Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons.

The use of genetically encoded 'self-labeling tags' with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.This work was supported by Howard Hughes Medical Institute (https://www.hhmi.org), the Medical Research Council (https://mrc.ukri.org; MRC file reference U105188491) and a European Research Council (https://erc.europa.eu) Consolidator grant (649111) to G.S.X.E.J., and a Royal Society (https://royalsociety.org) Dorothy Hodgkin Fellowship to S.C

Variant rs10911021 that associates with coronary heart disease in type 2 diabetes, is associated with lower concentrations of circulating HDL cholesterol and large HDL particles but not with amino acids.

AIMS: An intergenic locus on chromosome 1 (lead SNP rs10911021) was previously associated with coronary heart disease (CHD) in type 2 diabetes (T2D). Using data from the UCLEB consortium we investigated the relationship between rs10911021 and CHD in T2D, whether rs10911021 was associated with levels of amino acids involved in the γ-glutamyl cycle or any conventional risk factors (CRFs) for CHD in the T2D participants. METHODS: Four UCLEB studies (n = 6531) had rs10911021 imputation, CHD in T2D, CRF and metabolomics data determined using a nuclear magnetic resonance based platform. RESULTS: The expected direction of effect between rs10911021 and CHD in T2D was observed (1377 no CHD/160 CHD; minor allele OR 0.80, 95 % CI 0.60-1.06) although this was not statistically significant (p = 0.13). No association between rs10911021 and CHD was seen in non-T2D participants (11218 no CHD/1274 CHD; minor allele OR 1.00 95 % CIs 0.92-1.10). In T2D participants, while no associations were observed between rs10911021 and the nine amino acids measured, rs10911021 was associated with HDL-cholesterol (p = 0.0005) but the minor "protective" allele was associated with lower levels (-0.034 mmol/l per allele). Focusing more closely on the HDL-cholesterol subclasses measured, we observed that rs10911021 was associated with six large HDL particle measures in T2D (all p < 0.001). No significant associations were seen in non-T2D subjects. CONCLUSIONS: Our findings are consistent with a true association between rs10911021 and CHD in T2D. The protective minor allele was associated with lower HDL-cholesterol and reductions in HDL particle traits. Our results indicate a complex relationship between rs10911021 and CHD in T2D

Mendelian Randomisation study of the influence of eGFR on coronary heart disease.

Impaired kidney function, as measured by reduced estimated glomerular filtration rate (eGFR), has been associated with increased risk of coronary heart disease (CHD) in observational studies, but it is unclear whether this association is causal or the result of confounding or reverse causation. In this study we applied Mendelian randomisation analysis using 17 genetic variants previously associated with eGFR to investigate the causal role of kidney function on CHD. We used 13,145 participants from the UCL-LSHTM-Edinburgh-Bristol (UCLEB) Consortium and 194,427 participants from the Coronary ARtery DIsease Genome-wide Replication and Meta-analysis plus Coronary Artery Disease (CARDIoGRAMplusC4D) consortium. We observed significant association of an unweighted gene score with CHD risk (odds ratio = 0.983 per additional eGFR-increasing allele, 95% CI = 0.970-0.996, p = 0.008). However, using weights calculated from UCLEB, the gene score was not associated with disease risk (p = 0.11). These conflicting results could be explained by a single SNP, rs653178, which was not associated with eGFR in the UCLEB sample, but has known pleiotropic effects that prevent us from drawing a causal conclusion. The observational association between low eGFR and increased CHD risk was not explained by potential confounders, and there was no evidence of reverse causation, therefore leaving the remaining unexplained association as an open question

Identifying low density lipoprotein cholesterol associated variants in the Annexin A2 (ANXA2) gene.

