Publications by authors named "Timothy E Adams"

55 Publications

Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins.

Sci Rep 2021 Mar 4;11(1):5247. Epub 2021 Mar 4.

Biomedical Manufacturing, Commonwealth Scientific and Industrial Research Organisation, 343 Royal Parade, Parkville, Victoria, 3052, Australia.

Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.
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http://dx.doi.org/10.1038/s41598-021-84551-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933286PMC
March 2021

Molecular characterisation of ILRUN, a novel inhibitor of proinflammatory and antimicrobial cytokines.

Heliyon 2020 Jun 3;6(6):e04115. Epub 2020 Jun 3.

CSIRO Health & Biosecurity, Australian Centre for Disease Preparedness, Geelong, Victoria, 3220, Australia.

Regulation of type-I interferon (IFN) production is essential to the balance between antimicrobial defence and autoimmune disorders. The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains, previously C6orf106) was recently characterised as an inhibitor of antiviral and proinflammatory cytokine (interferon-alpha/beta and tumor necrosis factor alpha) transcription. Currently there is a paucity of information about the molecular characteristics of ILRUN, despite it being associated with several diseases including virus infection, coronary artery disease, obesity and cancer. Here, we characterise ILRUN as a highly phylogenetically conserved protein containing UBA-like and a NBR1-like domains that are both essential for inhibition of type-I interferon and tumor necrosis factor alpha) transcription in human cells. We also solved the crystal structure of the NBR1-like domain, providing insights into its potential role in ILRUN function. This study provides critical information for future investigations into the role of ILRUN in health and disease.
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http://dx.doi.org/10.1016/j.heliyon.2020.e04115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270589PMC
June 2020

Glycoengineered hepatitis B virus-like particles with enhanced immunogenicity.

Vaccine 2020 05 10;38(22):3892-3901. Epub 2020 Apr 10.

Royal Melbourne Institute of Technology (RMIT) University, School of Science, Melbourne, Victoria 3001, Australia; Victorian Infectious Diseases Reference Laboratory (VIDRL), Melbourne Health, The Peter Doherty Institute, Melbourne, Victoria 3000, Australia. Electronic address:

Virus-like particles (VLP) represent biological platforms for the development of novel products such as vaccines and delivery platforms for foreign antigenic sequences. VLPs composed of the small surface antigen (HBsAgS) derived from the hepatitis B virus (HBV) are the immunogenic components of a licensed, preventative vaccine, which contains aluminum hydroxide as adjuvant. Herein, we report that glycoengineering of N-glycosylated HBsAgS to generate hyper-glycosylated VLPs display an enhanced immunogenicity relative to the wild type (WT) HBsAgS VLPs when expressed in FreeStyle HEK 293F cells. Comparative mass spectrometry-based N-glycan profiling, gel electrophoresis, and immunoassays demonstrated that WT and hyper-glycosylated HBsAgS VLPs contain the same type and distribution of N-glycan structures, but the latter shows a higher glycan abundance per protein mass. The antigenic integrity of the modified VLPs was also shown to be retained. To assess whether hyper-glycosylated VLPs induce an enhanced immune response in the presence of the adjuvant aluminum hydroxide, the anti-HBV surface antigen (anti-HBsAgS) antibody response was monitored in BALB/c mice, subcutaneously injected with different VLP derivatives. In the absence and presence of adjuvant, hyper-glycosylated VLPs showed an enhanced immunogenicity compared to WT VLPs. The ability of hyper-glycosylated VLPs to promote potent anti-HBsAgS immune responses compared to VLPs with a native N-glycan level as well as non-glycosylated, yeast-derived HBsAgS VLPs opens exciting avenues for generating more efficacious VLP-based vaccines against hepatitis B and improved HBsAgS VLP carrier platforms using glycoengineering.
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http://dx.doi.org/10.1016/j.vaccine.2020.03.007DOI Listing
May 2020

Dual Site-Specific Labeling of an Antibody Fragment through Sortase A and π-Clamp Conjugation.

Bioconjug Chem 2019 10 3;30(10):2539-2543. Epub 2019 Oct 3.

CSIRO Manufacturing , Parkville , Victoria 3052 , Australia.

