Development of CVN424: A Selective and Novel GPR6 Inverse Agonist Effective in Models of Parkinson Disease.

GPR6 is an orphan G-protein-coupled receptor that has enriched expression in the striatopallidal, indirect pathway and medium spiny neurons of the striatum. This pathway is greatly impacted by the loss of the nigro-striatal dopaminergic neurons in Parkinson disease, and modulating this neurocircuitry can be therapeutically beneficial. In this study, we describe the in vitro and in vivo pharmacological characterization of (R)-1-(2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-3-((tetrahydrofuran-3-yl)amino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)ethan-1-one (CVN424), a highly potent and selective small-molecule inverse agonist for GPR6 that is currently undergoing clinical evaluation. CVN424 is brain-penetrant and shows dose-dependent receptor occupancy that attained brain 50% of receptor occupancy at plasma concentrations of 6.0 and 7.4 ng/ml in mice and rats, respectively. Oral administration of CVN424 dose-dependently increases locomotor activity and reverses haloperidol-induced catalepsy. Furthermore, CVN424 restored mobility in bilateral 6-hydroxydopamine lesion model of Parkinson disease. The presence and localization of GPR6 in medium spiny neurons of striatum postmortem samples from both nondemented control and patients with Parkinson disease were confirmed at the level of both RNA (using Nuclear Enriched Transcript Sort sequencing) and protein. This body of work demonstrates that CVN424 is a potent, orally active, and brain-penetrant GPR6 inverse agonist that is effective in preclinical models and is a potential therapeutic for improving motor function in patients with Parkinson disease. SIGNIFICANCE STATEMENT: CVN424 represents a nondopaminergic novel drug for potential use in patients with Parkinson disease.

"Watch Me Grow- Electronic (WMG-E)" surveillance approach to identify and address child development, parental mental health, and psychosocial needs: study protocol.

BACKGROUND: The COVID-19 pandemic and the associated economic recession has increased parental psychosocial stress and mental health challenges. This has adversely impacted child development and wellbeing, particularly for children from priority populations (culturally and linguistically diverse (CALD) and rural/regional communities) who are at an already increased risk of health inequality. The increased mental health and psychosocial needs were compounded by the closure of in-person preventive and health promotion programs resulting in health organisations embracing technology and online services. Watch Me Grow- Electronic (WMG-E) - developmental surveillance platform- exemplifies one such service. WMG-E was developed to monitor child development and guide parents towards more detailed assessments when risk is identified. This Randomised Controlled Trial (RCT) aims to expand WMG-E as a digital navigation tool by also incorporating parents' mental health and psychosocial needs. Children and families needing additional assessments and supports will be electronically directed to relevant resources in the 'care-as-usual' group. In contrast, the intervention group will receive continuity of care, with additional in-person assessment and 'warm hand over' by a 'service navigator' to ensure their needs are met. METHODS: Using an RCT we will determine: (1) parental engagement with developmental surveillance; (2) access to services for those with mental health and social care needs; and (3) uptake of service recommendations. Three hundred parents/carers of children aged 6 months to 3 years (recruited from a culturally diverse, or rural/regional site) will be randomly allocated to the 'care-as-usual' or 'intervention' group. A mixed methods implementation evaluation will be completed, with semi-structured interviews to ascertain the acceptability, feasibility and impact of the WMG-E platform and service navigator. CONCLUSIONS: Using WMG-E is expected to: normalise and de-stigmatise mental health and psychosocial screening; increase parental engagement and service use; and result in the early identification and management of child developmental needs, parental mental health, and family psychosocial needs. If effective, digital solutions such as WMG-E to engage and empower parents alongside a service navigator for vulnerable families needing additional support, will have significant practice and policy implications in the pandemic/post pandemic period. TRIAL REGISTRATION: The trial (Protocol No. 1.0, Version 3.1) was registered with ANZCTR (registration number: ACTRN12621000766819 ) on July 21st, 2021 and reporting of the trial results will be according to recommendations in the CONSORT Statement.

Investigating public perceptions of carbon dioxide utilisation (CDU) technology: a mixed methods study.

