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The aim of this article is to explore the practical complexity of accountability in health care by focusing on a particular crisis affecting one NHS trust in the UK, that of insufficient beds to meet demand. It is presented through the eyes of five middle managers with nursing backgrounds. Although the focus is on their words, their expressions of distress and their awareness of conflict, these lead to a commentary highlighting some of the relationships between theory and practice, policy making and implementation, and, in the final analysis, compulsion and choice. The managers seemed to work within four main patterns of provider accountability: public, professional, pecuniary and personal. These four Ps of accountability created incompatibilities in the accountability process, but the conclusion attempts to draw the threads together to suggest a possible way forward. In order to protect confidentiality, pseudonyms are used for the NHS trust and the interviewees, and some personal details have been disguised. [ABSTRACT FROM AUTHOR]

Abstract Background Nonmedical prescription opioid use has emerged as a major public health concern in recent years, particularly in rural Appalachia. Little is known about the routes of administration (ROA) involved in nonmedical prescription opioid use among rural and urban drug users. The purpose of this study was to describe rural-urban differences in ROA for nonmedical prescription opioid use. Methods A purposive sample of 212 prescription drug users was recruited from a rural Appalachian county (n = 101) and a major metropolitan area (n = 111) in Kentucky. Consenting participants were given an interviewer-administered questionnaire examining sociodemographics, psychiatric disorders, and self-reported nonmedical use and ROA (swallowing, snorting, injecting) for the following prescription drugs: buprenorphine, fentanyl, hydrocodone, hydromorphone, methadone, morphine, OxyContin® and other oxycodone. Results Among urban participants, swallowing was the most common ROA, contrasting sharply with substance-specific variation in ROA among rural participants. Among rural participants, snorting was the most frequent ROA for hydrocodone, methadone, OxyContin®, and oxycodone, while injection was most common for hydromorphone and morphine. In age-, gender-, and race-adjusted analyses, rural participants had significantly higher odds of snorting hydrocodone, OxyContin®, and oxycodone than urban participants. Urban participants had significantly higher odds of swallowing hydrocodone and oxycodone than did rural participants. Notably, among rural participants, 67% of hydromorphone users and 63% of morphine users had injected the drugs. Conclusions Alternative ROA are common among rural drug users. This finding has implications for rural substance abuse treatment and harm reduction, in which interventions should incorporate methods to prevent and reduce route-specific health complications of drug use.

Background: The Food and Drug Administration (FDA) in the US approved primary human papillomavirus (HPV) testing for speculum-based cervical cancer screening ten years ago and, in May 2024, approved the self-collection technique. Our study defines the kappa agreement between self- and speculum-based collection techniques for 15 types of high-risk HPV. Additionally, we describe the sensitivity and specificity ratios for HPV testing using both collection methods., Methods: Participants recruited in 2020-2022 included 97 colposcopy attendees and 96 routine primary care screening attendees aged 30-65, who agreed to self-sample before their clinically scheduled speculum-based exam. Prevalence-based kappa calculated agreement, sensitivity and specificity ratios calculated accuracy using the cervical intraepithelial neoplasia grade 2 or worse (CIN 2+) threshold., Results: The average ages were 45.9 (SD 10.5) and 46.2 (SD 11.0) years for the colposcopy and primary care attendees, respectively. HPV 16, 68, and 39 were the most common types detected. The lower bound of the 95 % Cl for kappa calculations was above 0.81, indicating almost perfect agreement across all HPV genotypes. The sensitivity and specificity ratios were consistent at 1.0 across both collection methods. The HPV positivity rate was significantly higher among colposcopy attendees at 66 % (64/97), compared to 14 % (13/96) among routine primary care screeners. The study identified 17 women with CIN2 + ., Conclusions: Primary HPV screening with self-collection is equivalent to speculum-based collection among people aged 30-65. The findings emphasize the utility of self-collection in identifying high-grade lesions and the consistency of HPV detection across different collection methods., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2025 The Authors. Published by Elsevier Inc.)

Microglia, the resident immune cells of the brain, regulate the balance of inflammation in the central nervous system under healthy and pathogenic conditions. Huntington's disease (HD) is a chronic neurodegenerative disease characterized by activated microglia and elevated concentrations of pro-inflammatory cytokines within the brain. Chronic hyperactivation of microglia is associated with brain pathology and eventual neuron death. However, it is unclear which specific cytokines are required for neuron death and whether HD neurons may be hypersensitive to neuroinflammation. We assessed the profile of microglia-secreted proteins in response to LPS and IFNγ, and a conditioned media paradigm was used to examine the effects of these secreted proteins on cultured neuronal cells. STHdh Q7/Q7 and STHdh Q111/Q111 neuronal cells were used to model wild-type and HD neurons, respectively. We determined that STHdh Q111/Q111 cells were hypersensitive to pro-inflammatory factors secreted by microglia, and that TNF was required to induce neuronal death. Microglia-mediated neuronal death could be effectively halted through the use of JAK-STAT or TNF inhibitors which supported the requirement for TNF as well as IFNγ in the process of secondary neurotoxicity. Further data derived from human HD patients as well as HD mice were suggestive of enhanced receptor density for TNF (TNFR1) and IFNγ (IFNGR) which could sensitize the HD brain to these cytokines. This highlights several potential mechanisms by which microglia may induce neuronal death and suggests that these mechanisms may be upregulated in the brain of HD patients., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Eileen M. Denovan-Wright reports financial support was provided by Dalhousie University Faculty of Medicine. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: PANX1 (pannexin 1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, the possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated., Method: We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1 MyHC6 )., Results: PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism and resulting glycolytic ATP production, with a concurrent decrease in oxidative phosphorylation, both in vivo and in vitro. In vitro, treatment of H9c2 (H9c2 rat myoblast cell line) cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knockdown of PANX1. To investigate nonischemic heart failure and the preceding cardiac hypertrophy, we administered isoproterenol, and we demonstrated that Panx1 MyHC6 mice were protected from systolic and diastolic left ventricle volume increases as a result of cardiomyocyte hypertrophy. Moreover, we found that Panx1 MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45 + ), particularly neutrophils (CD11b + [integrin subunit alpha M], Ly6g + [lymphocyte antigen 6 family member G]), to the myocardium., Conclusions: Together, these data demonstrate that PANX1 deficiency in cardiomyocytes increases glycolytic metabolism and protects against cardiac hypertrophy in nonischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in patients with heart failure., Competing Interests: None.

