Generating GBX2 antibodies: A useful tool in determining developmental mechanisms regulated by GBX2 [abstract]

Abstract only availableThe GBX class of homeobox genes is comprised of Gbx1 and Gbx2. Both loss-of-function and gain-of-function studies in mice have shown that Gbx2 is vital for normal anterior hindbrain development of mammalian organisms. To gain more insight into the developmental mechanisms regulated by GBX2, we are generating GBX2 antibodies. To accomplish this, we have subcloned Gbx2 into the pRSET A protein expression vector and transformed the construct into BL21(DE3)pLysS cells. Protein expression was induced by IPTG. The expressed protein was analyzed by SDS-PAGE as well as Western analysis. The purified protein will be used to elicit an immune response in chickens to generate the antibodies against GBX2. Preliminary results from the SDS-PAGE and Western analysis have suggested that the GBX2 protein is being expressed. However, further testing is necessary for confirmation. Currently, we are using Western analysis to specifically target the 6x His tagged GBX2 fusion protein in order to identify the protein for further analysis by mass spectroscopy. Generation of GBX2 antibodies will provide an important tool to enhance our knowledge of how GBX2 functions in development. Having these antibodies will allow for cellular localization. In addition, the antibodies will be used in chromatin immunoprecipitation assays, which will allow for the production of a library that contains genes directly regulated by GBX2. The identification of target genes will provide a way to enable the collection of valuable data that will be useful for more long-term research goals involving the specific signaling and genetic pathways in which this transcription factor is involved

Role of Gbx2 expression in cranial nerve V development

Abstract only availableGbx2 is a member of the Gbx family of homeobox genes that encodes for the transcriptional factors Gbx1 and Gbx2. The amino acid sequence of Gbx2 is highly conserved across multiple species (e.g. mice, zebrafish, chicken, and frogs) with nearly 100% sequence identity between the Gbx2 homeodomains of the aforementioned species. Expression studies have shown that Gbx2 is expressed early during gastrulation in the posterior neural plate (Bouillet et al., 1995) and prospective anterior hindbrain (Niss and Leutz, 1998). The function of Gbx2 in mice and zebrafish has been extensively studied with loss-of-function as well as hypomorphic models in both species. Results of these studies have shown that the Gbx2 homeobox gene is required for normal development of the isthmic organizer at the midbrain hindbrain boundary (MHB) that is responsible for patterning the midbrain and the anterior hindbrain. The hindbrain controls many basic life functions such as breathing and heartbeat. In early vertebrate development, the hindbrain is partitioned into seven or eight distinct segments called rhombomeres (r). Rhombomeres give rise to differentiated brain regions such as the cerebellum, pons, and medulla oblongata. Previous studies had shown that cranial nerve V (nV), derived from r2 and r3, fails to develop normally in Gbx2 hypomorphic mice. The nV motor axons innervate several muscles, including those in the jaw required to suckle and masticate. These Gbx2 hypomorphic mice die immediately after birth presumably due to an inability to nurse on their mother as wild-type mice do. Our preliminary studies have shown that zebrafish Gbx2 also affects nV development. Our research this summer was focused around examining if mouse Gbx2 can rescue the abnormalities in nV caused by injecting morpholino. To accomplish this, we will attempt to rescue the hindbrain phenotype in zebrafish embryos by simultaneously injecting zebrafish morpholino with synthesized mouse Gbx2 mRNA.Life Sciences Undergraduate Research Opportunity Progra

Generating GBX1 anitbodies: A useful tool in determining developmental mechanisms regulated by GBX1 [abstract]

Abstract only availableInactivation of Gbx1 in mice results in a locomotor phenotype specifically affecting hind-limb motion. GBX1 is a DNA-binding transcription factor that regulates the expression of its direct target genes. We are trying to create antibodies, which can be used as a reagent to identify target genes directly regulated by GBX1, in addition, these antibodies will be used for immunohistochemical analysis. The results from these studies will provide insight into the clarification of regulatory mechanisms controlling locomotion in mammals. In order to generate antibodies to GBX1, we will generate protein, which can be used elicit an immune response in chickens. To do this we have cloned the Gbx1 open reading frame (ORF) into the pBluescript II KS (+/-) vector, which allows for DNA sequencing. We then transformed the construct into DH5 cells. After sequencing, the Gbx1 ORF was subcloned into the protein expression vector pRSET B and transformed into BL21 bacterial cells. Protein expression was induced using Isopropyl -D-1-thiogalactopyranoside (IPTG). GBX1 protein will then be analyzed by SDS-PAGE and Western blot analysis. Upon confirmation of protein expression, GBX1 will be purified and used to elicit an immune response in chickens to generate GBX1 antibodies. Once antibodies have been generated, characterization will be carried out by analyzing the antibodies using Western blot analysis and immunohitochemistry

