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The efficient encapsulation of small molecule active ingredients has been a challenge for many decades across many commercial applications. Recently, successful attempts to address this issue have included deposition of thin metal shells onto liquid filled polymer microcapsules or emulsion droplets to provide an impermeable barrier to diffusion. In this work we have developed a novel method to protect small molecule active ingredients by deposition of thin mineral shells. Platinum nanoparticles are used to catalyse and direct growth of a calcium phosphate shell onto liquid filled polymer microcapsules under various reaction conditions. Findings indicate that a non-porous protective shell is formed on the majority of the microcapsule population, with small concentrations of the core material being released only from those microcapsules with defects, over a 7 days period, when conducting forced release studies into a solvent for the core oil. The resulting microcapsules show no significant cell toxicity when exposed to HEK 293 cells for 72 hours.

Patients with ataxia-telangiectasia (A-T) lack a functional ATM kinase protein and exhibit defective repair of DNA double-stranded breaks and response to oxidative stress. We show that CRISPR/Cas9-assisted gene correction combined with piggyBac (PB) transposon-mediated excision of the selection cassette enables seamless restoration of functional ATM alleles in induced pluripotent stem cells from an A-T patient carrying compound heterozygous exonic missense/frameshift mutations, and from a patient with a homozygous splicing acceptor mutation of an internal coding exon. We show that the correction of one allele restores expression of ~ 50% of full-length ATM protein and ameliorates DNA damage-induced activation (auto-phosphorylation) of ATM and phosphorylation of its downstream targets, KAP-1 and H2AX. Restoration of ATM function also normalizes radiosensitivity, mitochondrial ROS production and oxidative-stress-induced apoptosis levels in A-T iPSC lines, demonstrating that restoration of a single ATM allele is sufficient to rescue key ATM functions. Our data further show that despite the absence of a functional ATM kinase, homology-directed repair and seamless correction of a pathogenic ATM mutation is possible. The isogenic pairs of A-T and gene-corrected iPSCs described here constitute valuable tools for elucidating the role of ATM in ageing and A-T pathogenesis.

The effective management of pain is a longstanding public health concern. Although opioids have been frontline analgesics for decades, they also have well-known undesirable effects that limit their clinical utility, such as abuse liability and respiratory depression. The failure to develop better analgesics has, in some ways, contributed to the escalating opioid epidemic that has claimed tens of thousands of lives and has cost hundreds of billions of dollars in health-care expenses. A paradigm shift is needed in the pharmacotherapy of pain management that will require extensive efforts throughout biomedical science. The purpose of the present review is to highlight the critical role of the behavioral scientist to devise improved translational models of pain for drug development. Despite high heterogeneity of painful conditions that involve cortical-dependent pain processing, current models often feature an overreliance on simple reflex-based measures and an emphasis on the absence, rather than presence, of behavior as evidence of analgesic efficacy. Novel approaches should focus on the restoration of operant and other CNS-mediated behavior under painful conditions.

Esophageal adenocarcinoma (EAC) is one of the most frequent causes of cancer death, and yet compared to other common cancers, we know relatively little about the molecular composition of this tumor type. To further our understanding of this cancer, we have used open chromatin profiling to decipher the transcriptional regulatory networks that are operational in EAC. We have uncovered a transcription factor network that is usually found in primitive intestinal cells during embryonic development, centered on HNF4A and GATA6. These transcription factors work together to control the EAC transcriptome. We show that this network is activated in Barrett's esophagus, the putative precursor state to EAC, thereby providing novel molecular evidence in support of stepwise malignant transition. Furthermore, we show that HNF4A alone is sufficient to drive chromatin opening and activation of a Barrett's-like chromatin signature when expressed in normal human epithelial cells. Collectively, these data provide a new way to categorize EAC at a genome scale and implicate HNF4A activation as a potential pivotal event in its malignant transition from healthy cells.

