Iqbal, S, Parker, LM, Everest-Dass, AV, Moh, ESX, Sayyadi, N, Hutchinson, MR & Packer, NH 2020, 'Lipopolysaccharide and Morphine-3-Glucuronide-Induced Immune Signalling Increases the Expression of Polysialic Acid in PC12 Cells.', Molecular neurobiology, vol. 57, no. 2, pp. 964-975.View/Download from: Publisher's site
Polysialic acid (polySia), a long homopolymer of 2,8-linked sialic acids, is abundant in the embryonic brain and is restricted largely in adult brain to regions that exhibit neurogenesis and structural plasticity. In the central nervous system (CNS), polySia is highly important for cell-cell interactions, differentiation, migration and cytokine responses, which are critical neuronal functions regulating intercellular interactions that underlie immune signalling in the CNS. In recent reports, a metabolite of morphine, morphine-3-glucuronide (M3G), has been shown to cause immune signalling in the CNS. In this study, we compared the effects of neurite growth factor (NGF), lipopolysaccharide (LPS) and M3G exposure on the expression of polySia in PC12 cells using immunocytochemistry and Western blot analysis. PolySia was also extracted from stimulated cell proteins by endo-neuraminidase digestion and quantitated using fluorescent labelling followed by HPLC analysis. PolySia expression was significantly increased following NGF, M3G or LPS stimulation when compared with unstimulated cells or cells exposed to the TLR4 antagonist LPS-RS. Additionally, we analyzed the effects of test agent exposure on cell migration and the oxidative stress response of these cells in the presence and absence of polySia expression on their cell surface. We observed an increase in oxidative stress in cells without polySia as well as following M3G or LPS stimulation. Our study provides evidence that polySia expression in neuronal-like PC12 cells is influenced by M3G and LPS exposure alike, suggestive of a role of TLR4 in triggering these events.
Wang, Y, Sayyadi, N, Zheng, X, Woods, TA, Leif, RC, Shi, B, Graves, SW, Piper, JA & Lu, Y 2020, 'Time-resolved microfluidic flow cytometer for decoding luminescence lifetimes in the microsecond region.', Lab on a Chip: miniaturisation for chemistry, physics, biology, materials science and bioengineering.View/Download from: Publisher's site
Time-resolved luminescence detection using long-lived probes with lifetimes in the microsecond region have shown great potential in ultrasensitive and multiplexed bioanalysis. In flow cytometry, however, the long lifetime poses a significant challenge to measure wherein the detection window is often too short to determine the decay characteristics. Here we report a time-resolved microfluidic flow cytometer (tr-mFCM) incorporating an acoustic-focusing chip, which allows slowing down of the flow while providing the same detection conditions for every target, achieving accurate lifetime measurement free of autofluorescence interference. Through configuration of the flow velocity and detection aperture with respect to the time-gating sequence, a multi-cycle luminescence decay profile is captured for every event under maximum excitation and detection efficiency. A custom fitting algorithm is then developed to resolve europium-stained polymer microspheres as well as leukemia cells against abundant fluorescent particles, achieving counting efficiency approaching 100% and lifetime CVs (coefficient of variation) around 2-6%. We further demonstrate lifetime-multiplexed detection of prostate and bladder cancer cells stained with different europium probes. Our acoustic-focusing tr-mFCM offers a practical technique for rapid screening of biofluidic samples containing multiple cell types, especially in resource-limited environments such as regional and/or underdeveloped areas as well as for point-of-care applications.
Rzhevskiy, AS, Razavi Bazaz, S, Ding, L, Kapitannikova, A, Sayyadi, N, Campbell, D, Walsh, B, Gillatt, D, Ebrahimi Warkiani, M & Zvyagin, AV 2020, 'Rapid and Label-Free Isolation of Tumour Cells from the Urine of Patients with Localised Prostate Cancer Using Inertial Microfluidics.', Cancers, vol. 12, no. 1.View/Download from: Publisher's site
During the last decade, isolation of circulating tumour cells via blood liquid biopsy of prostate cancer (PCa) has attracted significant attention as an alternative, or substitute, to conventional diagnostic tests. However, it was previously determined that localised forms of PCa shed a small number of cancer cells into the bloodstream, and a large volume of blood is required just for a single test, which is impractical. To address this issue, urine has been used as an alternative to blood for liquid biopsy as a truly non-invasive, patient-friendly test. To this end, we developed a spiral microfluidic chip capable of isolating PCa cells from the urine of PCa patients. Potential clinical utility of the chip was demonstrated using anti-Glypican-1 (GPC-1) antibody as a model of the primary antibody in immunofluorescent assay for identification and detection of the collected tumour cells. The microchannel device was first evaluated using DU-145 cells in a diluted Dulbecco's phosphate-buffered saline sample, where it demonstrated >85 (±6) % efficiency. The microchannel proved to be functional in at least 79% of cases for capturing GPC1+ putative tumour cells from the urine of patients with localised PCa. More importantly, a correlation was found between the amount of the captured GPC1+ cells and crucial diagnostic and prognostic parameter of localised PCa-Gleason score. Thus, the technique demonstrated promise for further assessment of its diagnostic value in PCa detection, diagnosis, and prognosis.