Carbon dioxide utilisation (CDU) technologies hold promise for helping to limit atmospheric releases of CO2 while generating saleable products. However, while there is growing investment in the research and development required to bring CDU to the market, to date there has been very little systematic research into public perceptions of the technology. The current research reports upon the findings of a series of six qualitative focus groups (and an associated questionnaire) held with members of the UK public in order to discuss the perceived benefits and risks of CDU technology. The findings reveal that public awareness of CDU is currently very low and that there is a desire to learn more about the technology. While our participants did, on average, appear to develop an overall positive attitude towards CDU, this attitude was tentative and was associated with a number of caveats. The implications for the findings in terms of the development of communication and broader strategies of public engagements are outlined.

Biological effects of gold mine tailings on the intertidal marine environment in Nova Scotia, Canada.

From 1861 to the 1940s, gold was produced from 64 mining districts in Nova Scotia, where mercury amalgamation was the dominant method for the extraction of gold from ore until the 1880s. As a result, wastes (tailings) from the milling process were contaminated by mercury and were high in naturally occurring arsenic. In 2004 and 2005, sediments, water and mollusc tissues were collected from 29 sampling stations at nine former gold mining areas along the Atlantic coastline and were analysed for arsenic and mercury. The resulting data were compared with environmental quality guidelines. Samples indicated high potential risk of adverse effects in the intertidal environments of Seal Harbour, Wine Harbour and Harrigan Cove. Arsenic in Seal Harbour was bioavailable, resulting in high concentrations of arsenic in soft-shell clam tissues. Mercury concentrations in tissues were below guidelines. This paper presents results of the sampling programs and implications of these findings.

Analysis of human Nav1.8 expressed in SH-SY5Y neuroblastoma cells.

The tetrodotoxin-resistant voltage-gated sodium channel alpha-subunit Nav1.8 is expressed in nociceptors and has been implicated in chronic pain. Difficulties of heterologous expression have so far precluded analysis of the pharmacological properties of human Nav1.8. To address this we have introduced human Nav1.8 in neuroblastoma SH-SY5Y cells. Voltage-clamp analysis showed that human Nav1.8 generated an inward tetrodotoxin-resistant sodium current with an activating threshold around -50 mV, half maximal activation at -11+/-3 mV and a reversal potential of 67+/-4 mV. These properties closely match those of the endogenous rat tetrodotoxin-resistant sodium current in dorsal root ganglia suggesting that the expressed human channel is in a near physiological conformation. Human Nav1.8 was resistant to tetrodotoxin and activated by the pyrethroid toxin deltamethrin. Both voltage-activated and deltamethrin-activated human Nav1.8 were inhibited by the sodium channel blockers BIII 890 CL, NW-1029, and mexiletine. Inhibition of Nav1.8 by these compounds may underlie their known analgesic effects in animal models.

A high-throughput MS-PCR method on MADGE gels for ANG II type-1 receptor A1166C polymorphism.

We have developed a highly accurate, low-cost, single-step, mutagenically separated polymerase chain reaction (MS-PCR) method for the determination of angiotensin II type-1 receptor (AT(1)) A1166C gene polymorphism. The genotypes are determined using the microtiter array diagonal gel electrophoresis (MADGE) system. We have compared the MS-PCR method with allele-specific oligonucleotide hybridization and Dde I digestion techniques for determining the AT(1) A1166C genotype. The combination of MS-PCR and MADGE serves as a model for high-throughput single-nucleotide polymorphism genotyping in large population studies.

Polymorphisms in the angiotensinogen gene are associated with carotid intimal-medial thickening in females from a community-based population.

BACKGROUND: Polymorphisms within genes of the renin-angiotensin system have been associated with an increased risk of cardiovascular disease. We investigated the association of polymorphisms in the angiotensinogen (AGT) and angiotensin II receptor type 1 (AGTR1) genes with increased intima-media thickness (IMT) and the presence of plaques in carotid arteries. METHODS: Subjects (1111) from the Perth Carotid Ultrasound Disease Assessment Study (CUDAS) were genotyped for three polymorphisms: two in the promoter of the AGT gene, G-6A and A-20C; and one in the AGTR1 gene, A1166C. RESULTS: Using multivariate generalised linear models, the AGT-6A allele (P<0.001) and the AGT-20C allele (P<0.03) were significantly associated with increased mean carotid IMT in females but not in males when adjusted for conventional risk factors. The AGTR1 A1166C polymorphism did not show any significant relationship to mean IMT. Results suggest that the I allele of the angiotensin converting enzyme insertion/deletion polymorphism may interact with the AGT-6G allele to increase mean carotid IMT in the population as a whole. None of the polymorphisms investigated were significantly associated with the presence of carotid plaques. CONCLUSION: This study shows that polymorphisms in the angiotensinogen gene are associated with an increased risk of carotid intimal-medial wall thickening in females.