Summary There is evidence that ATM mutated in ataxia-telangiectasia (A-T) plays a key role in protecting against mitochondrial dysfunction, the mechanism for which remains unresolved. We demonstrate here that ATM-deficient cells are exquisitely sensitive to nutrient deprivation, which can be explained by defective cross talk between the endoplasmic reticulum (ER) and the mitochondrion. Tethering between these two organelles in response to stress was reduced in cells lacking ATM, and consistent with this, Ca2+ release and transfer between ER and mitochondria was reduced dramatically when compared with control cells. The impact of this on mitochondrial function was evident from an increase in oxygen consumption rates and a defect in mitophagy in ATM-deficient cells. Our findings reveal that ER-mitochondrial connectivity through IP3R1-GRP75-VDAC1, to maintain Ca2+ homeostasis, as well as an abnormality in mitochondrial fusion defective in response to nutrient stress, can account for at least part of the mitochondrial dysfunction observed in A-T cells., Graphical Abstract, Highlights • Hypersensitivity to glucose deprivation in ATM-deficient cells • Defective ER-mitochondrion cross talk after nutrient stress in these cells • Markedly reduced Ca2+ transfer between these two organelles in ATM-deficient cells • Mitochondrial dysfunction in response to nutrient stress in the absence of ATM, Cell Biology; Organizational Aspects of Cell Biology; Functional Aspects of Cell Biology

BACKGROUND: Daily use of marijuana is rising in adolescents, along with consumption of high potency marijuana products (high % Δ−9-tetrahydrocannabinol or THC). These dual, related trends have opened gaps in understanding the long-term effects of daily consumption of a high dose of THC in adolescents and whether a therapeutic dose of cannabidiol (CBD) modulates THC effects. METHODS: Adolescent squirrel monkeys (Saimiri boliviensis) were treated daily for four months with vehicle (n=4), a high THC dose (1 mg/kg i.m.; n=4), or THC + CBD (1 mg/kg + 3 mg/kg i.m.; n=4), to investigate whether: (1) a daily high THC dose affects performance in tasks of cognition (repeated acquisition, discrimination reversal); (2) a daily high THC dose affects spontaneous behavior and day/night activity (3) tolerance develops to the behavioral effects of THC; (4) whether CBD modulates THC effects. RESULTS: THC impaired performance of adolescent monkeys in a cognitive test initially, but not performance on a task of cognitive flexibility. THC reduced motor activity and increased sedentary behavior, with tolerance developing after weeks of daily treatment. Co-administered with THC, CBD did not modulate THC effects on cognitive performance, activity or tolerance, but prevented THC-induced emesis on the first day of daily treatment. CONCLUSIONS: Daily high dosing with THC compromised performance on a task of cognition, and reduced activity in adolescent primates, with tolerance developing within weeks. Whether our observations are relevant to a broader range of cognitive tasks vital for daily function in in human adolescents is uncertain.

SummaryInvestigations on the human germline and programming are challenging due to limited access to embryonic material. However, the pig as a model may provide insight on transcriptional network and epigenetic reprogramming applicable to both species. Here we show that during the pre- and early migratory stages pig primordial germ cells (PGCs) initiate large-scale epigenetic reprogramming, including DNA demethylation involving TET-mediated hydroxylation and potentially base excision repair (BER). There is also macroH2A1 depletion and increased H3K27me3, as well as X chromosome reactivation (XCR) in females. Concomitantly, there is dampening of glycolytic metabolism genes and re-expression of some pluripotency genes like those in preimplantation embryos. We identified evolutionarily young transposable elements and gene coding regions resistant to DNA demethylation in acutely hypomethylated gonadal PGCs, with potential for transgenerational epigenetic inheritance. Detailed insights into the pig germline will likely contribute significantly to advances in human germline biology, includingin vitrogametogenesis.