Moh, ESX, Sayyadi, N & Packer, NH 2019, 'Chemoenzymatic glycan labelling as a platform for site-specific IgM-antibody drug conjugates.', Analytical biochemistry, vol. 584.View/Download from: Publisher's site
Immunoglobulin M (IgM) type antibodies play a significant role in complement activation, cellular debris clearance and cell quality control, and have the potential to be used as a therapeutic or targeting/delivery antibody. However, this potential has not been explored thoroughly due to its high molecular weight, polymeric structure and large number of glycosylation sites. Site-specific antibody-drug-conjugates (ADC) are considered the next generation protein biotherapeutic drugs and currently all, in clinical trials and approved, are of the IgG isotype. As existing methods for the development and characterization of IgG-ADCs are not compatible with IgM-ADC, we describe a platform methodology suitable for site specific IgM-ADC using a chemoenzymatic method targeting the glycans on the IgM. Azide functionalized sialic acids were incorporated onto IgM glycans using sialyltransferase for biocompatible conjugation using "click" chemistry. The number of azide groups incorporated onto the IgM glycans were characterized by mass spectrometry of the enzymatically released glycans and glycopeptides. Quantitation of the azide incorporation showed an azide antibody ratio of 8 (glycan data) and 6-10 (glycopeptide data) which translates to a high drug antibody ratio based on IgG-ADC standards. This platform methodology can be readily adapted for any human IgM produced in a mammalian cell expression system.
Parker, LM, Sayyadi, N, Staikopoulos, V, Shrestha, A, Hutchinson, MR & Packer, NH 2019, 'Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization.', Journal of neuroinflammation, vol. 16, no. 1.View/Download from: Publisher's site
BACKGROUND:Neurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases. METHODS:In this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples. RESULTS:Significant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microgl...
Sayyadi, N, Connally, RE, Lawson, TS, Yuan, J, Packer, NH & Piper, JA 2019, 'Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic Staphylococcus aureus.', Molecules (Basel, Switzerland), vol. 24, no. 11.View/Download from: Publisher's site
We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, Staphylococcus aureus (S. aureus) was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific S. aureus was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to S. aureus via in situ hybridisation to differentiate S. aureus from a closely related but less pathogenic Staphylococcus species (S. epidermidis). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the S. aureus. After excitation (365 nm, Chelate λmax = 335 nm), the long-lived (Eu3+) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological samples. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained S. aureus with mean signal intensity 3.5 times higher than the threshold level of signal from S. epidermidis (with SNR 8 times higher). A positive control probe (EUB338-BHHTEGST-Eu3+) has mean signal intensity for S. aureus and S. epidermidis equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has gr...