Our lab is investigating the efficacy profiles of tropine analogs against opioid-induced respiratory depression. The companion manuscript reports that the cell-permeant tropeine, tropine ester (Ibutropin), produces a rapid and sustained reversal of the deleterious actions of fentanyl on breathing, alveolar-arterial (A-a) gradient (i.e., index of alveolar gas exchange), and arterial blood-gas (ABG) chemistry in freely-moving male Sprague Dawley rats, while not compromising fentanyl analgesia. We report here that in contrast to Ibutropin, the injection of the parent molecule, tropine (200 μmol/kg, IV), worsens the adverse actions of fentanyl (75 μg/kg, IV) on ventilatory parameters (e.g., frequency of breathing, tidal volume, minute ventilation, peak inspiratory and expiratory flows, and inspiratory and expiratory drives), A-a gradient, ABG chemistry (e.g., pH, pCO 2 , pO 2 , and sO 2 ), and sedation (i.e., the righting reflex), while not affecting fentanyl antinociception (i.e., the tail-flick latency) in freely-moving male Sprague Dawley rats. These data suggest that tropine augments opioid receptor-induced signaling events that mediate the actions of fentanyl on breathing and alveolar gas exchange. The opposite effects of Ibutropin and tropine may result from the ability of Ibutropin to readily enter peripheral and central cells. Of direct relevance is that tropine, resulting from the hydrolysis of Ibutropin, would combat the Ibutropin-induced reversal of the adverse effects of fentanyl. Because numerous drug classes, such as cocaine, atropine, and neuromuscular blocking drugs contain a tropine moiety, it is possible that their hydrolysis to tropine has unexpected/unintended consequences. Indeed, others have found that tropine exerts the same behavioral profile as cocaine upon central administration. Together, these data add valuable information about the pharmacological properties of tropine., Competing Interests: Author SB was employed by Galleon Pharmaceuticals, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Getsy, May, Young, Baby, Coffee, Bates, Hsieh and Lewis.)

We consider how finite-size scaling (FSS) is modified above the upper critical dimension, du=4, due to hyperscaling violations, which in turn arise from a dangerous irrelevant variable. In addition to the commonly studied case of periodic boundary conditions, we also consider new effects that arise with free boundary conditions. Some numerical results are presented in addition to theoretical arguments.

Microglia are resident immune cells of the central nervous system (CNS) and propagate inflammation following damage to the CNS, including the retina. Proliferative vitreoretinopathy (PVR) is a condition that can emerge following retinal detachment and is characterized by severe inflammation and microglial proliferation. The type 2 cannabinoid receptor (CB 2 ) is an emerging pharmacological target to suppress microglial-mediated inflammation when the eyes or brain are damaged. CB 2 -knockout mice have exacerbated inflammation and retinal pathology during experimental PVR. We aimed to assess the anti-inflammatory effects of CB 2 stimulation in the context of retinal damage and also explore the mechanistic roles of CB 2 in microglia function. To target CB 2 , we used a highly selective agonist, HU-308, as well as its enantiomer, HU-433, which is a putative selective agonist. First, β-arrestin2 and Gα i recruitment was measured to compare activation of human CB 2 in an in vitro heterologous expression system. Both agonists were then utilized in a mouse model of PVR, and the effects on retinal damage, inflammation, and cell death were assessed. Finally, we used an in vitro model of microglia to determine the effects of HU-308 and HU-433 on phagocytosis, cytokine release, migration, and intracellular signaling. We observed that HU-308 more strongly recruited both β-arrestin2 and Gα i compared to HU-433. Stimulation of CB 2 with either drug effectively blunted LPS- and IFNγ-mediated signaling as well as NO and TNF release from microglia. Furthermore, both drugs reduced IL-6 accumulation, total caspase-3 cleavage, and retinal pathology following the induction of PVR. Ultimately, this work supports that CB 2 is a valuable target for drugs to suppress inflammation and cell death associated with infection or sterile retinopathy, although the magnitude of effector recruitment may not be predictive of anti-inflammatory capacity., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Microglia, the resident immune cells of the brain, regulate neuroinflammation which can lead to secondary neuronal damage and cognitive impairment under pathological conditions. Two of the many molecules that can elicit an inflammatory response from microglia are lipopolysaccharide (LPS), a component of gram-negative bacteria, and interferon gamma (IFNγ), an endogenous pro-inflammatory cytokine. We thoroughly examined the concentration-dependent relationship between LPS from multiple bacterial species and IFNγ in cultured microglia and macrophages. We measured the effects that these immunostimulatory molecules have on pro-inflammatory activity of microglia and used a battery of signaling inhibitors to identify the pathways that contribute to the microglial response. We found that LPS and IFNγ interacted synergistically to induce a pro-inflammatory phenotype in microglia, and that inhibition of JAK1/2 completely blunted the response. We determined that this synergistic action of LPS and IFNγ was likely dependent on JNK and Akt signaling rather than typical pro-inflammatory mediators such as NF-κB. Finally, we demonstrated that LPS derived from Escherichia coli, Klebsiella pneumoniae, and Akkermansia muciniphila can elicit different inflammatory responses from microglia and macrophages, but these responses could be consistently prevented using ruxolitinib, a JAK1/2 inhibitor. Collectively, this work reveals a mechanism by which microglia may become hyperactivated in response to the combination of LPS and IFNγ. Given that elevations in circulating LPS and IFNγ occur in a wide variety of pathological conditions, it is critical to understand the pharmacological interactions between these molecules to develop safe and effective treatments to suppress this process., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

It is often assumed that future coastal cliff retreat rates will accelerate as global sea level rises, but few studies have investigated how SLR (sea level rise) might change cliff-front wave dynamics. Using a new simple numerical model, this study simulates the number and type (breaking, broken, or unbroken) of cliff-front waves under future SLR scenarios. Previous research shows breaking waves deliver more energy to cliffs than broken waves, and unbroken waves generate minimal impact. Here, we investigated six cliff-platform profiles from three regions (USA, New Zealand, and UK) with varied tidal ranges and wave climates. Model inputs included 2013-2100 hindcast/forecast incident wave height and tidal water level, and three future SLR scenarios. Results show the number of both cliff-front breaking and broken waves generally increase for a high-elevation (relative to tide) cliff-platform junction. In contrast, breaking/broken wave occurrence decrease by 38-92% for a near-horizontal shore platform with a low-elevation cliff-platform junction under a high SRL scenario, leading to high (96-97%) unbroken wave occurrence. Overall, results suggest the response of cliff-front waves to future SLR is complex and depends on shore platform geometries and SLR scenarios, indicating that future cliff retreat rates may not homogeneously accelerate under SLR., (© 2024. The Author(s).)

Pannexin 1 (PANX1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, a possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1 MyHC6 ). PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism both in vivo and in vitro . In vitro , treatment of H9c2 cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knock-down of PANX1. To investigate non-ischemic heart failure and the preceding cardiac hypertrophy we administered isoproterenol, and we demonstrate that Panx1 MyHC6 mice were protected from systolic and diastolic left ventricle volume increases and cardiomyocyte hypertrophy. Moreover, we found that Panx1 MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45 + ), particularly neutrophils (CD11b + , Ly6g + ), to the myocardium. Together these data demonstrate that PANX1 deficiency in cardiomyocytes impacts glycolytic metabolism and protects against cardiac hypertrophy in non-ischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in heart failure patients.