Overlapping selectivity of neurotoxin and dihydropyridine calcium channel blockers in cerebellar granule neurones.

Calcium (Ca(2+)) currents have been studied extensively in cerebellar granule neurones, but much of the whole-cell pharmacology is inconsistent. Ca(2+) channel currents were recorded from granule neurones to investigate whether the commonly used Ca(2+) channel blockers show overlapping selectivity. Using combinations of toxin channel blockers, 45% of the total current was shown to be carried by Ca(2+) channels susceptible to block by the combined, or cumulative application of, omega-agatoxin IVA, omega-conotoxin GVIA and omega-conotoxin MVIIC, thus representing P/Q- and N-type channel currents. However, sequential application of these toxins showed that substantial overlap occurred in the proportions of current sensitive to individual toxins. Application of the 1, 4-dihydropyridine nicardipine at 1 microM, a concentration reported to be selective for L-type channels, blocked 16% of the total current, without reducing the current sensitive to the toxins used. However, greater concentrations of nicardipine (>10 microM) blocked a proportion of the total current that could not be accounted for by L-type channels alone. These results demonstrate that a pharmacological approach based on the L, N, P/Q, and R classification does not adequately describe the Ca(2+) channel subtypes found in cerebellar granule neurones due to substantial cross-selectivity to the drugs and toxins used.

The involvement of multiple calcium channel sub-types in glutamate release from cerebellar granule cells and its modulation by GABAB receptor activation.

In this study, we have examined both the ability of various Ca2+ channel sub-types to support the release of [3H]glutamate from cerebellar granule neurons and the mechanism of action involved in the modulation of glutamate release by the GABAB receptor agonist, (-)-baclofen. Cerebellar granule neurons were stimulated to release newly synthesized [3H]glutamate by K(+)-evoked depolarization. Stimulated release was entirely calcium-dependent and abolished by the presence of 200 microM cadmium. Release of glutamate was not affected by either tetrodotoxin or 5-aminophosphonovaleric acid but was potentiated by dihydrokainate and inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione. Stimulated glutamate release was partially inhibited by both the L-type calcium channel blocker, nicardipine, and the N-type calcium channel blocker, omega-conotoxin GVIA; however, the P/Q-type calcium channel blocker omega-agatoxin IVA inhibited release of glutamate only after pre-incubation of cells with omega-conotoxin GVIA. K(+)-stimulated release of glutamate was observed when stimulated either in the presence of Ca2+ or of Ba2+ and similar inhibition of release by (-)-baclofen was seen under both conditions. In contrast to these results, ionomycin-evoked glutamate release was greatly reduced as compared to K(+)-evoked release and was not modulated by (-)-baclofen. In the presence of omega-conotoxin GVIA alone, inhibition of release by (-)-baclofen was attenuated but not abolished. Following block of nicardipine-sensitive channels, inhibition of release by (-)-baclofen was still present, and after prior block of omega-conotoxin GVIA-sensitive channels the presence of nicardipine restored the ability of (-)-baclofen to inhibit residual release of glutamate. Modulation of glutamate release by (-)-baclofen was unaffected by the presence of omega-agatoxin IVA alone; however, after block of both omega-conotoxin GVIA- and omega-agatoxin IVA-sensitive channels, inhibition of release by (-)-baclofen was completely abolished. These results indicate that multiple sub-types of voltage-dependent calcium channels are present on the presynaptic terminals of cerebellar granule neurons and support K(+)-stimulated release of [3H]glutamate. Modulation of release by GABAB receptor activation appears to be dependent upon interaction of this receptor with a number of voltage-sensitive calcium channels, including omega-conotoxin GVIA-sensitive and omega-agatoxin IVA-sensitive channels.

Emotionally related donors and renal transplantation.