The functionalization of proteins with different cargo molecules is highly desirable for a broad range of applications. However, the reproducible production of defined conjugates with multiple functionalities is a significant challenge. Herein, we report the dual site-specific labeling of an antibody fragment, utilizing the orthogonal Sortase A and π-clamp conjugation methods, and demonstrate that binding of the antibody fragment to its target receptor is retained after dual labeling.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00639DOI Listing
October 2019

Treatment of type 2 diabetes with the designer cytokine IC7Fc.

Nature 2019 10 25;574(7776):63-68. Epub 2019 Sep 25.

The Garvan Institute of Medical Research, Sydney, New South Wales, Australia.

The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.
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http://dx.doi.org/10.1038/s41586-019-1601-9DOI Listing
October 2019

Specific Sialoforms Required for the Immune Suppressive Activity of Human Soluble CD52.

Front Immunol 2019 27;10:1967. Epub 2019 Aug 27.

Department of Molecular Sciences, Macquarie University, Sydney, NSW, Australia.

Human CD52 is a small glycopeptide (12 amino acid residues) with one linked glycosylation site at asparagine 3 (Asn3) and several potential glycosylation serine/threonine sites. Soluble CD52 is released from the surface of activated T cells and mediates immune suppression via its glycan moiety. In suppressing activated T cells, it first sequesters the pro-inflammatory high mobility group Box 1 (HMGB1) protein, which facilitates its binding to the inhibitory sialic acid-binding immunoglobulin-like lectin-10 (Siglec-10) receptor. We aimed to identify the features of CD52 glycan that underlie its bioactivity. Analysis of native CD52 purified from human spleen revealed extensive heterogeneity in glycosylation and multi-antennary sialylated glycans with abundant polyLacNAc extensions, together with mainly di-sialylated glycosylation type structures. Glycomic (porous graphitized carbon-ESI-MS/MS) and glycopeptide (C8-LC-ESI-MS) analysis of recombinant soluble human CD52-immunoglobulin Fc fusion proteins revealed that CD52 bioactivity was correlated with a high abundance of tetra-antennary α-2,3/6 sialylated glycans. Removal of α-2,3 sialylation abolished bioactivity, which was restored by re-sialylation with α-2,3 sialyltransferases. When glycoforms of CD52-Fc were fractionated by anion exchange MonoQ-GL chromatography, bioactive fractions displayed mainly tetra-antennary, α-2,3 sialylated glycan structures and a lower relative abundance of bisecting GlcNAc structures compared to non-bioactive fractions. In addition, glycan core type-2 di-sialylated structures at Ser12 were more abundant in bioactive CD52 fractions. Understanding the structural features of CD52 glycan required for its bioactivity will aid its development as an immunotherapeutic agent.
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http://dx.doi.org/10.3389/fimmu.2019.01967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719568PMC
October 2020

Most clinical anti-EGFR antibodies do not neutralize both wtEGFR and EGFRvIII activation in glioma.

Neuro Oncol 2019 08;21(8):1016-1027

Brain Cancer Discovery Collaborative, New South Wales, Australia.

Background: Although epidermal growth factor receptor (EGFR) and its truncated, autoactive mutant EGFR variant (v)III are bona fide drivers of tumorigenesis in some gliomas, therapeutic antibodies developed to neutralize this axis have not improved patient survival in a limited number of trials. Previous studies using cells transduced to exogenously express EGFRvIII may have compromised mechanistic studies of anti-EGFR therapeutics. Therefore, we re-assessed the activity of clinical EGFR antibodies in patient-derived gliomaspheres that endogenously express EGFRvIII.

Methods: The antitumor efficacy of antibodies was assessed using in vitro proliferation assays and intracranial orthografts. Receptor activation status, antibody engagement, oncogenic signaling, and mechanism of action after antibody treatment were analyzed by immunoprecipitation and western blotting. Tracking of antibody receptor complexes was conducted using immunofluorescence.

Results: The EGFR domain III-targeting antibodies cetuximab, necitumumab, nimotuzumab, and matuzumab did not neutralize EGFRvIII activation. Chimeric monoclonal antibody 806 (ch806) neutralized EGFRvIII, but not wild-type (wt)EGFR activation. Panitumumab was the only antibody that neutralized both EGFRvIII and wtEGFR, leading to reduction of p-S6 signaling and superior in vitro and in vivo antitumor activity. Mechanistically, panitumumab induced recycling of receptor but not degradation as previously described. Panitumumab, via its unique avidity, stably cross-linked EGFRvIII to prevent its activation, while ch806 induced a marked reduction in the active EGFRvIII disulphide-bonded dimer.