L-cysteine ethylester (L-CYSee) is a membrane-permeable analogue of L-cysteine with a variety of pharmacological effects. The purpose of this study was to determine the effects of L-CYSee on morphine-induced changes in ventilation, arterial-blood gas (ABG) chemistry, Alveolar-arterial (A-a) gradient (i.e., a measure of the index of alveolar gas-exchange), antinociception and sedation in male Sprague Dawley rats. An injection of morphine (10 mg/kg, IV) produced adverse effects on breathing, including sustained decreases in minute ventilation. L-CYSee (500 μmol/kg, IV) given 15 min later immediately reversed the actions of morphine. Another injection of L-CYSee (500 μmol/kg, IV) after 15 min elicited more pronounced excitatory ventilatory responses. L-CYSee (250 or 500 μmol/kg, IV) elicited a rapid and prolonged reversal of the actions of morphine (10 mg/kg, IV) on ABG chemistry (pH, pCO 2 , pO 2 , sO 2 ) and A-a gradient. L-serine ethylester (an oxygen atom replaces the sulfur; 500 μmol/kg, IV), was ineffective in all studies. L-CYSee (500 μmol/kg, IV) did not alter morphine (10 mg/kg, IV)-induced sedation, but slightly reduced the overall duration of morphine (5 or 10 mg/kg, IV)-induced analgesia. In summary, L-CYSee rapidly overcame the effects of morphine on breathing and alveolar gas-exchange, while not affecting morphine sedation or early-stage analgesia. The mechanisms by which L-CYSee modulates morphine depression of breathing are unknown, but appear to require thiol-dependent processes., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that would have influenced the studies that are described in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Using an efficient polynomial-time ground-state algorithm we investigate the Ising spin glass state at zero temperature in two dimensions. For large sizes, we show that the spin state in a central region is independent of the interactions far away, indicating a "single-state" picture, presumably the droplet model. Surprisingly, a single power law describes corrections to this result down to the smallest sizes studied.

Changes in gene expression with age are typically normalised to constitutively expressed reference genes (RGs). However, RG expression may be affected by age or overall health and most studies use only male animals. We investigated whether expression of common RGs (Gapdh, Gusb, Rplp0, B2m, Tubb5, Rpl7l1, Hprt, Rer1) was affected by age, sex and/or overall health (frailty index) in skeletal muscle from young (4-mos) and aged (25-26-mos) mice. Standard RG selection programs recommended Gapdh (RefFinder/Genorm/NormFinder) or Rpl7l1 (BestKeeper) without considering age and sex. Analysis of raw Cq values showed only Rplp0 was stable in both sexes at both ages. When qPCR data were normalised to Rplp0, age affected RG expression, especially in females. For example, Hprt expression declined with age (Hprt=9.8 ×10 -2 ± 4.7 ×10 -2 vs. 6.5 ×10 -3 ± 8.8 ×10 -4 ; mean±SEM), while Gusb expression increased (6.0 ×10 -4 ± 5.5 ×10 -5 vs. 1.7 ×10 -3 ± 3.1 ×10 -4 ; n = 5/group; p < 0.05). These effects were not seen in males. Tubb5 and Gapdh were not affected by age or sex when normalised to Rplp0. Similar results were seen with normalisation by Gapdh or the Rplp0/Gapdh pair. Interestingly, RG expression was graded not only by age but by frailty. These data demonstrate that age, sex, and frailty of animals must be carefully considered when selecting RGs to normalise mRNA abundance data., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The radical S -adenosyl-l-methionine (SAM) enzyme TYW1 catalyzes the condensation of C-2 and C-3 atoms of pyruvate with N -methylguanosine containing tRNA Phe to form 4-demethylwyosine (imG-14) modified tRNA Phe . The fate of C-1 is not known, and either formate or carbon dioxide (CO 2 ) has been proposed. In this study, a coupled assay that transforms either CO 2 or formate to oxaloacetate (OAA) was used to determine the fate of C-1. In the presence of [1- 13 C 1 ]-pyruvate, 13 C-enriched OAA was observed in a process that is concomitant with the formation of imG-14, under conditions that preferentially transform CO 2 and not formate to OAA. These findings are discussed in the context of the cofactor content of TYW1 and a new role for the auxiliary cluster in catalyzing the oxidative cleavage of C-1-C-2 bond of pyruvate in the catalytic cycle of TYW1.

This study demonstrates that intravenous infusion of the cell-penetrant thiol ester, L-cysteine ethyl ester (L-CYSee), to adult male Sprague-Dawley rats elicited (a) minor alterations in frequency of breathing, expiratory time, tidal volume, minute ventilation, or expiratory drive but pronounced changes in inspiratory time, end-inspiratory and expiratory pauses, peak inspiratory and expiratory flows, EF 50 , relaxation time, apneic pause, inspiratory drive and non-eupneic breathing index, (b) minimal changes in arterial blood-gas (ABG) chemistry (pH, pCO 2 , pO 2 , SO 2 ) and Alveolar-arterial (A-a) gradient (index of alveolar gas exchange), and (c) minimal changes in antinociception (tail-flick latency). Subsequent injection of morphine (10 mg/kg, IV) elicited markedly smaller effects on the above parameters, ABG chemistry, and A-a gradient in rats receiving L-CYSee, whereas morphine antinociception was not impaired. Infusions of L-cysteine or L-serine ethyl ester (oxygen rather than sulfur moiety), did not affect morphine actions on ABG chemistry or A-a gradient. L-CYSee (250 μmol/kg, IV) injection elicited dramatic changes in ventilatory parameters given 15 min after injection of morphine in rats receiving L-CYSee. Our findings suggest that (a) L-CYSee acts in neurons that drive ventilation, (b) L-CYSee reversal of the adverse actions of morphine on ventilation, ABG chemistry and A-a gradient may be via modulation of intracellular signaling pathways activated by morphine rather than by direct antagonism of opioid receptors since morphine antinociception was not diminished by L-CYSee, and (c) the thiol moiety of L-CYSee is vital to efficacy, (d) intracellular conversion of L-CYSee to an S-nitrosylated form may be part of its mechanism of action., Competing Interests: Conflict of Interest Statement The authors declare that they have no competing financial interests or personal relationships that would have influenced the studies that are described in this manuscript., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Studies on the hydration properties, proton conductivity, and water content of perfluorinated ionomer thin films at temperatures relevant to fuel cell operation temperatures (around 80 °C) and the effect of ionomer chemistry are scarce. In this work, we report the water content and proton conductivity properties of thin-film ionomers (30 nm) at 80 °C over a wide range of relative humidity (0-90%) for seven different ionomers differing in the side-chain structure, including the number of protogenic groups, with the equivalent weight ranging from 620 to 1100 g/mol of sulfonic acid. The results show that the acid content or equivalent weight of the ionomer is the strongest determinant of both the swelling and the proton conductivity of ionomer films at a given relative humidity. The molar water content (λ) of ionomer films normalized to the molar protogenic group is observed to be equivalent-weight-dependent, implying that the affinity for water is acid-content-dependent. At high relative humidity conditions (>70%) pertinent to fuel cell operations, the proton conductivity of low-equivalent-weight ionomers was higher than that of higher-equivalent-weight ionomers. However, upon correlating the proton conductivity with molar water content (λ), the differences reduce dramatically, highlighting that water content is the controlling factor for proton conduction. Significantly higher values of both water content and proton conductivity are observed at 80 °C compared to those at 30 °C, implying that room temperature data are not reliable for estimating ionomer properties in the fuel cell catalyst layer.

