3D Printing Applications within Spectrophotometry

Ultraviolet-visible spectroscopy is a tool used throughout the field of chemistry and in chemical labs across the world. Spectrophotometers are a core technology in analytical chemistry, and are used to obtain accurate data on solution concentration. Unfortunately spectrophotometers can be difficult to obtain due to their high cost and low availability outside of a laboratory; this is especially true in either high school or lower grades where buying a spectrophotometer could be considered unreasonable due to price. There are ways to obtain cheaper spectrophotometers, but they can have a high initial cost or low overall quality. This paper will discuss and present a 3D printed spectrophotometer that is inexpensive to build, but displays an overall quality that will allow for accurate measurements of analyte. The design is relatively simple and allows for one time placement of both a blank and analyte cuvette, which makes for a more convenient and time efficient measurement

Genetic and Biochemical Approaches for <em>In Vivo</em> and <em>In Vitro</em> Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins

Structural and dielectric studies of the phase behaviour of the topological ferroelectric La1-xNdxTaO4

We thank the University of St Andrews and EPSRC (via DTG studentships to CALD and JG) for funding,The layered perovskite LaTaO4 has been prepared in its polar orthorhombic polymorphic form at ambient temperature. Although no structural phase transition is observed in the temperature interval 25° C < T < 500 °C, a very large axial thermal contraction effect is seen, which can be ascribed to an anomalous buckling of the perovskite octahedral layer. The non-polar monoclinic polymorph can be stabilised at ambient temperature by Nd-doping. A composition La0.90Nd0.10TaO4 shows a first-order monoclinic-orthorhombic (non-polar to polar) transition in the region 250° C < T < 350 °C. Dielectric responses are observed at both the above structural events but, despite the ‘topological ferroelectric’ nature of orthorhombic LaTaO4, we have not succeeded in obtaining ferroelectric P–E hysteresis behaviour. Structural relationships in the wider family of AnBnX3n+2 layered perovskites are discussed.Publisher PDFPeer reviewe

Characterisation of a novel interaction between the cardiac myosin binding protein-C and the ryanodine receptor/calcium release channel

Muscle excitation-contraction coupling (ECC) is a fundamental physiological process by which electrically-evoked sarcoplasmic reticulum (SR) Ca2+ release triggers cardiomyocyte contraction. Human ryanodine receptor 2 (RyR2) is an ion channel present in the SR membrane of cardiac myocytes, responsible for mediating calcium efflux. Cardiac myosin binding protein-C (cMyBP-C) is a modular protein anchored to the thick filament through its C-terminal region, whereas the N-terminal region of cMyBP-C is thought to regulate myocardial contractility by modifying actin-myosin association. Here, we present several lines of evidence indicating potential RyR2:cMyBP-C interaction, which could provide a novel retrograde regulation of SR Ca2+ release by the sarcomere. Firstly, co-immunoprecipitation (co-IP) experiments indicated that the detected putative association between RyR2 and cMyBP-C can be applicable to large mammalian species as well as to recombinant human proteins heterologously expressed in a mammalian cell line, with RyR2 N-terminus and cMyBP-C C-terminus being responsible for the binding. A 3D co-localisation immunocytochemistry study supported the possibility for the proteins to be readily available for protein-protein interaction, with recombinant human RyR2 and cMyBP-C shown to co-localise in HEK293 cells. Secondly, with the aid of two complementary techniques, namely co-IP and yeast 2-hybrid (Y2H), we showed that RyR2 residues 1-906 comprise the main cMyBP-C site, with residues 1-346 (corresponding to subdomain A and B of RyR2 NTD) and 654-906 (domain SPRY1 and P1) being important determinants for the binding with cMyBP-C C-terminus. Further mapping studies implied that cMyBP-C residues 820-972 and 1061-1274 are involved in the interaction with RyR2 N-terminus, with cMyBP-C Fn domain/s proposed to be directly responsible for the binding. Additionally, parallel experiments with the skeletal muscle RyR1 demonstrated that the corresponding N-terminus region (RyR2 1-906 amino acids) of mammalian isoform RyR1 can also interact with MyBP-C, which could indicate that the detected association can also be relevant in the regulation of ECC in skeletal muscle. Thirdly, we pursued the possible functional significance of RyR2:cMyBP-C interaction. The results from single cell Ca2+ imaging indicated that cMyBP-C interaction with RyR2 may have an inhibitory effect on Ca2+ release channel function, with cMyBP-C binding diminishing RyR2 channel activity. In particular, HEK293 cells co-expressing RyR2 and cMyBP-C had a reduced frequency of spontaneous intracellular calcium oscillations compared to cells expressing only RyR2. Lastly, further co-IP data demonstrated that cMyBP-C:RyR2 binding is not affected by four different RyR2 single point mutation variants (S166C, R176Q, R420Q and L433P) associated with different pathological phenotypes. Thus, while a physical and functional association between RyR2 and cMyBP-C was shown to possibly regulate normal cardiac physiology, it is unlikely to be involved in RyR2-related cardiac disease. In order to unravel the pathological significance of the detected cMyBP-C:RyR2 association, more studies are needed to understand what role cMyBP-C plays in RyR2 channel dysfunction that could potentially affect muscle contraction/relaxation

