Microwave-assisted synthesis of a MK2 inhibitor by Suzuki-Miyaura coupling for study in Werner syndrome cells

Microwave-assisted Suzuki-Miyaura cross-coupling reactions have been employed towards the synthesis of three different MAPKAPK2 (MK2) inhibitors to study accelerated aging in Werner syndrome (WS) cells, including the cross-coupling of a 2-chloroquinoline with a 3-pyridinylboronic acid, the coupling of an aryl bromide with an indolylboronic acid and the reaction of a 3-amino-4-bromopyrazole with 4-carbamoylphenylboronic acid. In all of these processes, the Suzuki-Miyaura reaction was fast and relatively efficient using a palladium catalyst under microwave irradiation. The process was incorporated into a rapid 3-step microwave-assisted method for the synthesis of a MK2 inhibitor involving 3-aminopyrazole formation, pyrazole C-4 bromination using N-bromosuccinimide (NBS), and Suzuki-Miyaura cross-coupling of the pyrazolyl bromide with 4-carbamoylphenylboronic acid to give the target 4-arylpyrazole in 35% overall yield, suitable for study in WS cells

Building molecules for the modulation of age-related diseases

Improved understanding of normal human ageing will provide important insights into major risk factors for many age-related diseases. Using the progeroid disease Werner syndrome (WS) as a model for accelerated ageing, this project aimed to investigate how modulation of protein function in the p38α stress-signalling kinase cascade could affect ageing on the cellular level. New rapid routes towards p38α and MK2 inhibitors have been developed to evaluate their use as chemical probes to investigate the mechanisms of cellular ageing. The p38α inhibitor RO3201195 has been synthesized and used as a probe to corroborate previously published evidence that inhibition of the p38α protein kinase reverses the aged morphology of WS fibroblasts. A new route for the synthesis of the MK2 inhibitor PF-3644022 was developed, exploring new methods for building the heterocyclic scaffold through a number of retrosynthetic strategies. Microwave-assisted organic synthesis was used extensively as a tool towards halogenated benzothiophenes and fused quinoline systems, and in rapid Suzuki-Miyaura coupling and Buchwald-Hartwig N-arylation chemistry. This MK2 inhibitor can now be used as a readily accessible probe to further investigate the role of kinases downstream of p38α and the role of MK2 in the premature ageing of WS cells. The final part of the project focussed on the potential of a known p38α inhibitor, BIRB 796, in the development of new chemical probes. BIRB 796 binds allosterically outside of the ATPbinding pocket of p38α MAPK, which drives a conformational change of the kinase into an inactive form. Using rapid synthetic methods towards pyrazole and urea formation, the selectivity elements of the inhibitor were reviewed and a new library of analogues was synthesized. Upon testing against a small panel of kinases, it was found that through small changes the inhibitor potency for p38α could be dramatically altered, and binding affinity for alternative kinases could be selectively enhanced

Microwave-assisted synthesis of 3-aminobenzo[b]thiophene scaffolds for the preparation of kinase inhibitors

Microwave irradiation of 2-halobenzonitriles and methyl thioglycolate in the presence of triethylamine in DMSO at 130 °C provides rapid access to 3-aminobenzo[b]thiophenes in 58–96% yield. This transformation has been applied in the synthesis of the thieno[2,3-b]pyridine core motif of LIMK1 inhibitors, the benzo[4,5]thieno[3,2-e][1,4]diazepin-5(2H)-one scaffold of MK2 inhibitors and a benzo[4,5]thieno[3,2-d]pyrimidin-4-one inhibitor of the PIM kinases

The effect of RO3201195 and a pyrazolyl ketone P38 MAPK inhibitor library on the proliferation of Werner syndrome cells

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The impact of gastrointestinal parasitism on the behaviour and welfare of weaned housed lambs