Perturbation of the endocannabinoid system can have profound effects on immune function and synaptic plasticity. Microglia are one of few cell types with a self-contained endocannabinoid system and are positioned at the interface between the immune system and the central nervous system. Past work has produced conflicting results with respect to the effects of pro-inflammatory conditions on the microglial endocannabinoid system. Thus, we systematically investigated the relationship between the concentration of two distinct pro-inflammatory stimuli, lipopolysaccharide and interferon gamma, on the abundance of components of the endocannabinoid system within microglia. Here we show that lipopolysaccharide and interferon gamma influence messenger RNA abundances of the microglial endocannabinoid system in a concentration-dependent manner. Furthermore, we demonstrate that the efficacy of different synthetic cannabinoid treatments with respect to inhibition of microglia nitric oxide release is dependent on the concentration and type of pro-inflammatory stimuli presented to the microglia. This indicates that different pro-inflammatory stimuli influence the capacity of microglia to synthesize, degrade, and respond to cannabinoids which has implications for the development of cannabinoid-based treatments for neuroinflammation., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to report., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Microglia are resident immune cells of the brain that survey the microenvironment, provide trophic support to neurons, and clear debris to maintain homeostasis and healthy brain function. Microglia are also drivers of neuroinflammation in several neurodegenerative diseases. Microglia produce endocannabinoids and express both cannabinoid receptor subtypes suggesting that this system is a target to suppress neuroinflammation. We tested whether cannabinoid type 1 (CB 1 ) or type 2 (CB 2 ) receptors could be targeted selectively or in combination to dampen the pro-inflammatory behavior of microglia, and whether this would have functional relevance to decrease secondary neuronal damage. We determined that components of the endocannabinoid system were altered when microglia are treated with lipopolysaccharide and interferon-gamma and shift to a pro-inflammatory phenotype. Furthermore, pro-inflammatory microglia released cytotoxic factors that induced cell death in cultured STHdh Q7/Q7 neurons. Treatment with synthetic cannabinoids that were selective for CB 1 receptors (ACEA) or CB 2 receptors (HU-308) dampened the release of nitric oxide (NO) and pro-inflammatory cytokines and decreased levels of mRNA for several pro-inflammatory markers. A nonselective agonist (CP 55,940) exhibited similar influence over NO release but to a lesser extent relative to ACEA or HU-308. All three classes of synthetic cannabinoids ultimately reduced the secondary damage to the cultured neurons. The mechanism for the observed neuroprotective effects appeared to be related to cannabinoid-mediated suppression of MAPK signaling in microglia. Taken together, the data indicate that activation of CB 1 or CB 2 receptors interfered with the pro-inflammatory activity of microglia in a manner that also reduced secondary damage to neurons., (Copyright © 2022 Elsevier Inc. All rights reserved.)

S-nitrosothiols exert multiple effects on neural processes in the central and peripheral nervous system. This study shows that intravenous infusion of S-nitroso-L-cysteine (SNO-L-CYS, 1 μmol/kg/min) in anesthetized male Sprague Dawley rats elicits (a) sustained increases in minute ventilation, via increases in frequency of breathing and tidal volume, (b) a decrease in Alveolar-arterial (A-a) gradient, thus improving alveolar gas-exchange, (c) concomitant changes in arterial blood-gas chemistry, such as an increase in pO 2 and a decrease in pCO 2 , (d) a decrease in mean arterial blood pressure (MAP), and (e) an increase in tail-flick (TF) latency (antinociception). Infusion of S-nitroso-D-cysteine (SNO-D-CYS, 1 μmol/kg/min, IV), did not elicit similar responses, except for a sustained decrease in MAP equivalent to that elicited by SNO-L-CYS. A bolus injection of morphine (2 mg/kg, IV) in rats receiving an infusion of vehicle elicited (a) sustained decreases in frequency of breathing tidal volume, and therefore minute ventilation, (b) a sustained decrease in MAP, (c) sustained decreases in pH, pO 2 and maximal sO 2 with sustained increases in pCO 2 and A-a gradient, and (d) a sustained increase in TF latency. In rats receiving SNO-L-CYS infusion, morphine elicited markedly smaller changes in minute ventilation, arterial blood gas chemistry, A-a gradient and MAP. In contrast, the antinociceptive effects of morphine were enhanced in rats receiving the infusion of SNO-L-CYS. The morphine-induced responses in rats receiving SNO-D-CYS infusion were similar to vehicle-infused rats. These data are the first to demonstrate that infusion of an S-nitrosothiol, such as SNO-L-CYS, can stereoselectively ameliorate the adverse effects of morphine on breathing and alveolar gas exchange while promoting antinociception., Competing Interests: Declaration of conflicting interests The authors declare that they have no competing financial interests or personal relationships that would have influenced the studies that are described in this manuscript., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