BACKGROUND AND AIMS: Annexin-A2 (AnxA2) is an endogenous inhibitor of proprotein convertase subtilisin/kexin type-9 (PCSK9). The repeat-one (R1) domain of AnxA2 binds to PCSK9, blocking its ability to promote degradation of low-density lipoprotein cholesterol-receptors (LDL-R) and thereby regulate low-density lipoprotein cholesterol (LDL-C) levels. Here we identify variants in ANXA2 influencing LDL-C levels and we determine the molecular mechanisms of their effects. RESULTS: The ANXA2 single nucleotide polymorphism (SNP) genotype-phenotype association was examined using the Second-Northwick-Park Heart Study (NPHSII) (n∼2700) and the UCL-LSHTM-Edinburgh-Bristol (UCLEB) consortium (n∼14,600). The ANXA2-R1 domain coding-SNP rs17845226 (V98L) associated with LDL-C, homozygotes for the minor allele having ≈18.8% higher levels of LDL-C (p = 0.004), and higher risk of coronary heart disease (CHD) (p = 0.04). The SNP is in modest linkage disequilibrium (r(2) > 0.5) with two intergenic SNPs, rs17191344 and rs11633032. Both SNPs showed allele-specific protein binding, and the minor alleles caused significant reduction in reporter gene expression (≈18%, p < 0.001). In the expression quantitative trait loci (eQTL) study, minor allele homozygotes have significantly lower levels of ANXA2-mRNA expression (p = 1.36 × 10(-05)). CONCLUSIONS: Both rs11633032 and rs17191344 SNPs are functional variants, where the minor alleles create repressor-binding protein sites for transcription factors that contribute to reduced ANXA2 gene expression. Lower AnxA2 levels could increase plasma levels of PCSK9 and thus increase LDL-C levels and risk of CHD. This supports, for the first time in humans, previous observations in mouse models that changes in the levels of AnxA2 directly influence plasma LDL-C levels, and thus implicate this protein as a potential therapeutic target for LDL-C lowering

Functional Analysis of the Coronary Heart Disease Risk Locus on Chromosome 21q22.

Background. The coronary heart disease (CHD) risk locus on 21q22 (lead SNP rs9982601) lies within a "gene desert." The aim of this study was to assess if this locus is associated with CHD risk factors and to identify the functional variant(s) and gene(s) involved. Methods. A phenome scan was performed with UCLEB Consortium data. Allele-specific protein binding was studied using electrophoretic mobility shift assays. Dual-reporter luciferase assays were used to assess the impact of genetic variation on expression. Expression quantitative trait analysis was performed with Advanced Study of Aortic Pathology (ASAP) and Genotype-Tissue Expression (GTEx) consortium data. Results. A suggestive association between QT interval and the locus was observed (rs9982601  p = 0.04). One variant at the locus, rs28451064, showed allele-specific protein binding and its minor allele showed 12% higher luciferase expression (p = 4.82 × 10-3) compared to the common allele. The minor allele of rs9982601 was associated with higher expression of the closest upstream genes (SLC5A3 1.30-fold increase p = 3.98 × 10-5; MRPS6 1.15-fold increase p = 9.60 × 10-4) in aortic intima media in ASAP. Both rs9982601 and rs28451064 showed a suggestive association with MRPS6 expression in relevant tissues in the GTEx data. Conclusions. A candidate functional variant, rs28451064, was identified. Future work should focus on identifying the pathway(s) involved

Comparative analysis of genome-wide association studies signals for lipids, diabetes, and coronary heart disease: Cardiovascular Biomarker Genetics Collaboration

To evaluate the associations of emergent genome-wide-association study-derived coronary heart disease (CHD)-associated single nucleotide polymorphisms (SNPs) with established and emerging risk factors, and the association of genome-wide-association study-derived lipid-associated SNPs with other risk factors and CHD events

322. Evaluation of the BioFire® Bone and Joint Infection (BJI) Panel for the Detection of Microorganisms and Antimicrobial Resistance Genes in Synovial Fluid Specimens