(&minus, )-N-Phenethyl analogs of optically pure N-norhydromorphone were synthesized and pharmacologically evaluated in several in vitro assays (opioid receptor binding, stimulation of [35S]GTP&gamma, S binding, forskolin-induced cAMP accumulation assay, and MOR-mediated &beta, arrestin recruitment assays). &ldquo, Body&rdquo, and &ldquo, tail&rdquo, interactions with opioid receptors (a subset of Portoghese&rsquo, s message-address theory) were used for molecular modeling and simulations, where the &ldquo, address&rdquo, can be considered the &ldquo, body&rdquo, of the hydromorphone molecule and the &ldquo, message&rdquo, delivered by the substituent (tail) on the aromatic ring of the N-phenethyl moiety. One compound, N-p-chloro-phenethynorhydromorphone ((7aR,12bS)-3-(4-chlorophenethyl)-9-hydroxy-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one, 2i), was found to have nanomolar binding affinity at MOR and DOR. It was a potent partial agonist at MOR and a full potent agonist at DOR with a &delta, /&mu, potency ratio of 1.2 in the ([35S]GTP&gamma, S) assay. Bifunctional opioids that interact with MOR and DOR, the latter as agonists or antagonists, have been reported to have fewer side-effects than MOR agonists. The p-chlorophenethyl compound 2i was evaluated for its effect on respiration in both mice and squirrel monkeys. Compound 2i did not depress respiration (using normal air) in mice or squirrel monkeys. However, under conditions of hypercapnia (using air mixed with 5% CO2), respiration was depressed in squirrel monkeys.

Ataxia-telangiectasia (A-T) mutated (ATM) plays a central role in the response to DNA double strand breaks (DNA DSB) but there is increasing evidence that it has a key role in protecting against mitochondrial dysfunction, the mechanism for which remains unresolved. We demonstrate here that A-T cells are also exquisitely sensitive to metabolic stress which can be explained by defective cross-talk between the endoplasmic reticulum (ER) and the mitochondrion through the mitochondrial–associated membrane (MAM). We showed that formation of a molecular bridge between the voltage-dependent calcium channel (VDAC1) and the inositol 1,4,5 trisphosphate receptor type 1 (IP3R1), mediated by the mitochondrial chaperone GRP75, is defective in A-T cells after nutrient stress. Tethering between these two organelles in response to stress, as determined by number of ER-mitochondrial contact sites, was reduced in A-T cells and consistent with this, Ca2+ release and transfer between ER and mitochondria was reduced dramatically compared to wild type cells. The impact of this on mitochondrial function was evident from an increase in oxygen consumption rates and a defect in mitophagy in ATM-deficient cells after glucose deprivation. Our findings reveal that ER- mitochondrial connectivity through IP3R1-GRP75-VDAC1, to maintain Ca2+ homeostasis, as well as an abnormality in mitochondrial fusion is defective in A-T cells in response to nutrient stress which can account for at least part of the mitochondrial dysfunction observed in A-T cells.

Evidence suggests that the α4β2, but not the α7, subtype of the nicotinic acetylcholine receptor (nAChR) plays a key role in mediating the behavioral effects of nicotine and related drugs. However, the importance of other nAChR subtypes remains unclear. The present studies were conducted to examine the involvement of nAChR subtypes by determining the effects of selected nicotinic agonists and antagonists in squirrel monkeys either 1) responding for food reinforcement or 2) discriminating the nicotinic agonist (+)-epibatidine (0.001 mg/kg) from vehicle. In food-reinforcement studies, nicotine, (+)-epibatidine, varenicline and cytisine all produced dose-dependent decreases in rates of food-maintained responding. The rate-decreasing effects of nicotine were antagonized by mecamylamine (nonselective), not appreciably altered by dihydro-β-erythroidine (α4β2 selective), and exacerbated by the nicotinic partial agonists, varenicline and cytisine. Results from discrimination studies show that non-nicotinic drugs did not substitute for (+)-epibatidine, and that except for lobeline, the nicotinic agonists produced either full [(+)-epibatidine, (−)-epibatidine, and nicotine] or partial (varenicline, cytisine, anabaseine, and isoarecolone) substitution for (+)-epibatidine. In interaction studies with antagonists differing in selectivity, (+)-epibatidine discrimination was substantively antagonized by mecamylamine, slightly attenuated by hexamethonium (peripherally restricted) or dihydro-β-erythroidine, and not altered by methyllycaconitine (α7 selective). Varenicline and cytisine enhanced (+)-epibatidine’s discriminative-stimulus effects. Correlational analysis revealed a close correspondence between relative behavioral potencies of nicotinic agonists in both studies and their published relative binding affinities at α4β2 and α3β4, but not α7 nAChR, subtypes. Collectively, these results are consistent with the idea that the α4β2 and α3β4, but not α7 nAChR subtypes play a role in the behavioral effects of nicotinic agonists.

