Biochemistry and Pharmacology - Research Publications

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    FRET-enhanced photoluminescence of perylene diimides by combining molecular aggregation and insulation
    Zhang, B ; Lyskov, I ; Wilson, LJ ; Sabatini, RP ; Manian, A ; Soleimaninejad, H ; White, JM ; Smith, TA ; Lakhwani, G ; Jones, DJ ; Ghiggino, KP ; Russo, SP ; Wong, WWH (Royal Society of Chemistry, 2020-07-14)
    The photoluminescence quantum yield (ϕPL) of perylene diimide derivatives (PDIs) is often limited by aggregation caused quenching (ACQ) at high concentration or in the neat solid-state. Energy transfer in high dye concentration systems is also a key factor in determining ϕPL as a result of energy funneling to trap sites in the sample. By tuning the substituents, we present two classes of PDIs with aggregation and insulation of the PDI core. By combining these fluorophores in a polymer film, we demonstrate highly emissive samples (85% ϕPL) at high concentration (140 mM or 20% w/w). Experimental and theoretical studies provide insight into why such a combination is necessary to achieve high ϕPL. While insulated fluorophores maintain respectable ϕPL at high concentration, an improved ϕPL can be achieved in the presence of appropriately oriented fluorophore aggregates as emissive traps. The theoretical calculations show that the relative orientation of aggregated monomers can result in energetic separation of localized states from the charge-transfer and bi-excitonic states thereby enabling high ϕPL.
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    Next Generation Cell Culture Tools Featuring Micro‐ and Nanotopographies for Biological Screening (Adv. Funct. Mater. 3/2022)
    Carthew, J ; Abdelmaksoud, HH ; Cowley, KJ ; Hodgson‐Garms, M ; Elnathan, R ; Spatz, JP ; Brugger, J ; Thissen, H ; Simpson, KJ ; Voelcker, NH ; Frith, JE ; Cadarso, VJ (Wiley, 2022-01)
    In article number 2100881, Nicolas H. Voelcker, Jessica E. Frith, Victor J. Cadarso, and co-workers demonstrate a novel approach to imprint micro and nanoscaled topographical features into conventional cell cultureware, facilitating its compatibility with standard biological techniques. This enables high-throughput screening to integrate the effects of surface topographies into unique cell specific responses and fate determination.
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    Extracellular vesicular lipids as biomarkers for the diagnosis of Alzheimer’s disease
    Su, H ; Rustam, YH ; Masters, CL ; Makalic, E ; McLean, C ; Hill, AF ; Barnham, KJ ; Reid, GE ; Vella, LJ (Wiley, 2021-12-31)
    An increasing number of studies have revealed that dysregulated lipid homeostasis is associated with the pathological processes that lead to Alzheimer’s disease (AD). If changes in key lipid species could be detected in the periphery, it would advance our understanding of the disease and facilitate biomarker discovery. Global lipidomic profiling of sera/blood however has proved challenging with limited disease or tissue specificity. Small extracellular vesicles (EV) in the central nervous system, can pass the blood-brain barrier and enter the periphery, carrying a subset of lipids that could reflect lipid homeostasis in brain. This makes EVs uniquely suited for peripheral biomarker exploration.
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    AMPK CA(R)Sts a new light on amino acid sensing
    Brown, KK (WILEY, 2021-11-02)
    AMP-activated protein kinase (AMPK) is recognized as a critical regulator of cellular energy metabolism impacted by AMP/ATP and ADP/ATP ratios, or glucose- and fatty acid-derived metabolites. However, its ability to sense alterations in amino acid levels is poorly understood. Recent work by Yuan et al (2021) identifies a novel mechanism of AMPK regulation responsive to changes in availability of the sulfur-containing amino acid cysteine.
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    Complementation in trans of Porphyromonas gingivalis Lipopolysaccharide Biosynthetic Mutants Demonstrates Lipopolysaccharide Exchange
    Glew, MD ; Gorasia, DG ; McMillan, PJ ; Butler, CA ; Veith, PD ; Reynolds, EC ; Comstock, LE (American Society for Microbiology, 2021-04-21)
    Porphyromonas gingivalis, a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface, enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to anionic lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled in situ for correct secretion and A-LPS modification of cargo proteins, combinations of nonpigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Reacquisition of pigmentation occurred only between an A-LPS mutant and a T9SS mutant, which coincided with A-LPS modification of cargo proteins detected by Western blotting and coimmunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis, leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two-strain crosses involving only the five T9SS OM component mutants produced black pigmentation, implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation, and hence, a more ordered temporal in situ assembly of T9SS components may be required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host. IMPORTANCE: Porphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions.