We determined whether intravenous injections of the membrane-permeable ventilatory stimulants, D-cysteine ethyl ester (ethyl (2 S)- 2-amino-3-sulfanylpropanoate) (D-CYSee) and D-cystine dimethyl ester (methyl (2 S)- 2-amino-3-[[(2 S)- 2-amino-3-methoxy-3-oxopropyl]disulfanyl] propanoate) (D-CYSdime), could overcome the deleterious actions of intravenous morphine on arterial blood pH, pCO 2 , pO 2 and sO 2 , and Alveolar-arterial (A-a) gradient (i.e., the measure of exchange of gases in the lungs) in Sprague Dawley rats anesthetized with isoflurane. Injection of morphine (2 mg/kg, IV) caused pronounced reductions in pH, pO 2 and sO 2 accompanied by elevations in pCO 2 , all which are suggestive of diminished ventilation, and elevations in A-a gradient, which suggests a mismatch of ventilation-perfusion. Subsequent boluses of D-cysteine ethyl ester (2 ×100 μmol/kg, IV) or D-cystine dimethyl ester (2 ×50 μmol/kg, IV) rapidly reversed of the negative actions of morphine on pH, pCO 2 , pO 2 and sO 2 , and A-a gradient. Similar injections of D-cysteine (2 ×100 μmol/kg, IV) were without effect, whereas injections of D-cystine (2 ×50 μmol/kg, IV) produced a modest reversal. Our data show that D-cysteine ethyl ester and D-cystine dimethyl ester readily overcome the deleterious effects of morphine on arterial blood gas (ABG) chemistry and A-a gradient by mechanisms that may depend upon their ability to rapidly enter cells. As a result of their known ability to enter the brain, lungs, muscles of the chest wall, and most likely the major peripheral chemoreceptors (i.e., carotid bodies), the effects of the thiolesters on changes in ABG chemistry and A-a gradient elicited by morphine likely involve central and peripheral mechanisms. We are employing target prediction methods to identify an array of in vitro and in vivo methods to test potential functional proteins by which D-CYSee and D-CYSdime modulate the effects of morphine on breathing., (Published by Elsevier B.V.)

Cell-penetrant thiol esters including the disulfides, D-cystine diethyl ester and D-cystine dimethyl ester, and the monosulfide, L-glutathione ethyl ester, prevent and/or reverse the deleterious effects of opioids, such as morphine and fentanyl, on breathing and gas exchange within the lungs of unanesthetized/unrestrained rats without diminishing the antinociceptive or sedative effects of opioids. We describe here the effects of the monosulfide thiol ester, D-cysteine ethyl ester (D-CYSee), on intravenous morphine-induced changes in ventilatory parameters, arterial blood-gas chemistry, alveolar-arterial (A-a) gradient (i.e., index of gas exchange in the lungs), and sedation and antinociception in freely-moving rats. The bolus injection of morphine (10 mg/kg, IV) elicited deleterious effects on breathing, including depression of tidal volume, minute ventilation, peak inspiratory flow, and inspiratory drive. Subsequent injections of D-CYSee (2 × 500 μmol/kg, IV, given 15 min apart) elicited an immediate and sustained reversal of these effects of morphine. Morphine (10 mg/kg, IV) also A-a gradient, which caused a mismatch in ventilation perfusion within the lungs, and elicited pronounced changes in arterial blood-gas chemistry, including pronounced decreases in arterial blood pH, pO 2 and sO 2 , and equally pronounced increases in pCO 2 (all responses indicative of decreased ventilatory drive). These deleterious effects of morphine were immediately reversed by the injection of a single dose of D-CYSee (500 μmol/kg, IV). Importantly, the sedation and antinociception elicited by morphine (10 mg/kg, IV) were minimally affected by D-CYSee (500 μmol/kg, IV). In contrast, none of the effects of morphine were affected by administration of the parent thiol, D-cysteine (1 or 2 doses of 500 μmol/kg, IV). Taken together, these data suggest that D-CYSee may exert its beneficial effects via entry into cells that mediate the deleterious effects of opioids on breathing and gas exchange. Whether D-CYSee acts as a respiratory stimulant or counteracts the inhibitory actions of µ-opioid receptor activation remains to be determined. In conclusion, D-CYSee and related thiol esters may have clinical potential for the reversal of the adverse effects of opioids on breathing and gas exchange, while largely sparing antinociception and sedation., Competing Interests: SB was employed by Galleon Pharmaceuticals, Inc., during the performance of the studies in this manuscript. SL declares that this study was funded in part by Galleon Pharmaceuticals, Inc. The leadership of the company was not directly involved in this study as a commercial entity. Only the principal scientist of the company (SB) was involved in the design of the studies, the collection, analysis, interpretation of data, the writing of the manuscript, and the ultimate decision to submit the manuscript for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Getsy, Baby, May, Young, Gaston, Hodges, Forster, Bates, Wilson, Lewis, Hsieh and Lewis.)

Microglia, the resident immune cells of the brain, can take on a range of pro- or anti-inflammatory phenotypes to maintain homeostasis. However, the sustained activation of pro-inflammatory microglia can lead to a state of chronic neuroinflammation characterized by high concentrations of neurotoxic soluble factors throughout the brain. In healthy brains, the inflammatory processes cease and microglia transition to an anti-inflammatory phenotype, but failure to halt the pro-inflammatory processes is a characteristic of many neurological disorders. The endocannabinoid system has been identified as a promising therapeutic target for chronic neuroinflammation as there is evidence that synthetic and endogenously produced cannabinoids temper the pro-inflammatory response of microglia and may encourage a switch to an anti-inflammatory phenotype. Activation of cannabinoid type 2 (CB 2 ) receptors has been proposed as the mechanism of action responsible for these effects. The abundance of components of the endocannabinoid system in microglia also change dynamically in response to several brain pathologies. This can impact the ability of microglia to synthesize and degrade endocannabinoids or react to endogenous and exogenous cannabinoids. Cannabinoid receptors also participate in the formation of receptor heteromers which influences their function specifically in cells that express both receptors, such as microglia. This creates opportunities for drug-drug interactions between CB 2 receptor-targeted therapies and other classes of drugs. In this article, we review the roles of pro- and anti-inflammatory microglia in the development and resolution of neuroinflammation. We also discuss the fluctuations observed in the components of the endocannabinoid in microglia and examine the potential of CB 2 receptors as a therapeutic target in this context., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Young and Denovan-Wright.)

Ribosomally synthesized post-translationally modified peptides (RiPPs) are ubiquitous and represent a structurally diverse class of natural products. The ribosomally encoded precursor polypeptides are often extensively modified post-translationally by enzymes that are encoded by coclustered genes. Radical S -adenosyl-l-methionine (SAM) enzymes catalyze numerous chemically challenging transformations. In RiPP biosynthetic pathways, these transformations include the formation of C-H, C-C, C-S, and C-O linkages. In this paper, we show that the Geobacter lovleyi sbtM gene encodes a radical SAM protein, SbtM, which catalyzes the cyclization of a Cys/SeCys residue in a minimal peptide substrate. Biochemical studies of this transformation support a mechanism involving H-atom abstraction at the C-3 of the substrate Cys to initiate the chemistry. Several possible cyclization products were considered. The collective biochemical, spectroscopic, mass spectral, and computational observations point to a thiooxazole as the product of the SbtM-catalyzed modification. To our knowledge, this is the first example of a radical SAM enzyme that catalyzes a transformation involving a SeCys-containing peptide and represents a new paradigm for formation of oxazole-containing RiPP natural products.