Future direction of pathogenesis and treatment for rheumatic disorders

After the breakthrough in the treatment of rheumatoid arthritis and numerous related disorders with biological therapies targeting TNFa at the Kennedy Institute in London Millions of patients have tremendously benefitted. However, we cannot cure these diseases yet and have to search for additional therapeutic targets. Since it was shown that synovial fibroblasts (SF) are not only effector cells responding to inflammatory stimuli, but appear endogenously activated and potentially involved into spreading the disease [1], we searched for the epigenetic modifications leading to the activated phenotype of these cells. Epigenetics in its scientific definition “is the study of all heritable and potentially reversible changes in genome function that do not alter the nucleotide sequence within the DNA”, but might be considered in simpler terms as the regulation of gene expression. Epigenetic modifications include: Acetylation

Overexpression of toll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoid arthritis: Toll-like receptor expression in early and longstanding arthritis

OBJECTIVE: To analyze the expression, regulation, and biologic relevance of Toll-like receptors (TLRs) 1-10 in synovial and skin fibroblasts and to determine the expression levels of TLRs 2, 3, and 4 in synovial tissues from patients with early rheumatoid arthritis (RA), longstanding RA, and osteoarthritis (OA). METHODS: Expression of TLRs 1-10 in RA synovial fibroblasts (RASFs), OASFs, and skin fibroblasts was analyzed by real-time polymerase chain reaction (PCR). Fibroblasts were stimulated with tumor necrosis factor alpha, interleukin-1beta (IL-1beta), bacterial lipopeptide, poly(I-C), lipopolysaccharide, and flagellin. Production of IL-6 was determined by enzyme-linked immunosorbent assay and induction of TLRs 2-5, matrix metalloproteinases (MMPs) 3 and 13 messenger RNA by real-time PCR. Expression of TLRs 2-4 in synovial tissues was analyzed by immunohistochemistry. RESULTS: Synovial fibroblasts expressed TLRs 1-6, but not TLRs 7-10. Among the expressed TLRs, TLR-3 and TLR-4 were the most abundant in synovial fibroblasts, and stimulation of synovial fibroblasts with the TLR-3 ligand poly(I-C) led to the most pronounced increase in IL-6, MMP-3, and MMP-13. In contrast, skin fibroblasts did not up-regulate MMP-3 or MMP-13 after stimulation with any of the tested stimuli. In synovial tissues from patients with early RA, TLR-3 and TLR-4 were highly expressed and were comparable to the levels of patients with longstanding RA. These expression levels were elevated as compared with those in OA. CONCLUSION: Our findings of high expression of TLRs, particularly TLRs 3 and 4, at an early stage of RA and the reactivity of synovial fibroblasts in vitro to TLR ligands suggest that TLR signaling pathways resulting in persistent inflammation and joint destruction are activated early in the disease process

Association of functional polymorphisms in CYP19A1 with aromatase inhibitor associated arthralgia in breast cancer survivors

INTRODUCTION: Aromatase inhibitor-associated arthralgia (AIAA) is a common and often debilitating symptom in breast cancer survivors. Since joint symptoms have been related to estrogen deprivation through the menopausal transition, we hypothesized that genetic polymorphisms in CYP19A1, the final enzyme in estrogen synthesis, may be associated with the occurrence of AIAA. METHODS: We performed a cross-sectional study of postmenopausal women with stage 0 to III breast cancer receiving adjuvant aromatase inhibitor (AI) therapy. Patient-reported AIAA was the primary outcome. DNA was genotyped for candidate CYP19A1 polymorphisms. Serum estrogen levels were evaluated by radioimmunoassay. Multivariate analyses were performed to examine associations between AIAA and genetic variants controlling for possible confounders. RESULTS: Among 390 Caucasian participants, 50.8% reported AIAA. Women carrying at least one 8-repeat allele had lower odds of AIAA (adjusted odds ratio (AOR) 0.41, 95% confidence interval (CI) 0.21 to 0.79, P = 0.008) after adjusting for demographic and clinical covariates. Estradiol and estrone were detectable in 47% and 86% of subjects on AIs, respectively. Although these post-AI levels were associated with multiple genotypes, they were not associated with AIAA. In multivariate analyses, women with more recent transition into menopause (less than five years) were significantly more likely to report AIAA than those greater than ten years post-menopause (AOR 3.31, 95% CI 1.72 to 6.39, P < 0.001). CONCLUSIONS: Functional polymorphism in CYP19A1 and time since menopause are associated with patient-reported AIAA, supporting the hypothesis that the host hormonal environment contributes to the pathophysiology of AAIA. Prospective investigation is needed to further delineate relationships between host genetics, changing estrogen levels and AIAA