Nicotine can produce antinociception in preclinical pain models; however, the ability of nicotine to augment the antinociceptive effects of opioid agonists has not been investigated. The present experiments were conducted to determine how nicotine modifies the effects of opioid agonists differing in efficacy. Male squirrel monkeys responded for the delivery of milk under a fixed ratio 10 schedule of reinforcement. During the 30-second timeout period following each milk delivery, the subject's tail was immersed in 35, 50, 52, or 55°C water, and the latency to remove the tail was recorded. Dose-response functions for tail-withdrawal latency and operant performance were determined for fentanyl, oxycodone, buprenorphine, and nalbuphine alone and after treatment with nicotine. Excepting nalbuphine, all opioids produced dose-related disruptions in food-maintained responding and increases in tail-withdrawal latency at each water temperature. Nicotine did not exacerbate the behaviorally disruptive effects of the μ-opioids on operant performance but produced a significant mecamylamine-sensitive enhancement of the antinociceptive potency of each opioid. Failure of arecoline to augment the antinociceptive effects of oxycodone and antagonism by mecamylamine suggests this nicotine-induced augmentation of prescription opioid antinociception was nicotinic acetylcholine receptor (nAChR) mediated. This was reflected in leftward shifts in the antinociceptive dose-response curve of each opioid, ranging from 2- to 7-fold increases in the potency of oxycodone across all water temperatures to an approximately 70-fold leftward shift in the antinociceptive dose-response curve of nalbuphine at the lower and intermediate water temperatures. These results suggest that nicotine may enhance μ-opioid antinociceptive effects without concomitantly exacerbating their behaviorally disruptive effects. SIGNIFICANCE STATEMENT: Prescription opioids remain the most effective pain-management pharmacotherapeutics but are limited by their adverse effects. The present results indicate that nicotine enhances antinociceptive effects of various opioid agonists in nonhuman primates without increasing their disruptive effects on operant performance. These results suggest that nicotine might function as an opioid adjuvant for pain management by enabling decreased clinically effective analgesic doses of prescription opioids without exacerbating their adverse behavioral effects.

Buprenorphine, a partial agonist at the μ-opioid receptor, is commonly prescribed for the management of opioid addiction. Notwithstanding buprenorphine's clinical popularity, the relationship between its effectiveness in attenuating relapse-related behavior and its opioid efficacy is poorly understood. Furthermore, changes in the antinociceptive potency or effectiveness of opioid drugs that might occur during buprenorphine treatment have not been characterized. Here, we address these questions by assessing the ability of daily buprenorphine treatment to protect against the reinstatement of drug-seeking behavior by six opioids differing in efficacy (methadone, heroin, oxycodone, buprenorphine, butorphanol, nalbuphine) and, in separate experiments, by determining how such treatment may modify their antinociceptive effects. In one set of experiments, squirrel monkeys were trained to respond under concurrent schedules (choice) of food or intravenous oxycodone presentations. The priming strength of different opioids during sessions in which saline, rather than oxycodone, was available for intravenous self-administration was determined before and during chronic buprenorphine treatment (0.1 or 0.32 mg/kg per day). In other subjects, antinociceptive effects of the different opioids were assessed using cumulative dosing procedures in a modified warm-water tail withdrawal procedure before and during buprenorphine treatment. Results show that, notwithstanding some tolerance, full agonists retain high efficacy in producing priming and antinociceptive effects. In contrast, both the priming strength and antinociceptive effectiveness of partial agonists were decreased. These results suggest that the utility of buprenorphine in the management of opioid addiction, and how it alters the analgesic effects of opioids, can vary depending on the efficacy of the abused or prescribed opioid. SIGNIFICANCE STATEMENT: Our findings indicate that the pharmacological efficacy of abused opioids may predict the ability of buprenorphine to attenuate their relapse-related priming and analgesia-related antinociceptive effects. This information can help inform physicians as to the effectiveness and limitations of buprenorphine as a pharmacotherapy for opioid addiction.