Professor Chia-Kuang (Frank) Tsung made his scientific impact primarily through the atomic-level design of nanoscale materials for application in heterogeneous catalysis. He approached this challenge from two directions: above and below the material surface. Below the surface, Prof. Tsung synthesized finely controlled nanoparticles, primarily of noble metals and metal oxides, tailoring their composition and surface structure for efficient catalysis. Above the surface, he was among the first to leverage the tunability and stability of metal-organic frameworks (MOFs) to improve heterogeneous, molecular, and biocatalysts. This article, written by his former students, seeks first to commemorate Prof. Tsung's scientific accomplishments in three parts: (1) rationally designing nanocrystal surfaces to promote catalytic activity; (2) encapsulating nanocrystals in MOFs to improve catalyst selectivity; and (3) tuning the host-guest interaction between MOFs and guest molecules to inhibit catalyst degradation. The subsequent discussion focuses on building on the foundation laid by Prof. Tsung and on his considerable influence on his former group members and collaborators, both inside and outside of the lab.

Understanding the low-temperature pure state structure of spin glasses remains an open problem in the field of statistical mechanics of disordered systems. Here we study Monte Carlo dynamics, performing simulations of the growth of correlations following a quench from infinite temperature to a temperature well below the spin-glass transition temperature T&#95;{c} for a one-dimensional Ising spin-glass model with diluted long-range interactions. In this model, the probability P&#95;{ij} that an edge {i,j} has nonvanishing interaction falls as a power law with chord distance, P&#95;{ij}∝1/R&#95;{ij}^{2σ}, and we study a range of values of σ with 1/2<σ<1. We consider a correlation function C&#95;{4}(r,t). A dynamic correlation length that shows power-law growth with time ξ(t)∝t^{1/z} can be identified in the data and, for large time t, C&#95;{4}(r,t) decays as a power law r^{-α&#95;{d}} with distance r when r≪ξ(t). The calculation can be interpreted in terms of the maturation metastate averaged Gibbs state, or MMAS, and the decay exponent α&#95;{d} differentiates between a trivial MMAS (α&#95;{d}=0), as expected in the droplet picture of spin glasses, and a nontrivial MMAS (α&#95;{d}≠0), as in the replica-symmetry-breaking (RSB) or chaotic pairs pictures. We find nonzero α&#95;{d} even in the regime σ>2/3 which corresponds to short-range systems below six dimensions. For σ<2/3, the decay exponent α&#95;{d} follows the RSB prediction for the decay exponent α&#95;{s}=3-4σ of the static metastate, consistent with a conjectured statics-dynamics relation, while it approaches α&#95;{d}=1-σ in the regime 2/3<σ<1; however, it deviates from both lines in the vicinity of σ=2/3.

TYW1 is a radical S -adenosyl-l-methionine (SAM) enzyme that catalyzes the condensation of pyruvate and N -methylguanosine-containing tRNA Phe , forming 4-demethylwyosine-containing tRNA Phe . Homologues of TYW1 are found in both archaea and eukarya; archaeal homologues consist of a single domain, while eukaryal homologues contain a flavin binding domain in addition to the radical SAM domain shared with archaeal homologues. In this study, TYW1 from Saccharomyces cerevisiae ( Sc TYW1) was heterologously expressed in Escherichia coli and purified to homogeneity. Sc TYW1 is purified with 0.54 ± 0.07 and 4.2 ± 1.9 equiv of flavin mononucleotide (FMN) and iron, respectively, per mole of protein, suggesting the protein is ∼50% replete with Fe-S clusters and FMN. While both NADPH and NADH are sufficient for activity, significantly more product is observed when used in combination with flavin nucleotides. Sc TYW1 is the first example of a radical SAM flavoenzyme that is active with NAD(P)H alone.

The human brain requires adequate cerebral blood flow to meet the high demand for nutrients and to clear waste products. With age, there is a chronic reduction in cerebral blood flow in small resistance arteries that can eventually limit proper brain function. The endothelin system is a key mediator in the regulation of cerebral blood flow, but the contributions of its constituent receptors in the endothelial and vascular smooth muscle layers of cerebral arteries have not been well defined in the context of aging. We isolated posterior cerebral arteries from young and aged Fischer 344 rats, as well as ET B receptor knock-out rats and mounted the vessels in plexiglass pressure myograph chambers to measure myogenic tone in response to increasing pressure and targeted pharmacological treatments. We used an ET A receptor antagonist (BQ-123), an ET B receptor antagonist (BQ-788), endothelin-1, an endothelin-1 synthesis inhibitor (phosphoramidon), and vessel denudation to dissect the roles of each receptor in aging vasculature. Aged rats exhibited a higher myogenic tone than young rats, and the tone was sensitive to the ET A antagonist, BQ-123, but insensitive to the ET B antagonist, BQ-788. By contrast, the tone in the vessels from young rats was raised by BQ-788 but unaffected by BQ-123. When the endothelial layer that is normally enriched with ET B1 receptors was removed from young vessels, myogenic tone increased. However, denudation of the endothelial layer did not influence vessels from aged animals. This indicated that endothelial ET B1 receptors were not functional in the vessels from aged rats. There was also an increase in ET A receptor expression with age, whereas ET B receptor expression remained constant between young and aged animals. These results demonstrate that in young vessels, ET B1 receptors maintain a lower myogenic tone, but in aged vessels, a loss of ET B receptor activity allows ET A receptors in vascular smooth muscle cells to raise myogenic tone. Our findings have potentially important clinical implications for treatments to improve cerebral perfusion in older adults with diseases characterized by reduced cerebral blood flow.

We have identified thiolesters that reverse the negative effects of opioids on breathing without compromising antinociception. Here we report the effects of D-cystine diethyl ester (D-cystine diEE) or D-cystine dimethyl ester (D-cystine diME) on morphine-induced changes in ventilation, arterial-blood gas chemistry, A-a gradient (index of gas-exchange in the lungs) and antinociception in freely moving rats. Injection of morphine (10 mg/kg, IV) elicited negative effects on breathing (e.g., depression of tidal volume, minute ventilation, peak inspiratory flow, and inspiratory drive). Subsequent injection of D-cystine diEE (500 μmol/kg, IV) elicited an immediate and sustained reversal of these effects of morphine. Injection of morphine (10 mg/kg, IV) also elicited pronounced decreases in arterial blood pH, pO 2 and sO 2 accompanied by pronounced increases in pCO 2 (all indicative of a decrease in ventilatory drive) and A-a gradient (mismatch in ventilation-perfusion in the lungs). These effects of morphine were reversed in an immediate and sustained fashion by D-cystine diME (500 μmol/kg, IV). Finally, the duration of morphine (5 and 10 mg/kg, IV) antinociception was augmented by D-cystine diEE. D-cystine diEE and D-cystine diME may be clinically useful agents that can effectively reverse the negative effects of morphine on breathing and gas-exchange in the lungs while promoting antinociception. Our study suggests that the D-cystine thiolesters are able to differentially modulate the intracellular signaling cascades that mediate morphine-induced ventilatory depression as opposed to those that mediate morphine-induced antinociception and sedation.