Cordina, NM, Sayyadi, N, Parker, LM, Everest-Dass, A, Brown, LJ & Packer, NH 2018, 'Reduced background autofluorescence for cell imaging using nanodiamonds and lanthanide chelates', SCIENTIFIC REPORTS, vol. 8.View/Download from: Publisher's site
Ma, K, Zhang, F, Sayyadi, N, Chen, W, Anwer, AG, Care, A, Xu, B, Tian, W, Goldys, EM & Liu, G 2018, '"Turn-on" Fluorescent Aptasensor Based on AIEgen Labeling for the Localization of IFN-gamma in Live Cells', ACS SENSORS, vol. 3, no. 2, pp. 320-326.View/Download from: Publisher's site
Sayyadi, N, Care, A, Connally, RE, Try, AC, Bergquist, PL & Sunna, A 2016, 'A Novel Universal Detection Agent for Time-Gated Luminescence Bioimaging', SCIENTIFIC REPORTS, vol. 6.View/Download from: Publisher's site
Sayyadi, N, Connally, RE & Try, A 2016, 'A novel biocompatible europium ligand for sensitive time-gated immunodetection', CHEMICAL COMMUNICATIONS, vol. 52, no. 6, pp. 1154-1157.View/Download from: Publisher's site
Sayyadi, N, Justiniano, I, Connally, RE, Zhang, R, Shi, B, Kautto, L, Everest-Dass, AV, Yuan, J, Walsh, BJ, Jin, D, Willows, RD, Piper, JA & Packer, NH 2016, 'Sensitive Time-Gated Immunoluminescence Detection of Prostate Cancer Cells Using a TEGylated Europium Ligand', ANALYTICAL CHEMISTRY, vol. 88, no. 19, pp. 9564-9571.View/Download from: Publisher's site
Shi, Y, Shi, B, Dass, AVE, Lu, Y, Sayyadi, N, Kautto, L, Willows, RD, Chung, R, Piper, J, Nevalainen, H, Walsh, B, Jin, D & Packer, NH 2016, 'Stable Upconversion Nanohybrid Particles for Specific Prostate Cancer Cell Immunodetection.', Scientific Reports, vol. 6, pp. 1-11.View/Download from: Publisher's site
Prostate cancer is one of the male killing diseases and early detection of prostate cancer is the key for better treatment and lower cost. However, the number of prostate cancer cells is low at the early stage, so it is very challenging to detect. In this study, we successfully designed and developed upconversion immune-nanohybrids (UINBs) with sustainable stability in a physiological environment, stable optical properties and highly specific targeting capability for early-stage prostate cancer cell detection. The developed UINBs were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and luminescence spectroscopy. The targeting function of the biotinylated antibody nanohybrids were confirmed by immunofluorescence assay and western blot analysis. The UINB system is able to specifically detect prostate cancer cells with stable and background-free luminescent signals for highly sensitive prostate cancer cell detection. This work demonstrates a versatile strategy to develop UCNPs based sustainably stable UINBs for sensitive diseased cell detection.
Jia, H, Gao, X, Shi, Y, Sayyadi, N, Zhang, Z, Zhao, Q, Meng, Q & Zhang, R 2015, 'Fluorescence detection of Fe3+ ions in aqueous solution and living cells based on a high selectivity and sensitivity chemosensor', SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY, vol. 149, pp. 674-681.View/Download from: Publisher's site
Panahi, Y, Izadi, M, Sayyadi, N, Rezaee, R, Jonaidi-Jafari, N, Beiraghdar, F, Zamani, A & Sahebkar, A 2015, 'Comparative trial of Aloe vera/olive oil combination cream versus phenytoin cream in the treatment of chronic wounds', JOURNAL OF WOUND CARE, vol. 24, no. 10, pp. 459-465.View/Download from: Publisher's site
Sayyadi, N, Taleski, D, Leesch, S & Jolliffe, KA 2014, 'Investigating the scope of pseudoproline assisted peptide cyclization', TETRAHEDRON, vol. 70, no. 42, pp. 7700-7706.View/Download from: Publisher's site
Fairweather, KA, Sayyadi, N, Luck, IJ, Clegg, JK & Jolliffe, KA 2010, 'Synthesis of All-L Cyclic Tetrapeptides Using Pseudoprolines as Removable Turn Inducers', ORGANIC LETTERS, vol. 12, no. 14, pp. 3136-3139.View/Download from: Publisher's site
Parker, LM, Staikopoulos, V, Cordina, NM, Sayyadi, N, Hutchinson, MR & Packer, NH 2016, 'Fluorescent nanodiamond and lanthanide labelled in situ hybridization for the identification of RNA transcripts in fixed and CLARITY-cleared central nervous system tissues(Conference Presentation)', Clinical and Translational Neurophotonics; Neural Imaging and Sensing; and Optogenetics and Optical Manipulation, Neural Imaging and Sensing, SPIE.View/Download from: Publisher's site
Clegg, JK, Cochrane, JR, Sayyadi, N, Skropeta, D, Turner, P & Jolliffe, KA 2008, 'Solid-State and Solution-Phase Conformations of Pseudoproline-Containing Dipeptides', AUSTRALIAN JOURNAL OF CHEMISTRY, 23rd RACI Organic Division Conference 2008, CSIRO PUBLISHING, Hobart, AUSTRALIA, pp. 711-719.View/Download from: Publisher's site
Sayyadi, N & Jolliffe, KA 2007, 'ORGN 844-Exploring the scope of pseudoproline-assisted cyclization', ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, AMER CHEMICAL SOC.