Background Daily use of marijuana is rising in adolescents, along with consumption of high potency marijuana products (high % Δ-9-tetrahydrocannabinol or THC). These dual, related trends have opened gaps in understanding the long-term effects of daily consumption of a high dose of THC in adolescents and whether a therapeutic dose of cannabidiol (CBD) modulates THC effects. Methods Adolescent squirrel monkeys (Saimiri boliviensis) were treated daily for four months with vehicle (n = 4), a high THC dose (1 mg/kg i.m.; n = 4), or THC + CBD (1 mg/kg +3 mg/kg i.m.; n = 4), to investigate whether: (1) a daily high THC dose affects performance in tasks of cognition (repeated acquisition, discrimination reversal); (2) a daily high THC dose affects spontaneous behavior and day/night activity (3) tolerance develops to the behavioral effects of THC; (4) whether CBD modulates THC effects. Results THC impaired performance of adolescent monkeys in a cognitive test initially, but not performance on a task of cognitive flexibility. THC reduced motor activity and increased sedentary behavior, with tolerance developing after weeks of daily treatment. Co-administered with THC, CBD did not modulate THC effects on cognitive performance, activity or tolerance, but prevented THC-induced emesis on the first day of daily treatment. Conclusions Daily high dosing with THC compromised performance on a task of cognition, and reduced activity in adolescent primates, with tolerance developing within weeks. Whether our observations are relevant to a broader range of cognitive tasks vital for daily function in human adolescents is uncertain.

Recent studies in rodents suggest that non-nicotine constituents of tobacco smoke (e.g., minor tobacco alkaloids) may promote tobacco consumption—either through their own pharmacological effects or by augmenting the effects of nicotine. However, there is scant information on the behavioral pharmacology of minor tobacco alkaloids in primate species. The present studies were conducted to determine whether the minor tobacco alkaloids nornicotine, anabasine, anatabine, myosmine, and cotinine exhibit nicotine-like behavioral effects in squirrel monkeys. Initial experiments were conducted to determine the effects of nicotine (0.032–1.0 mg/kg) and the minor tobacco alkaloids nornicotine (1–1.8 mg/kg), anabasine (0.1–1.0 mg/kg), anatabine (10–32 mg/kg), myosmine (0.32–1.8 mg/kg), and cotinine (10–180 mg/kg) on food-maintained performance (n = 4). Next, the ability of tobacco alkaloids to substitute for the α4β2-selective nicotinic agonist (+)-epibatidine in drug discrimination experiments was evaluated in a separate group of monkeys (n = 4). Results show that nicotine and each minor tobacco alkaloid except cotinine (a) produced dose-related decreases in food-maintained responding; (b) substituted for (+)-epibatidine and, in additional experiments, produced additive effects when combined with nicotine; (c) induced emesis or tremor at doses that reduced food-maintained responding and had (+)-epibatidine-like discriminative-stimulus effects; and (d) based on correlation with reported receptor binding affinities, likely produced their behavioral effects through α4β2 receptor mechanisms. Selected minor tobacco alkaloids have nicotinic-like effects that may contribute to tobacco consumption and addiction.