The standard short-range two-dimensional Ising spin glass is numerically well accessible, in particular, because there are polynomial-time ground-state algorithms. On the other hand, in contrast to higher dimensional spin glasses, it does not exhibit a rich behavior, i.e., no ordered phase at finite temperature. Here, we investigate whether long-range correlated bonds change this behavior. This would still keep the model numerically well accessible while exhibiting a more interesting behavior. The bonds are drawn from a Gaussian distribution with a two-point correlation for bonds at distance r that decays as (1+r^{2})^{-a/2}, a≥0. We study numerically with exact algorithms the ground-state and domain-wall excitations. Our results indicate that the inclusion of bond correlations still does not lead to a spin-glass order at any finite temperature. A further analysis reveals that bond correlations have a strong effect at local length scales, inducing ferro- and antiferromagnetic domains into the system. The length scale of ferro- and antiferromagnetic order diverges exponentially as the correlation exponent approaches a critical value, a→a&#95;{crit}=0. Thus, our results suggest that the system becomes a ferro- or antiferromagnet only in the limit a→0.

There is an urgent need to develop novel compounds that prevent the deleterious effects of opioids such as fentanyl on minute ventilation while, if possible, preserving the analgesic actions of the opioids. We report that L-glutathione ethyl ester (GSHee) may be such a novel compound. In this study, we measured tail flick latency (TFL), arterial blood gas (ABG) chemistry, Alveolar-arterial gradient, and ventilatory parameters by whole body plethysmography to determine the responses elicited by bolus injections of fentanyl (75 μg/kg, IV) in male adult Sprague-Dawley rats that had received a bolus injection of GSHee (100 μmol/kg, IV) 15 min previously. GSHee given alone had minimal effects on TFL, ABG chemistry and A-a gradient whereas it elicited changes in some ventilatory parameters such as an increase in breathing frequency. In vehicle-treated rats, fentanyl elicited (1) an increase in TFL, (2) decreases in pH, pO 2 and sO 2 and increases in pCO 2 (all indicative of ventilatory depression), (3) an increase in Alveolar-arterial gradient (indicative of a mismatch in ventilation-perfusion in the lungs), and (4) changes in ventilatory parameters such as a reduction in tidal volume, that were indicative of pronounced ventilatory depression. In GSHee-pretreated rats, fentanyl elicited a more prolonged analgesia, relatively minor changes in ABG chemistry and Alveolar-arterial gradient, and a substantially milder depression of ventilation. GSHee may represent an effective member of a novel class of thiolester drugs that are able to prevent the ventilatory depressant effects elicited by powerful opioids such as fentanyl and their deleterious effects on gas-exchange in the lungs without compromising opioid analgesia.

Background: Methylenetetrahydrofolate reductase, the encoded by the MTHFR gene, plays a crucial role in converting the amino acid homocysteine to methionine. Two polymorphisms of the MTHFR gene, C677T and A1298C, reportedly reduce enzyme activity, resulting in hyperhomocysteinemia. Patients with C677T and A1298C polymorphisms may be at higher risk for developing abnormal hyperhomocysteinemia, which has been linked to catastrophic neurological including fatal outcomes., Objective: Determine the prevalence of the MTHFR gene variants C677T and A1298C among pediatric dental patients treated at King Abdulaziz University Hospital., Design: Cross-sectional., Setting: Clinics of pediatric dentistry department., Subjects and Methods: Healthy Saudi children 6-12 years old with no known allergies were screened for eligibility between May and December 2019. A single investigator collected saliva samples. The MTHFR C677T and A1298C polymorphisms were analyzed using polymerase chain reaction and restriction fragment length polymorphism., Main Outcome Measure: The prevalence of MTHFR gene variants (C677T and A1298C) among the subjects., Sample Size: 138., Results: MTHFR C677T polymorphism was present in 36.2% of the sample and 90.0% of children carrying this allele were heterozygotes. MTHFR A1298C polymorphism was present in 91.3% of the sample and 77.0% of the children carrying this allele were heterozygotes. No linkage disequilibrium between MTHFR C677T and MTHFR A1298C was observed within this sample., Conclusions: Our study found a high frequency of the MTHFR A1298C genotype, which was substantially more abundant than expected based on a Hardy-Weinberg distribution. Therefore, caution is advised in using N 2 O in Saudi children as the increased prevalence of this MTHFR allele may increase the incidence of serious adverse effects among these children., Limitations: Further studies are recommended with a larger sample size from randomly selected hospitals from different regions of Saudi Arabia., Conflict of Interest: None.

The cannabinoid type 1 (CB 1 ) receptor and the dopamine type 2 (D 2 ) receptor are co-localized on medium spiny neuron terminals in the globus pallidus where they modulate neural circuits involved in voluntary movement. Physical interactions between the two receptors have been shown to alter receptor signaling in cell culture. The objectives of the current study were to identify the presence of CB 1 /D 2 heteromers in the globus pallidus of C57BL/6J male mice, define how CB 1 /D 2 heteromer levels are altered following treatment with cannabinoids and/or antipsychotics, and determine if fluctuating levels of CB 1 /D 2 heteromers have functional consequences. Using in situ proximity ligation assays, we observed CB 1 /D 2 heteromers in the globus pallidus of C57BL/6J mice. The abundance of the heteromers increased following treatment with the nonselective cannabinoid receptor agonist, CP55,940. In contrast, treatment with the typical antipsychotic haloperidol reduced the number of CB 1 /D 2 heteromers, whereas the atypical antipsychotic olanzapine treatment had no effect. Co-treatment with CP55,940 and haloperidol had similar effects to haloperidol alone, whereas co-treatment with CP55,940 and olanzapine had similar effects to CP55,940. The observed changes were found to have functional consequences as the differential effects of haloperidol and olanzapine also applied to γ-aminobutyric acid release in STHdh Q7/Q7 cells and motor function in C57BL/6J male mice. This work highlights the clinical relevance of co-exposure to cannabinoids and different antipsychotics over acute and prolonged time periods., (© 2020 Wiley Periodicals LLC.)