Published medical and psychological evidence now available strongly supports the widening of donor sources to include the emotionally related donor. Irrespective of the benefit conferred by a living related over an unrelated donor, it now seems indefensible to refuse the donation of a kidney from an emotionally related individual to a recipient who meets the required criteria. An absence of other donor sources, the existence of preformed antibodies, or priority on the transplant list all seem to us to be compelling reasons to consider the use of the emotionally related donor. This is especially true when the psycho-social assessment reveals a true volunteer who is likely to derive psychological benefit from the donation and where "donation consensus" exists within the family.

Medicinal plants of Seberida (Riau Province, Sumatra, Indonesia).

Field enquiries on the plants used to treat diseases in villages of Seberida Municipality indicated that a large number of plant species (at least 100) are being used in therapy. Many of the uses, however, are magical in nature. Those in which a cause-effect relationship may be established (56) are presented in this paper. A review of the ethnomedical and experimental literature showed that medicinal plant uses in Seberida fall into three categories: those for which uses are corroborated by similar medicinal uses for the same plant or different species of the same genus in other cultures, those for which uses of the plant or species of the same genus are corroborated by evidence of relevant pharmacological activity in the experimental literature and those for which the medicinal uses are not corroborated. A discussion of these categories is presented. Taken as a whole, the medicinal uses of plants in Seberida are characterized by a remarkably high proportion of plants used to treat fevers and malaria and by a high proportion of species of which the leaves are used (externally or internally) for medicinal purposes. Comparison with other studies reported in the literature seems to indicate that a high frequency of the use of leaves in therapy may be a part of a larger cultural phenomenon among the tropical forest tribes of Southeast Asia and the southern Pacific Islands. Possible rationales for this type of use are offered.

A comparison of teaching strategies for integrating information technology into clinical nursing education.

As health care becomes more information-intensive and diverse, there is a need to integrate information technology (IT) into clinical education. Little is known, however, about how to design instructional strategies for integrating information technology into clinical nursing education. This article outlines the instructional strategies used by faculty in five nursing programs who taught students to use a point-of-care information technology system. The article also reports students' computer acceptance and summarizes IT clinical teaching recommendations.

Amorphous trehalose dihydrate by cryogenic milling.

Trehalose dihydrate is a safe, naturally occurring disaccharide used as a food ingredient and pharmaceutical excipient. It has been reported that room temperature milling does not lead to the formation of amorphous trehalose dihydrate. This paper reports the behaviour of trehalose dihydrate upon milling at cryogenic temperatures as studied by DSC, TGA, XRPD and Raman spectroscopy. We have demonstrated that the crystal to glass transformation for trehalose dihydrate is possible using cryogenic milling. This is the first reported example of cryogenic milling (a mild and widely applicable technique) applied to generating amorphous hydrates.

Synthesis and SAR of novel 2-arylthiazolidinones as selective analgesic N-type calcium channel blockers.

A series of new N-type (Ca(v)2.2) calcium channel blockers derived from the 'hit' structures 2-(3-bromo-4-fluorophenyl)-3-(2-pyridin-2-ylethyl)thiazolidin-4-one 9 and its 2-[4-(4-bromophenyl)pyridin-3-yl]-3-isobutyl analogue 10 is described. Extensive SAR studies using a range of synthetic approaches resulted in novel, patented compounds with IC50 values of up to 0.2 microM in an in vitro IMR32 assay, and selectivities for N/L of up to 30-fold. The new compounds described have potential in treatment of neuropathic pain.

Intergranular giant magnetoresistance in a spontaneously phase separated perovskite oxide.

We present small-angle neutron scattering data proving that, on the insulating side of the metal-insulator transition, the doped perovskite cobaltite La(1-x)Sr(x)CoO(3) phase separates into ferromagnetic metallic clusters embedded in a nonferromagnetic matrix. This induces a hysteretic magnetoresistance, with temperature and field dependence characteristic of intergranular giant magnetoresistance (GMR). We argue that this system is a natural analog to the artificial structures fabricated by depositing nanoscale ferromagnetic particles in a metallic or insulating matrix; i.e., this material displays a GMR effect without the deliberate introduction of chemical interfaces.

The ethics of therapeutic and reproductive human cloning.