Breaking Tolerance to Double Stranded DNA, Nucleosome, and Other Nuclear Antigens Is Not Required for the Pathogenesis of Lupus Glomerulonephritis

In lupus-prone NZM2328 mice, a locus Cgnz1 on chromosome 1 was linked to chronic glomerulonephritis, severe proteinuria, and early mortality in females. A locus Adnz1 on chromosome 4 was linked to antinuclear antibody (ANA) and anti–double stranded DNA (dsDNA) antibody (Ab) production. In this investigation, two congenic strains, NZM2328.C57L/Jc1 (NZM.C57Lc1) and NZM2328.C57L/Jc4 (NZM.C57Lc4), were generated by replacing the respective genetic intervals containing either Cgnz1 or Adnz1 with those from C57L/J, a nonlupus-prone strain. The NZM.C57Lc1 females had markedly reduced incidence of chronic glomerulonephritis and severe proteinuria. NZM.C57Lc4 females had chronic glomerulonephritis and severe proteinuria without circulating ANA, anti-dsDNA, and antinucleosome Ab. These data confirm the linkage analysis. Unexpectedly, NZM.C57Lc1 females had little anti-dsDNA and related Ab, suggesting the presence of a second locus Adnz2 on chromosome 1. The diseased NZM.C57Lc4 kidneys had immune complexes by immunofluorescence and electron microscopy. The eluates from these kidneys did not contain ANA, anti-dsDNA, and antinucleosome Ab, indicative of the presence of non–anti-dsDNA nephritogenic Ab. Thus, breaking tolerance to dsDNA and chromatin is not required for the pathogenesis of lupus nephritis. These results reaffirm that anti-dsDNA and related Ab production and chronic glomerulonephritis are under independent genetic control. These findings have significant implications in the pathogenesis of systemic lupus erythematosus

Quantifying the Evolution of Vascular Barrier Disruption in Advanced Atherosclerosis with Semipermeant Nanoparticle Contrast Agents

Acute atherothrombotic occlusion in heart attack and stroke implies disruption of the vascular endothelial barrier that exposes a highly procoagulant intimal milieu. However, the evolution, severity, and pathophysiological consequences of vascular barrier damage in atherosclerotic plaque remain unknown, in part because quantifiable methods and experimental models are lacking for its in vivo assessment.To develop quantitative nondestructive methodologies and models for detecting vascular barrier disruption in advanced plaques.Sustained hypercholesterolemia in New Zealand White (NZW) rabbits for >7-14 months engendered endothelial barrier disruption that was evident from massive and rapid passive penetration and intimal trapping of perfluorocarbon-core nanoparticles (PFC-NP: ∼250 nm diameter) after in vivo circulation for as little as 1 hour. Only older plaques (>7 mo), but not younger plaques (<3 mo) demonstrated the marked enhancement of endothelial permeability to these particles. Electron microscopy revealed a complex of subintimal spongiform channels associated with endothelial apoptosis, superficial erosions, and surface-penetrating cholesterol crystals. Fluorine ((19)F) magnetic resonance imaging and spectroscopy (MRI/MRS) enabled absolute quantification (in nanoMolar) of the passive permeation of PFC-NP into the disrupted vascular lesions by sensing the unique spectral signatures from the fluorine core of plaque-bound PFC-NP.The application of semipermeant nanoparticles reveals the presence of profound barrier disruption in later stage plaques and focuses attention on the disrupted endothelium as a potential contributor to plaque vulnerability. The response to sustained high cholesterol levels yields a progressive deterioration of the vascular barrier that can be quantified with fluorine MRI/MRS of passively permeable nanostructures. The possibility of plaque classification based on the metric of endothelial permeability to nanoparticles is suggested