Conclusions: We discovered a previously unknown major resistance mechanism in glioma in that most EGFR domain III-targeting antibodies do not neutralize EGFRvIII. The superior in vitro and in vivo antitumor activity of panitumumab supports further clinical testing of this antibody against EGFRvIII-stratified glioma.
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http://dx.doi.org/10.1093/neuonc/noz073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682217PMC
August 2019

Investigation onto the correlation between systemic antibodies to surface glycoproteins of infectious laryngotracheitis virus (ILTV) and protective immunity.

Vet Microbiol 2019 Jan 11;228:252-258. Epub 2018 Dec 11.

Asia Pacific Centre for Animal Health, The University of Melbourne, Werribee, VIC, 3030, Australia.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes upper respiratory tract disease in chickens and significant losses to the poultry industry worldwide. Both antibody and cell-mediated responses are generated against ILTV infection; however, the correlation of humoral immune response with protection against ILTV infection is debatable. To examine if whether antibody responses to individual ILTV glycoproteins are correlated with disease and protection, four ILTV glycoproteins (gD, gE, gG and gJ) were expressed as recombinant proteins and used in conjunction with commercially available recombinant gC and gI in indirect ELISAs to measure post-vaccination and/or post-challenge chicken serum antibodies. Serum optical density (OD) values detected by the whole virus, gC, gI and gJ were significantly higher in birds vaccinated with the Serva vaccine strain compared to the SA2 vaccine strain. However, the mean ODs detected by gD, gE and gG were not significantly different between the vaccine strains. Examination of post-ILTV vaccination sera found that gE was the most antigenic glycoprotein and that gC ODs were strongly correlated with those of gI and gJ, while ODs to gG had a relatively poor correlation with those of other glycoproteins. Moderate to poor correlations were found between microscopic tracheal lesion scores and ODs to individual glycoproteins. Examination of post-vaccination pre-challenge antibodies to individual glycoproteins did not find a strong correlation with protective immunity as measured by the severity of clinical signs, gross lesions, and tracheal viral load. Results from this study demonstrated that systemic antibody titers to individual ILTV glycoproteins C, D, E, G, I and J had a relatively poor correlation to protective immunity.
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http://dx.doi.org/10.1016/j.vetmic.2018.12.010DOI Listing
January 2019

Koala and Wombat Gammaherpesviruses Encode the First Known Viral NTPDase Homologs and Are Phylogenetically Divergent from All Known Gammaherpesviruses.

J Virol 2019 03 5;93(6). Epub 2019 Mar 5.

Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia.

There is a large taxonomic gap in our understanding of mammalian herpesvirus genetics and evolution corresponding to those herpesviruses that infect marsupials, which diverged from eutherian mammals approximately 150 million years ago (mya). We compare the genomes of two marsupial gammaherpesviruses, (PhaHV1) and (VoHV1), which infect koalas () and wombats (), respectively. The core viral genomes were approximately 117 kbp and 110 kbp in length, respectively, sharing 69% pairwise nucleotide sequence identity. Phylogenetic analyses showed that PhaHV1 and VoHV1 formed a separate branch, which may indicate a new gammaherpesvirus genus. The genomes contained 60 predicted open reading frames (ORFs) homologous to those in eutherian herpesviruses and 20 ORFs not yet found in any other herpesvirus. Seven of these ORFs were shared by the two viruses, indicating that they were probably acquired prespeciation, approximately 30 to 40 mya. One of these shared genes encodes a putative nucleoside triphosphate diphosphohydrolase (NTPDase). NTPDases are usually found in mammals and higher-order eukaryotes, with a very small number being found in bacteria. This is the first time that an NTPDase has been identified in any viral genome. Interrogation of public transcriptomic data sets from two koalas identified PhaHV1-specific transcripts in multiple host tissues, including transcripts for the novel NTPDase. PhaHV1 ATPase activity was also demonstrated , suggesting that the encoded NTPDase is functional during viral infection. In mammals, NTPDases are important in downregulation of the inflammatory and immune responses, but the role of the PhaHV1 NTPDase during viral infection remains to be determined. The genome sequences of the koala and wombat gammaherpesviruses show that the viruses form a distinct branch, indicative of a novel genus within the Their genomes contain several new ORFs, including ORFs encoding a β-galactoside α-2,6-sialyltransferase that is phylogenetically closest to poxvirus and insect homologs and the first reported viral NTPDase. NTPDases are ubiquitously expressed in mammals and are also present in several parasitic, fungal, and bacterial pathogens. In mammals, these cell surface-localized NTPDases play essential roles in thromboregulation, inflammation, and immune suppression. In this study, we demonstrate that the virus-encoded NTPDase is enzymatically active and is transcribed during natural infection of the host. Understanding how these enzymes benefit viruses can help to inform how they may cause disease or evade host immune defenses.
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http://dx.doi.org/10.1128/JVI.01404-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401436PMC
March 2019