Gastrointestinal (GI) parasitism is a health and production concern in sheep, yet its impact on animal welfare remains unclear. The impact of subclinical infections is especially ambiguous as GI parasitism often remains undiagnosed until clinical signs such as diarrhoea are evident. This study applied quantitative and qualitative methods to examine the effects of subclinical Teladorsagia circumcincta infection on the behaviour and welfare of 96 Suffolk-cross lambs (24 pens of 4 lambs) weaned at 10 weeks old. The hypothesis that parasitism causes negative affective states was tested. Lambs were divided into three groups at the pen level: ad-lib fed control (AC), restricted-fed control (RC), and ad-lib fed parasitised (AP). Parasitised lambs (AP) were dosed three times weekly with 7000 third stage T. circumcincta larvae (L 3) from 16 weeks of age. Lambs in the RC group were pair fed to match AP feed intake to separate the effects of infection-induced anorexia from the potential direct impacts of infection. From 7 days pre-infection to 23 days post-infection, scan and behaviour samples were taken from video recordings to quantitatively monitor behaviour, and animal-based measures such as faecal soiling score (FSS) were recorded as welfare indicators. Lying, standing, eating, play and social behaviour were monitored. Qualitative behaviour assessment (QBA) was conducted weekly using the AWIN (2015) protocol to gain insight into the lambs’ affective states over the onset of infection. Parasitised lambs were more likely to stand inactive than AC lambs as the infection progressed (P=0.006). They were also less likely to display eating behaviour in the third daily scan sample than RC lambs (P&lt;0.001). Principal Component Analysis of the QBA data revealed that the first dimension (PC1) described arousal levels, the second (PC2) described the valence of the animals’ affective states, and the third (PC3) described fearfulness and aggression levels. Parasitised lambs (est=10.64,SE=0.33) scored higher than RC lambs (est=9.42, SE=0.33) on PC3, the fearfulness dimension (P=0.030). There were no differences between fearfulness scores of AC and AP lambs or RC lambs and treatment group had no significant impact on the distribution of scores on PC1 or PC2. These findings demonstrate that subclinical GI parasitism negatively impacts lamb welfare not only in the health domain but in the behaviour and mental domains as well. This has implications for welfare assessments and early disease detection in lambs. Future research could explore remote monitoring of the indicators of parasitism identified in this study.</p

The impact of gastrointestinal parasitism on the behaviour and welfare of weaned housed lambs

Gastrointestinal (GI) parasitism is a health and production concern in sheep, yet its impact on animal welfare remains unclear. The impact of subclinical infections is especially ambiguous as GI parasitism often remains undiagnosed until clinical signs such as diarrhoea are evident. This study applied quantitative and qualitative methods to examine the effects of subclinical Teladorsagia circumcincta infection on the behaviour and welfare of 96 Suffolk-cross lambs (24 pens of 4 lambs) weaned at 10 weeks old. The hypothesis that parasitism causes negative affective states was tested. Lambs were divided into three groups at the pen level: ad-lib fed control (AC), restricted-fed control (RC), and ad-lib fed parasitised (AP). Parasitised lambs (AP) were dosed three times weekly with 7000 third stage T. circumcincta larvae (L 3) from 16 weeks of age. Lambs in the RC group were pair fed to match AP feed intake to separate the effects of infection-induced anorexia from the potential direct impacts of infection. From 7 days pre-infection to 23 days post-infection, scan and behaviour samples were taken from video recordings to quantitatively monitor behaviour, and animal-based measures such as faecal soiling score (FSS) were recorded as welfare indicators. Lying, standing, eating, play and social behaviour were monitored. Qualitative behaviour assessment (QBA) was conducted weekly using the AWIN (2015) protocol to gain insight into the lambs’ affective states over the onset of infection. Parasitised lambs were more likely to stand inactive than AC lambs as the infection progressed (P=0.006). They were also less likely to display eating behaviour in the third daily scan sample than RC lambs (P&lt;0.001). Principal Component Analysis of the QBA data revealed that the first dimension (PC1) described arousal levels, the second (PC2) described the valence of the animals’ affective states, and the third (PC3) described fearfulness and aggression levels. Parasitised lambs (est=10.64,SE=0.33) scored higher than RC lambs (est=9.42, SE=0.33) on PC3, the fearfulness dimension (P=0.030). There were no differences between fearfulness scores of AC and AP lambs or RC lambs and treatment group had no significant impact on the distribution of scores on PC1 or PC2. These findings demonstrate that subclinical GI parasitism negatively impacts lamb welfare not only in the health domain but in the behaviour and mental domains as well. This has implications for welfare assessments and early disease detection in lambs. Future research could explore remote monitoring of the indicators of parasitism identified in this study.</p