Background Prescription opioid abuse continues to be a public health concern of epidemic proportions. Notwithstanding the extensive literature regarding opioid action, there has been little systematic research regarding the effects of opioid dependence and withdrawal on aspects of cognition-related behavior in laboratory animals. The present studies examined the effects of the prescription opioid oxycodone on learning processes in nonhuman primates. Methods The ability of subjects to repeatedly learn novel touchscreen-based visual discriminations was examined during three conditions of opioid exposure. Discrimination learning was examined, first, during oxycodone self-administration (3-hr sessions, 0.1 mg/kg/injection) and, next, during non-contingent chronic treatment with oxycodone (10 mg/kg/day). Finally, discrimination learning was re-examined during antagonist-precipitated opioid withdrawal (0.001-0.1 mg/kg naltrexone) and, subsequently, following abrupt discontinuation of oxycodone treatment. Results Although motoric behavior was disrupted by oxycodone, neither the development of discrimination learning nor steady-state performance were impaired following oxycodone self-administration or during non-contingent chronic oxycodone treatment. However, discrimination learning was substantially impaired during oxycodone withdrawal, whether elicited by naltrexone or by abrupt oxycodone discontinuation. Moreover, these learning impairments were concordant with autonomic signs of opioid withdrawal. Conclusions Taken together, the present studies indicate that impairment of learning processes can accompany the unconditioned signs of opioid withdrawal.

Oesophageal adenocarcinoma (OAC) is one of the most frequent causes of cancer deaths and yet compared to other common cancers, we know relatively little about the molecular composition of this tumour type. To further our understanding of this cancer we have used open chromatin profiling to decipher the transcriptional regulatory networks that are operational in OAC. We have uncovered a transcription factor network that is usually found in primitive intestinal cells during embryonic development, centred on HNF4A and GATA6. These transcription factors work together to control the OAC transcriptome. Importantly, we show that this network is activated in Barrett’s oesophagus, the putative precursor state to OAC thereby providing novel molecular evidence in support of stepwise malignant transition. Furthermore, we show that HNF4A alone, is sufficient to drive chromatin opening and activation of a Barrett’s-like chromatin signature when expressed in normal human epithelial cells. Collectively, these data provide a new way to categorise OAC at a genome scale and implicate HNF4A activation as a potential pivotal event in regulating its malignant transition from healthy cells.

High-resolution molecular programs delineating the cellular foundations of mammalian embryogenesis have emerged recently. Similar analysis of human embryos is limited to pre-implantation stages, since early post-implantation embryos are inaccessible. Notwithstanding, we previously suggested conserved principles of pig and human early development. For further insight on pluripotent states and lineage delineation, we analysed pig embryos at single cell resolution. Here we show progressive segregation of inner cell mass and trophectoderm in early blastocysts, and then of epiblast and hypoblast in late blastocysts. We detected distinct pluripotent states, first as a short ‘naïve’ state followed by a protracted primed state. Dosage compensation with respect to the X-chromosome in females is attained via X-inactivation in late epiblasts. Detailed human-pig comparison is a basis towards comprehending early human development and a foundation for further studies of human pluripotent stem cell differentiation in pig interspecies chimeras.

Consumption of ethanol is a considerable risk factor for death in heroin overdose. We sought to determine whether a mildly intoxicating concentration of ethanol could alter morphine tolerance at the cellular level. In rat locus coeruleus (LC) neurons, tolerance to morphine was reversed by acute exposure of the brain slice to ethanol (20 mM). Tolerance to the opioid peptide [d-Ala(2),N-MePhe(4),Gly-ol]-enkephalin was not reversed by ethanol. Previous studies in LC neurons have revealed a role for protein kinase C (PKC)α in μ-opioid receptor (MOPr) desensitization by morphine and in the induction and maintenance of morphine tolerance, but we have been unable to demonstrate that 20 mM ethanol produces significant inhibition of PKCα. The ability of ethanol to reverse cellular tolerance to morphine in LC neurons was absent in the presence of the phosphatase inhibitor okadaic acid, indicating that dephosphorylation is involved. In human embryonic kidney 293 cells expressing the MOPr, ethanol reduced the level of MOPr phosphorylation induced by morphine. Ethanol reversal of tolerance did not appear to result from a direct effect on MOPr since acute exposure to ethanol (20 mM) did not modify the affinity of binding of morphine to the MOPr or the efficacy of morphine for G-protein activation as measured by guanosine 5'-O-(3-[(35)S]thio)triphosphate binding. Similarly, ethanol did not affect MOPr trafficking. We conclude that acute exposure to ethanol enhances the effects of morphine by reversing the processes underlying morphine cellular tolerance.

