparable to the adult group against early-pandemic SARS-CoV-2 and more recent variants. More specifically, the older cohort has lower neutralizing capacity at 7-14 days following the second dose but equilibrates with the younger cohort after 2-3 months. While long-term vaccination responses remain to be determined, our results implicate vaccine-induced protection in older adults against SARS-CoV-2 variants and inform thinking about boost vaccination.
Agassiz's desert tortoise (Gopherus agassizii) is a long-lived species native to the Mojave Desert and is listed as threatened under the US Endangered Species Act. To aid conservation efforts for ...preserving the genetic diversity of this species, we generated a whole genome reference sequence with an annotation based on deep transcriptome sequences of adult skeletal muscle, lung, brain, and blood. The draft genome assembly for G. agassizii has a scaffold N50 length of 252 kbp and a total length of 2.4 Gbp. Genome annotation reveals 20,172 protein-coding genes in the G. agassizii assembly, and that gene structure is more similar to chicken than other turtles. We provide a series of comparative analyses demonstrating (1) that turtles are among the slowest-evolving genome-enabled reptiles, (2) amino acid changes in genes controlling desert tortoise traits such as shell development, longevity and osmoregulation, and (3) fixed variants across the Gopherus species complex in genes related to desert adaptations, including circadian rhythm and innate immune response. This G. agassizii genome reference and annotation is the first such resource for any tortoise, and will serve as a foundation for future analysis of the genetic basis of adaptations to the desert environment, allow for investigation into genomic factors affecting tortoise health, disease and longevity, and serve as a valuable resource for additional studies in this species complex.
Sampling fecal droppings (scat) to genetically identify individual animals is an established method for monitoring mammal populations and could be highly useful for monitoring reptile populations. ...Whereas existing protocols for obtaining DNA from reptile scat focus on analyses of whole, fresh scat deposited during animal handling, the collection of scat naturally deposited by reptiles in situ, as required for non‐invasive population monitoring, requires protocols to extract highly degraded DNA. Using surface swabs from such scats can reduce PCR inhibition and increase genotyping success. We report on three related but independently designed studies of DNA analyses from scat swabs of herbivorous reptiles under natural desert conditions: two free‐ranging desert tortoise species (Agassiz's desert tortoise, Gopherus agassizii, California, US, and Morafka's desert tortoise, G. morafkai, Arizona, US) and the common chuckwalla (Sauromalus atar) (Arizona, US, and Sonora, MX). We analyzed samples from both tortoise species with the same set of 16 microsatellites and chuckwalla samples with four mtDNA markers; studies also varied in swab preservation medium and DNA extraction method. Microsatellite amplification success per sample, defined as ≥9 loci with amplification, was 15% for the study of Agassiz's desert tortoise and for the study of 42% Morafka's desert tortoise. For chuckwallas, we successfully amplified and sequenced 50% of samples. We recovered fragments up to 400 bp for tortoises and 980 bp for chuckwallas from scat swab samples. This study indicates that genotypes can successfully be obtained from swabs of scat from herbivorous reptiles collected in the field under natural environmental conditions and emphasizes that repeat amplifications are necessary for the genetic identification of individuals from non‐invasive samples.
Non‐invasive methods for monitoring herbivorous reptile populations would benefit our knowledge of their ecology, space use, and conservation status. Current methods of obtaining fecal DNA for these species have only been demonstrated to work well on fresh scats rather than those found under field conditions. We present a compilation of three independently conducted but similar studies of fecal DNA collected from the common chuckwalla, Agassiz's desert tortoise, and Morafka's desert tortoise to show that swabs of scats obtained under natural conditions have the potential to be used to obtain genotypes for population monitoring.
Chronic pain is a growing public health crisis that requires exigent and efficacious therapeutics. GPR171 is a promising therapeutic target that is widely expressed through the brain, including ...within the descending pain modulatory regions. Here, we explore the therapeutic potential of the GPR171 agonist, MS15203, in its ability to alleviate chronic pain in male and female mice using a once-daily systemic dose (10 mg/kg, i.p.) of MS15203 over the course of 5 days. We found that in our models of Complete Freund's Adjuvant (CFA)-induced inflammatory pain and chemotherapy-induced peripheral neuropathy (CIPN), MS15203 did not alleviate thermal hypersensitivity and allodynia, respectively, in female mice. On the other hand, MS15203 treatment decreased the duration of thermal hypersensitivity in CFA-treated male mice following 3 days of once-daily administration. MS15203 treatment also produced an improvement in allodynia in male mice, but not female mice, in neuropathic pain after 5 days of treatment. Gene expression of GPR171 and that of its endogenous ligand BigLEN, encoded by the gene PCSK1N, were unaltered within the periaqueductal gray (PAG) in both male and female mice following inflammatory and neuropathic pain. However, following neuropathic pain in male mice, the protein levels of GPR171 were decreased in the PAG. Treatment with MS15203 then rescued the protein levels of GPR171 in the PAG of these mice. Taken together, our results identify GPR171 as a GPCR that displays sexual dimorphism in alleviation of chronic pain. Further, our results suggest that GPR171 and MS15203 have demonstrable therapeutic potential in the treatment of chronic pain.
Two experiments were conducted to evaluate the effects of slow-release urea (SRU) versus feed-grade urea on ruminal metabolite characteristics in steers and DMI, gain, and G:F in growing beef steers. ...Experiment 1 used 12 ruminally cannulated steers (529 ± 16 kg of BW) to monitor the behavior of SRU in the ruminal environment. Compared with feed-grade urea, SRU decreased ruminal ammonia concentration (P = 0.02) and tended to increase ruminal urease activity (P = 0.06) without affecting ruminal VFA molar proportions or total concentrations (P > 0.20). After 35 d of feeding, the in situ degradation rate of SRU was not different between animals fed urea or SRU (P = 0.48). Experiment 2 used 180 Angus-cross steers (330 ± 2.3 kg) fed corn silage-based diets supplemented with urea or SRU for 56 d to evaluate the effects on feed intake, gain, and G:F. The design was a randomized complete block with a 2 x 4 + 1 factorial arrangement of treatments. Treatments included no supplemental urea (control) or urea or SRU at 0.4, 0.8, 1.2, or 1.6% of diet DM. Over the entire 56 d experiment, there were interactions of urea source x concentration for gain (P = 0.04) and G:F (P = 0.01) because SRU reduced ADG and G:F at the 0.4 and 1.6% supplementation concentrations but was equivalent to urea at the 0.8 and 1.2% supplementation concentrations; these effects were due to urea source x concentration interactions for gain (P = 0.06) and G:F (P = 0.05) during d 29 to 56 of the experiment. The SRU reduced DMI during d 29 to 56 (P = 0.01) but not during d 0 to 28, so that over the entire experiment there was no difference in DMI for urea source (P = 0.19). These collective results demonstrate that SRU releases N slowly in the rumen with no apparent adaptation within 35 d. Supplementation of SRU may limit N availability at low (0.4%) concentrations but is equivalent to urea at 0.8 and 1.2% concentrations.
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