Mapping male circumcision for HIV prevention efforts in sub-Saharan Africa

Background HIV remains the largest cause of disease burden among men and women of reproductive age in sub-Saharan Africa. Voluntary medical male circumcision (VMMC) reduces the risk of female-to-male transmission of HIV by 50–60%. The World Health Organization (WHO) and Joint United Nations Programme on HIV/AIDS (UNAIDS) identified 14 priority countries for VMMC campaigns and set a coverage goal of 80% for men ages 15–49. From 2008 to 2017, over 18 million VMMCs were reported in priority countries. Nonetheless, relatively little is known about local variation in male circumcision (MC) prevalence. Methods We analyzed geo-located MC prevalence data from 109 household surveys using a Bayesian geostatistical modeling framework to estimate adult MC prevalence and the number of circumcised and uncircumcised men aged 15–49 in 38 countries in sub-Saharan Africa at a 5 × 5-km resolution and among first administrative level (typically provinces or states) and second administrative level (typically districts or counties) units. Results We found striking within-country and between-country variation in MC prevalence; most (12 of 14) priority countries had more than a twofold difference between their first administrative level units with the highest and lowest estimated prevalence in 2017. Although estimated national MC prevalence increased in all priority countries with the onset of VMMC campaigns, seven priority countries contained both subnational areas where estimated MC prevalence increased and areas where estimated MC prevalence decreased after the initiation of VMMC campaigns. In 2017, only three priority countries (Ethiopia, Kenya, and Tanzania) were likely to have reached the MC coverage target of 80% at the national level, and no priority country was likely to have reached this goal in all subnational areas. Conclusions Despite MC prevalence increases in all priority countries since the onset of VMMC campaigns in 2008, MC prevalence remains below the 80% coverage target in most subnational areas and is highly variable. These mapped results provide an actionable tool for understanding local needs and informing VMMC interventions for maximum impact in the continued effort towards ending the HIV epidemic in sub-Saharan Africa

Cardiovascular risk factors before and during pregnancy: Does pregnancy unmask or initiate risk?

Objectives: To understand if pregnancy unmasks previously silent cardiovascular (CV) adverse factors, or initiates lasting injury.Methods: Pre-pregnancy and during pregnancy CV risk factors (blood pressure, fasting lipids, and glucose) from 296 women belonging to studies in the International Childhood Cardiovascular Cohort (i3C) Consortium, a group of studies assessing the relationship between child and adolescent CV risk factors and adult outcomes, were used. Correlation coefficients between the pre- and during pregnancy measures were calculated, and the mean difference between the measures was modeled with adjustment for age, body mass index, race, smoking, and study.Results: Measures were strongly correlated at pre- and during-pregnancy visits (p Conclusions: Pre- and during-pregnancy CV risk factors are moderately well correlated. This may indicate that susceptible women enter pregnancy with higher risk rather than pregnancy inducing new vascular or metabolic effects.</p

A Soluble Form of the High Affinity IgE Receptor, Fc-Epsilon-RI, Circulates in Human Serum

Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI), the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum

Diet quality trajectories and cardiovascular phenotypes/metabolic syndrome risk by 11-12 years

Objective To investigate associations between early-life diet trajectories and preclinical cardiovascular phenotypes and metabolic risk by age 12 years. Methods Participants were 1861 children (51% male) from the Longitudinal Study of Australian Children. At five biennial waves from 2-3 to 10-11 years: Every 2 years from 2006 to 2014, diet quality scores were collected from brief 24-h parent/self-reported dietary recalls and then classified using group-based trajectory modeling as 'never healthy' (7%), 'becoming less healthy' (17%), 'moderately healthy' (21%), and 'always healthy' (56%). At 11-12 years: During children's physical health Child Health CheckPoint (2015-2016), we measured cardiovascular functional (resting heart rate, blood pressure, pulse wave velocity, carotid elasticity/distensibility) and structural (carotid intima-media thickness, retinal microvasculature) phenotypes, and metabolic risk score (composite of body mass index z-score, systolic blood pressure, high-density lipoproteins cholesterol, triglycerides, and glucose). Associations were estimated using linear regression models (n = 1100-1800) adjusted for age, sex, and socioeconomic position. Results Compared to 'always healthy', the 'never healthy' trajectory had higher resting heart rate (2.6 bpm, 95% CI 0.4, 4.7) and metabolic risk score (0.23, 95% CI 0.01, 0.45), and lower arterial elasticity (-0.3% per 10 mmHg, 95% CI -0.6, -0.1) and distensibility (-1.2%, 95% CI -1.9, -0.5) (all effect sizes 0.3-0.4). Heart rate, distensibility, and diastolic blood pressure were progressively poorer for less healthy diet trajectories (linear trends p Conclusions Children following the least healthy diet trajectory had poorer functional cardiovascular phenotypes and metabolic syndrome risk, including higher resting heart rate, one of the strongest precursors of all-cause mortality. Structural phenotypes were not associated with diet trajectories, suggesting the window to prevent permanent changes remains open to at least late childhood.</p