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during weeks 2-3 of early post-implantation development. Using in vitro models of hPGC induction, recent studies have suggested that there are marked mechanistic differences in the specification of human and mouse PGCs. This may be due in part to the divergence in their pluripotency networks and early post-implantation development. As early human embryos are not accessible for direct study, we considered alternatives including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs originate from the posterior pre-primitive-streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. We use this model together with human and monkey in vitro models simulating peri-gastrulation development to show the conserved principles of epiblast development for competency for primordial germ cell fate. This process is followed by initiation of the epigenetic program and regulated by a balanced SOX17-BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models provides synthetic insights into early human development.

There is ongoing debate about the role of G protein-coupled receptor kinases (GRKs) in agonist-induced desensitization of the μ-opioid receptor (MOPr) in brain neurons. In the present paper, we have used a novel membrane-permeable, small-molecule inhibitor of GRK2 and GRK3, Takeda compound 101 (Cmpd101; 3-[[[4-methyl-5-(4-pyridyl)-4H-1,2,4-triazole-3-yl] methyl] amino]-N-[2-(trifuoromethyl) benzyl] benzamidehydrochloride), to study the involvement of GRK2/3 in acute agonist-induced MOPr desensitization. We observed that Cmpd101 inhibits the desensitization of the G protein-activated inwardly-rectifying potassium current evoked by receptor-saturating concentrations of methionine-enkephalin (Met-Enk), [d-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), endomorphin-2, and morphine in rat and mouse locus coeruleus (LC) neurons. In LC neurons from GRK3 knockout mice, Met-Enk-induced desensitization was unaffected, implying a role for GRK2 in MOPr desensitization. Quantitative analysis of the loss of functional MOPrs following acute agonist exposure revealed that Cmpd101 only partially reversed MOPr desensitization. Inhibition of extracellular signal-regulated kinase 1/2, protein kinase C, c-Jun N-terminal kinase, or GRK5 did not inhibit the Cmpd101-insensitive component of desensitization. In HEK 293 cells, Cmpd101 produced almost complete inhibition of DAMGO-induced MOPr phosphorylation at Ser(375), arrestin translocation, and MOPr internalization. Our data demonstrate a role for GRK2 (and potentially also GRK3) in agonist-induced MOPr desensitization in the LC, but leave open the possibility that another, as yet unidentified, mechanism of desensitization also exists.

Summary Investigations of the human germline and programming are challenging because of limited access to embryonic material. However, the pig as a model may provide insights into transcriptional network and epigenetic reprogramming applicable to both species. Here we show that, during the pre- and early migratory stages, pig primordial germ cells (PGCs) initiate large-scale epigenomic reprogramming, including DNA demethylation involving TET-mediated hydroxylation and, potentially, base excision repair (BER). There is also macroH2A1 depletion and increased H3K27me3 as well as X chromosome reactivation (XCR) in females. Concomitantly, there is dampening of glycolytic metabolism genes and re-expression of some pluripotency genes like those in preimplantation embryos. We identified evolutionarily young transposable elements and gene coding regions resistant to DNA demethylation in acutely hypomethylated gonadal PGCs, with potential for transgenerational epigenetic inheritance. Detailed insights into the pig germline will likely contribute significantly to advances in human germline biology, including in vitro gametogenesis., Graphical Abstract, Highlights • Gene expression profiles of pig and human primordial germ cells are closely aligned • Pre-migratory pig PGCs undergo DNA demethylation, XCR, and histone remodeling • Identification of DNA demethylation-resistant loci in the pig germline, Zhu et al. show that pig primordial germ cells (PGCs) undergo DNA demethylation, histone remodeling, and X chromosome reactivation after specification. Pig PGCs retain few methylated loci after genome-wide demethylation, with potential for transgenerational inheritance. Species comparisons shows close similarities in transcriptional profiles of pig and human PGCs.