The endothelin receptor A (ETA) and endothelin receptor B (ETB) are G protein-coupled receptors that are co-expressed in vascular smooth muscle cells. Endothelin-1 (ET-1) activates endothelin receptors to cause microvascular vasoconstriction. Previous studies have shown that heteromerization between ETA and ETB prolongs Ca 2+ transients, leading to prolongation of Gα q -dependent signaling and sustained vasoconstriction. We hypothesized that these effects are in part mediated by the resistance of ETA/ETB heteromers to β-arrestin recruitment and subsequent desensitization. Using bioluminescence resonance energy transfer 2 (BRET 2 ), we found that ETB has a relatively equal affinity to form either homomers or heteromers with ETA when co-expressed in the human embryonic kidney 293 (HEK293) cells. When co-expressed, activation of ETA and ETB by ET-1 caused a heteromer-specific reduction and delay in β-arrestin-2 recruitment with a corresponding reduction and delay in ET-1-induced ETA/ETB co-internalization. Furthermore, the co-expression of ETA and ETB inhibited ET-1-induced β-arrestin-1-dependent extracellular signal-regulated kinase (ERK) phosphorylation while prolonging ET-1-induced Gα q -dependent ERK phosphorylation. ETA/ETB heteromerization mediates the long-lasting vasoconstrictor response to ET-1 by the prolongation of Gα q -dependent signaling and inhibition of β-arrestin function.

We apply generalizations of the Swendson-Wang and Wolff cluster algorithms, which are based on the construction of Fortuin-Kasteleyn clusters, to the three-dimensional ±1 random-bond Ising model. The behavior of the model is determined by the temperature T and the concentration p of negative (antiferromagnetic) bonds. The ground state is ferromagnetic for 0≤p0, our data suggest that the percolation transition is universal, irrespective of whether the ground state exhibits ferromagnetic or spin-glass order, and is in the universality class of standard percolation. This shows that correlations in the bond occupancy of the Fortuin-Kasteleyn clusters are irrelevant, except for p=0 where the clusters are strictly tied to Ising correlations so the percolation transition is in the Ising universality class.

Controlling the surface composition of shaped bimetallic nanoparticles could offer precise tunability of geometric and electronic surface structure for new nanocatalysts. To achieve this goal, a platform for studying the intermixing process in a shaped nanoparticle was designed, using multilayered Pd-Ni-Pt core-shell nanocubes as precursors. Under mild conditions, the intermixing between Ni and Pt could be tuned by changing layer thickness and number, triggering intermixing while preserving nanoparticle shape. Intermixing of the two metals is monitored using transmission electron microscopy. The surface structure evolution is characterized using electrochemical methanol oxidation. DFT calculations suggest that the low-temperature mixing is enhanced by shorter diffusion lengths and strain introduced by the layered structure. The platform and insights presented are an advance toward the realization of shape-controlled multimetallic nanoparticles tailored to each potential application., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

RNA-fluorescence in situ hybridization (FISH) is a powerful tool to visualize target messenger RNA transcripts in cultured cells, tissue sections or whole-mount preparations. As the technique has been developed over time, an ever-increasing number of divergent protocols have been published. There is now a broad selection of options available to facilitate proper tissue preparation, hybridization, and post-hybridization background removal to achieve optimal results. Here we review the technical aspects of RNA-FISH, examining the most common methods associated with different sample types including cytological preparations and whole-mounts. We discuss the application of commonly used reagents for tissue preparation, hybridization, and post-hybridization washing and provide explanations of the functional roles for each reagent. We also discuss the available probe types and necessary controls to accurately visualize gene expression. Finally, we review the most recent advances in FISH technology that facilitate both highly multiplexed experiments and signal amplification for individual targets. Taken together, this information will guide the methods development process for investigators that seek to perform FISH in organisms that lack documented or optimized protocols., Competing Interests: The authors declare that they have no competing interests., (© 2020 Young et al.)

The presence of a capping agent (CTAB) on Pd nanoparticles produces a strong static disorder in the surface region. This results in a surface softening, which contributes to an overall increase in the Debye-Waller coefficient measured by X-ray powder diffraction. Molecular dynamics and density functional theory simulations show that the adsorption-induced surface disorder is strong enough to overcome the effects of nanoparticle size and shape.

Reverse transcription quantitative PCR (RT-qPCR) is a robust technique for the quantification and comparison of gene expression. To obtain reliable results with this method, one or more reference genes must be employed to normalize expression measurements among treatments or tissue samples. Candidate reference genes must be validated to ensure that they are stable prior to use in qPCR experiments. The pond snail ( Lymnaea stagnalis ) is a common research organism, particularly in the areas of learning and memory, and is an emerging model for the study of biological asymmetry, biomineralization, and evolution and development. However, no systematic assessment of qPCR reference genes has been performed in this animal. Therefore, the aim of our research was to identify stable reference genes to normalize gene expression data from several commonly studied tissues in L. stagnalis as well as across the entire body. We evaluated a panel of seven reference genes across six different tissues in L. stagnalis with RT-qPCR. The genes included: elongation factor 1-alpha , glyceraldehyde-3-phosphate dehydrogenase , beta-actin , beta-tubulin , ubiquitin , prenylated rab acceptor protein 1 , and a voltage gated potassium channel. These genes exhibited a wide range of expression levels among tissues. The tissue-specific stability of each of the genes was consistent when measured by the standard stability assessment algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Our data indicate that the most stable reference genes vary among the tissues that we examined (central nervous system, tentacles, lips, penis, foot, mantle). Our results were generally congruent with those obtained from similar studies in other molluscs. Given that a minimum of two reference genes are recommended for data normalization, we provide suggestions for strong pairs of reference genes for single- and multi-tissue analyses of RT-qPCR data in L. stagnalis ., Competing Interests: The authors declare that they have no competing interests., (© 2019 Young et al.)

Theories of memory must account for memory performance during both the acquisition (i.e., ongoing learning) and retention (i.e., following disuse) stages of training. One factor affecting both stages is whether repeated encounters with a set of material occur with no delay between blocks (massed) or alternating with another intervening task (spaced). Whereas the retention advantage for spaced over massed practice is well accounted for by some current theories of memory, theories of decay or general interference predict massed, rather than spaced, advantages during acquisition. In a series of 3 experiments, we show that the effects of spacing on acquisition depend on the relationship between primary and delay tasks. Specifically, massed acquisition advantages occur only in the presence of code-specific interference (the engagement in two alternating tasks both emphasizing the same processing code, such as verbal or spatial processing codes; e.g., learning letter-number pairs and reading text), whereas spaced acquisition advantages are observed only when code-specific interference is absent. These results present a challenge for major theories of memory. Furthermore, we argue that code-specific interference is important for researchers of the spacing and interleaving effects to take into consideration, as the relationship between the alternating tasks used has a substantial impact on acquisition performance.

Pronuclear microinjection remains the most widely used method for the germline modification of mice and other species. The method is conceptually quite simple and at least in rodents can produce transgenic offspring with relatively high efficiency. Here, we describe the various components of the production of transgenic mice including a detailed list of materials and equipment. We outline in detail the preparation of animals, the retrieval, culture and transfer of embryos, the preparation of DNA, and the microinjection process. We have added a substantial collection of notes with helpful suggestions that reflect the many years of experience we have using this technology and our continuing efforts to improve animal welfare and the efficiency of producing transgenics.