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    Goat milk skin products may cause the development of goat milk allergy
    De Luca, JF ; Mackay, GA ; Chatelier, JW ; Chan, SS-Y ; Zhang, SS ; Godsell, J ; Spriggs, K ; Slade, C ; Douglass, JA (WILEY, 2022-05)
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    Familial ALS-associated SFPQ variants promote the formation of SFPQ cytoplasmic aggregates in primary neurons
    Widagdo, J ; Udagedara, S ; Bhembre, N ; Tan, JZA ; Neureiter, L ; Huang, J ; Anggono, V ; Lee, M (ROYAL SOC, 2022-09-28)
    Splicing factor proline- and glutamine-rich (SFPQ) is a nuclear RNA-binding protein that is involved in a wide range of physiological processes including neuronal development and homeostasis. However, the mislocalization and cytoplasmic aggregation of SFPQ are associated with the pathophysiology of amyotrophic lateral sclerosis (ALS). We have previously reported that zinc mediates SFPQ polymerization and promotes the formation of cytoplasmic aggregates in neurons. Here we characterize two familial ALS (fALS)-associated SFPQ variants, which cause amino acid substitutions in the proximity of the SFPQ zinc-coordinating centre (N533H and L534I). Both mutants display increased zinc-binding affinities, which can be explained by the presence of a second zinc-binding site revealed by the 1.83 Å crystal structure of the human SFPQ L534I mutant. Overexpression of these fALS-associated mutants significantly increases the number of SFPQ cytoplasmic aggregates in primary neurons. Although they do not affect the density of dendritic spines, the presence of SFPQ cytoplasmic aggregates causes a marked reduction in the levels of the GluA1, but not the GluA2 subunit of AMPA-type glutamate receptors on the neuronal surface. Taken together, our data demonstrate that fALS-associated mutations enhance the propensity of SFPQ to bind zinc and form aggregates, leading to the dysregulation of AMPA receptor subunit composition, which may contribute to neuronal dysfunction in ALS.
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    Paraspeckle subnuclear bodies depend on dynamic heterodimerisation of DBHS RNA-binding proteins via their structured domains
    Lee, PW ; Marshall, AC ; Knott, GJ ; Kobelke, S ; Martelotto, L ; Cho, E ; McMillan, PJ ; Lee, M ; Bond, CS ; Fox, AH (ELSEVIER, 2022-11)
    RNA-binding proteins of the DBHS (Drosophila Behavior Human Splicing) family, NONO, SFPQ, and PSPC1 have numerous roles in genome stability and transcriptional and posttranscriptional regulation. Critical to DBHS activity is their recruitment to distinct subnuclear locations, for example, paraspeckle condensates, where DBHS proteins bind to the long noncoding RNA NEAT1 in the first essential step in paraspeckle formation. To carry out their diverse roles, DBHS proteins form homodimers and heterodimers, but how this dimerization influences DBHS localization and function is unknown. Here, we present an inducible GFP-NONO stable cell line and use it for live-cell 3D-structured illumination microscopy, revealing paraspeckles with dynamic, twisted elongated structures. Using siRNA knockdowns, we show these labeled paraspeckles consist of GFP-NONO/endogenous SFPQ dimers and that GFP-NONO localization to paraspeckles depends on endogenous SFPQ. Using purified proteins, we confirm that partner swapping between NONO and SFPQ occurs readily in vitro. Crystallographic analysis of the NONO-SFPQ heterodimer reveals conformational differences to the other DBHS dimer structures, which may contribute to partner preference, RNA specificity, and subnuclear localization. Thus overall, our study suggests heterodimer partner availability is crucial for NONO subnuclear distribution and helps explain the complexity of both DBHS protein and paraspeckle dynamics through imaging and structural approaches.
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    Making microbial genomics work for clinical and public health microbiology
    Azarian, T ; Sherry, NL ; Baker, K ; Holt, KE ; Okeke, IN (MICROBIOLOGY SOC, 2022-09)
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    Pulmonary function testing during SARS-CoV-2: An ANZSRS/TSANZ position statement
    Borg, BM ; Osadnik, C ; Adam, K ; Chapman, DG ; Farrow, CE ; Glavas, V ; Hancock, K ; Lanteri, CJ ; Morris, EG ; Romeo, N ; Schneider-Futschik, EK ; Selvadurai, H (WILEY, 2022-09)
    The Thoracic Society of Australia and New Zealand (TSANZ) and the Australian and New Zealand Society of Respiratory Science (ANZSRS) commissioned a joint position paper on pulmonary function testing during coronavirus disease 2019 (COVID-19) in July 2021. A working group was formed via an expression of interest to members of both organizations and commenced work in September 2021. A rapid review of the literature was undertaken, with a 'best evidence synthesis' approach taken to answer the research questions formed. This allowed the working group to accept findings of prior relevant reviews or societal document where appropriate. The advice provided is for providers of pulmonary function tests across all settings. The advice is intended to supplement local infection prevention and state, territory or national directives. The working group's key messages reflect a precautionary approach to protect the safety of both healthcare workers (HCWs) and patients in a rapidly changing environment. The decision on strategies employed may vary depending on local transmission and practice environment. The advice is likely to require review as evidence grows and the COVID-19 pandemic evolves. While this position statement was contextualized specifically to the COVID-19 pandemic, the working group strongly advocates that any changes to clinical/laboratory practice, made in the interest of optimizing the safety and well-being of HCWs and patients involved in pulmonary function testing, are carefully considered in light of their potential for ongoing use to reduce transmission of other droplet and/or aerosol borne diseases.