Neither therapeutic cloning nor reproductive cloning necessarily pose insurmountable ethical obstacles. Two defences of therapeutic cloning are considered. The first defence, the argument from property, is rejected because it entails morally counter-intuitive consequences. We should prefer a 'balance of reasons' defence which leaves room for the view that human life has intrinsic value. Reproductive cloning is best defended by an appeal to the right to procreative autonomy. The sorts of harms it is feared clones will suffer are also suffered by children conceived through natural means, even when these harms were preventable. The right to reproductive autonomy disallows state control of any form of reproduction for the reasons of child welfare discussed.

Human cloning and child welfare.

In this paper we discuss an objection to human cloning which appeals to the welfare of the child. This objection varies according to the sort of harm it is expected the clone will suffer. The three formulations of it that we will consider are: 1. Clones will be harmed by the fearful or prejudicial attitudes people may have about or towards them (H1); 2. Clones will be harmed by the demands and expectations of parents or genotype donors (H2); 3. Clones will be harmed by their own awareness of their origins, for example the knowledge that the genetic donor is a stranger (H3). We will show why these three versions of the child welfare objection do not necessarily supply compelling reasons to ban human reproductive cloning. The claim that we will develop and defend in the course of our discussion is that even if it is the case that a cloned child will suffer harms of the type H1-H3, it is none the less permissible to conceive by cloning so long as these cloning-induced welfare deficits are not such as to blight the existence of the resultant child, whoever this may be.

A modulatory role for protein phosphatase 2B (calcineurin) in the regulation of Ca2+ entry.

The Ca2+/calmodulin-dependent protein phosphatase 2B (PP2B) also known as calcineurin (CN) has been implicated in the Ca2+-dependent inactivation of Ca2+ channels in several cell types. To study the role of calcineurin in the regulation of Ca2+-channel activity, phosphatase expression was altered in NG108-15 cells by transfection of sense and antisense plasmid constructs carrying the catalytic subunit of human PP2Bbeta3. Relative to mock-transfected (wild-type) controls, cells overexpressing calcineurin showed dramatically reduced high-voltage-activated Ca2+ currents which were recoverable by the inclusion of 1 microM FK506 in the patch pipette. Conversely, in cells with reduced calcineurin expression, high-voltage-activated Ca2+ currents were larger relative to controls. Additionally in these cells, low-voltage-activated currents were significantly reduced. Analysis of high-voltage-activated Ca2+ currents revealed that the kinetics of inactivation were significantly accelerated in cells overexpressing calcineurin. Following the delivery of a train of depolarizing pulses in experiments designed to produce large-scale Ca2+ influx across the cell membrane, Ca2+-dependent inactivation of high-voltage-activated Ca2+ currents was increased in sense cells, and this increase could be reduced by intracellular application of 1 mM BAPTA or 1 microM FK506. These data support a role of calcineurin in the negative feedback regulation of Ca2+ entry through voltage-operated Ca2+ channels.

Functional expression of rat brain cloned alpha1E calcium channels in COS-7 cells.

The properties of the rat brain alpha1E Ca2+ channel subunit and its modulation by accessory rat brain alpha2-delta and beta1b subunits were studied by transient transfection in a mammalian cell line in order to attempt to reconcile the debate as to whether alpha1E forms a low-voltage-activated (LVA) or high-voltage-activated (HVA) Ca2+ channel and to examine its pharmacology in detail. alpha1E alone was capable of forming an ion-conducting pore in COS-7 cells. The properties of heteromultimeric alpha1E/alpha2-delta/beta1b channels were largely dictated by the presence of the beta1b subunit, which increased current density and tended to produce a hyperpolarizing shift in the voltage dependence of activation and inactivation. alpha1E/alpha2-delta/beta1b channels did not appear to be regulated by Ca2+-induced inactivation. alpha1E was shown to exhibit a unique pharmacological profile. omega-Agatoxin IVA blocked the current in a dose-dependent manner with an IC50 of approximately 50 nM and a maximum inhibition of about 80%, whilst omega-conotoxin MVIIC was without effect. The 1,4-dihydropyridine (DHP) antagonist nicardipine (1 micro;M) produced an inhibition of 51 +/- 7%, whereas the DHP agonist S-(-)BAY K 8644 was without effect. Our findings suggest a re-evaluation of the classification of the alpha1E Ca2+ channel subunit; we propose that rat brain alpha1E forms a novel Ca2+ channel with properties more similar to a subtype of LVA than HVA Ca2+ current.