Current Welfare Problems Facing Horses in Great Britain as Identified by Equine Stakeholders

Despite growing concerns about the welfare of horses in Great Britain (GB) there has been little surveillance of the welfare status of the horse population. Consequently we have limited knowledge of the range of welfare problems experienced by horses in GB and the situations in which poor welfare occurs. Thirty-one in-depth interviews were conducted with a cross -section of equine stakeholders, in order to explore their perceptions of the welfare problems faced by horses in GB. Welfare problems relating to health, management and riding and training were identified, including horses being under or over weight, stabling 24 hours a day and the inappropriate use of training aids. The interviewees also discussed broader contexts in which they perceived that welfare was compromised. The most commonly discussed context was where horses are kept in unsuitable environments, for example environments with poor grazing. The racing industry and travellers horses were identified as areas of the industry where horse welfare was particularly vulnerable to compromise. Lack of knowledge and financial constraints were perceived to be the root cause of poor welfare by many interviewees. The findings give insight into the range of welfare problems that may be faced by horses in GB, the contexts in which these may occur and their possible causes. Many of the problems identified by the interviewees have undergone limited scientific investigation pointing to areas where further research is likely to be necessary for welfare improvement. The large number of issues identified suggests that some form of prioritisation may be necessary to target research and resources effectively

Quantitative cardiovascular magnetic resonance for molecular imaging

Cardiovascular magnetic resonance (CMR) molecular imaging aims to identify and map the expression of important biomarkers on a cellular scale utilizing contrast agents that are specifically targeted to the biochemical signatures of disease and are capable of generating sufficient image contrast. In some cases, the contrast agents may be designed to carry a drug payload or to be sensitive to important physiological factors, such as pH, temperature or oxygenation. In this review, examples will be presented that utilize a number of different molecular imaging quantification techniques, including measuring signal changes, calculating the area of contrast enhancement, mapping relaxation time changes or direct detection of contrast agents through multi-nuclear imaging or spectroscopy. The clinical application of CMR molecular imaging could offer far reaching benefits to patient populations, including early detection of therapeutic response, localizing ruptured atherosclerotic plaques, stratifying patients based on biochemical disease markers, tissue-specific drug delivery, confirmation and quantification of end-organ drug uptake, and noninvasive monitoring of disease recurrence. Eventually, such agents may play a leading role in reducing the human burden of cardiovascular disease, by providing early diagnosis, noninvasive monitoring and effective therapy with reduced side effects

Genetic Variants of APOL1 Are Major Determinants of Kidney Failure in People of African Ancestry With HIV

INTRODUCTION: Variants of the APOL1 gene are associated with chronic kidney disease (CKD) in people of African ancestry, although evidence for their impact in people with HIV are sparse. METHODS: We conducted a cross-sectional study investigating the association between APOL1 renal risk alleles and kidney disease in people of African ancestry with HIV in the UK. The primary outcome was end-stage kidney disease (ESKD; estimated glomerular filtration rate [eGFR] of 30 mg/mmol), and biopsy-proven HIV-associated nephropathy (HIVAN). Multivariable logistic regression was used to estimate the associations between APOL1 high-risk genotypes (G1/G1, G1/G2, G2/G2) and kidney disease outcomes. RESULTS: A total of 2864 participants (mean age 48.1 [SD 10.3], 57.3% female) were genotyped, of whom, 354 (12.4%) had APOL1 high-risk genotypes, and 99 (3.5%) had ESKD. After adjusting for demographic, HIV, and renal risk factors, individuals with APOL1 high-risk genotypes were at increased odds of ESKD (odds ratio [OR] 10.58, 95% CI 6.22–17.99), renal impairment (OR 5.50, 95% CI 3.81–7.95), albuminuria (OR 3.34, 95% CI 2.00–5.56), and HIVAN (OR 30.16, 95% CI 12.48–72.88). An estimated 49% of ESKD was attributable to APOL1 high-risk genotypes. CONCLUSION: APOL1 high-risk genotypes were strongly associated with kidney disease in people of African ancestry with HIV and accounted for approximately half of ESKD cases in this cohort