Background Bone and Joint Infections (BJIs) present with non-specific symptoms that may include pain, swelling, and fever and are associated with high morbidity and significant risk of mortality. BJIs can be caused by a variety of bacteria and fungi, including anaerobes and microorganisms that can be challenging to culture or identify by traditional microbiological methods. Clinicians primarily rely on culture to identify the pathogen(s) responsible for infection. The BioFire® Bone and Joint Infection (BJI) Panel (BioFire Diagnostics, Salt Lake City, UT) is designed to detect 15 gram-positive bacteria (including seven anaerobes), 14 gram-negative bacteria (including one anaerobe), two yeast, and eight antimicrobial resistance (AMR) genes from synovial fluid specimens in about an hour. The objective of this study was to evaluate the performance of an Investigational Use Only (IUO) version of the BioFire BJI Panel compared to various reference methods. Methods Remnant synovial fluid specimens, which were collected for routine clinical care at 13 study sites in the US and Europe, underwent testing using an IUO version of the BioFire BJI Panel. Performance of this test was determined by comparison to Standard of Care (SoC) consisting of bacterial culture performed at each study site according to their routine procedures. Results A total of 1544 synovial fluid specimens were collected and tested with the BioFire BJI Panel. The majority of specimens were from knee joints (77.9%) and arthrocentesis (79.4%) was the most common collection method. Compared to SoC culture, overall sensitivity was 90.2% and specificity was 99.8%. The BioFire BJI Panel yielded a total of 268 Detected results, whereas SoC yielded a total of 215 positive results for on-panel analytes. Conclusion The BioFire BJI Panel is a sensitive, specific, and robust test for rapid detection of a wide range of analytes in synovial fluid specimens. The number of microorganisms and resistance genes included in the BioFire BJI Panel, together with a reduced time-to-result and increased diagnostic yield compared to culture, is expected to aid in the timely diagnosis and appropriate management of BJIs

Monoamine content in operationally matched 6-OHDA lesioned rats at >70% TH protein loss.

<p>Representation of relative impact of our 6-OHDA lesion protocol on monoamine tissue content (expressed as ng monoamine per mg protein) in striatum with at least 70% confirmed loss of dopamine (top panel). As shown in the middle panel, there was no loss of NE in the 6-OHDA lesion method employed, but there was a trend toward a decrease in 5-HT (bottom panel). Statistics: DA, ***<i>p</i><0.0001, t = 10.87. <i>n</i> = 7 paired observations for all monoamines.</p

Impact of L-DOPA on monoamine uptake A. Dopamine uptake in lesioned striatum in presence of L-DOPA.

<p>1 µM L-DOPA was added in striatal synaptosomes prepared from at least 70% lesioned striatum and from the operationally-matched contralateral control. After 5 min preincubation period, 500 nM [7-, 8- ³H-DA] was added and uptake was determined for 2 min. In the lesioned striatal synaptosomes, L-DOPA was significantly more (77% above inhibition in control) effective to inhibit DA uptake. Statistics: *<i>p</i><0.05, t = 3.31, two-tailed Student’s paired t-test, <i>n = </i>5 paired observations. <b>B.</b> Impact of L-DOPA on DA versus NE uptake. 1 µM L-DOPA was added to naïve (unlesioned) striatal synaptosomes and after 5 min preincubation, either 500 nM ³H-DA or 250 nM [³H] NE was added and uptake was determined for 2 min. Statistics (*<i>p</i><0.001, t = 12.75, unpaired two-tailed Student’s t-test, <i>n</i> = 4 for NE, 5 for DA).</p

Norepinephrine Transporter (NET) striatal protein expression and function in 6-OHDA lesioned rats at >70% TH protein loss. A

<p>. NET protein expression. Quantification of NET showed that NET was significantly increased in lesioned striatum compared to its matched non-lesioned control. Statsitics: NET, *<i>p</i><.05, t = 3.251, n = 4 paired observations. <b>B</b>. Representative western blot of NET expression. Relative expression of NET (∼80 kDa band) between contralateral, control, striatum (<b>C</b>) and 6-OHDA-lesioned striatum (<b>L</b>). <b>C</b>. Increased NET function in 6-OHDA lesioned striatum. Desipramine (DMI)-mediated inhibition of [³H] NE uptake between contralateral (control) and lesioned striatum (mean TH loss = 66%). Statistics <i>p</i><0.05, t = 3.127, <i>n</i> = 3 observations.</p