APOMAB Antibody-Drug Conjugates Targeting Dead Tumor Cells are Effective .

Mol Cancer Ther 2019 02 9;18(2):335-345. Epub 2018 Nov 9.

Translational Oncology Laboratory, Centre for Cancer Biology, SA Pathology and University of South Australia, Adelaide, SA, Australia.

Antibody-drug conjugates (ADC) have revolutionized the field of cancer therapy. ADCs combine the high specificity of tumor-targeting monoclonal antibodies with potent cytotoxic drugs, which cannot be used alone because of their high toxicity. Till date, all ADCs have either targeted cell membrane proteins on tumors or the tumor vasculature and microenvironment. Here, we investigate ADCs of APOMAB (DAB4, or its chimeric derivative, chDAB4), which is a mAb targeting the La/SSB protein, which is only accessible for binding in dying or dead cancer cells. We show that DAB4-labeled dead cells are phagocytosed by macrophages, and that the apoptotic/necrotic areas within lung tumor xenografts are bound by DAB4 and are infiltrated with macrophages. We show that only DAB4-ADCs with a cleavable linker and diffusible drug are effective in two lung cancer models, particularly when given after chemotherapy. These results are consistent with other recent studies showing that direct internalization of ADCs by target cells is not essential for ADC activity because the linker can be cleaved extracellularly or through other mechanisms. Rather than targeting a tumor cell type specific antigen, DAB4-ADCs have the advantage of targeting a common trait in most solid tumors: an excess of post-apoptotic, necrotic cells either adjacent to hypoxic tumor regions or distributed more generally after cytotoxic therapy. Consequently, any antitumor effects are solely the result of bystander killing, either through internalization of the dead, ADC-bound tumor cells by macrophages, or extracellular cleavage of the ADC in the tumor microenvironment.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0842DOI Listing
February 2019

Activation of ERBB4 in Glioblastoma Can Contribute to Increased Tumorigenicity and Influence Therapeutic Response.

Cancers (Basel) 2018 Jul 25;10(8). Epub 2018 Jul 25.

Oncogenic Signalling Group, Hudson Institute of Medical Research, 21⁻37 Wright Street, Clayton, VIC 3168, Australia.

Glioblastoma (GBM) is often resistant to conventional and targeted therapeutics. ErbB2 Receptor Tyrosine Kinase 4 (ERBB4) is expressed throughout normal brain and is an oncogene in several pediatric brain cancers; therefore, we investigated ERBB4 as a prognostic marker and therapeutic target in GBM. Using RT-qPCR, we quantified mRNA encoding total ERBB4 and known ERBB4 variants in GBM and non-neoplastic normal brain (NNB) samples. Using immunohistochemistry, we characterized the localization of total and phosphorylated ERBB4 (p-ERBB4) and EGFR protein in archived GBM samples and assessed their association with patient survival. Furthermore, we evaluated the effect of ERBB4 phosphorylation on angiogenesis and tumorigenicity in GBM xenograft models. Total mRNA was significantly lower in GBM than NNB samples, with the juxtamembrane JM-a and cytoplasmic CYT-2 variants predominating. ERBB4 protein was ubiquitously expressed in GBM but was not associated with patient survival. However, high p-ERBB4 in 11% of archived GBM samples, independent of p-EGFR, was associated with shorter patient survival (12.0 ± 3.2 months) than was no p-ERBB4 (22.5 ± 9.5 months). Increased ERBB4 activation was also associated with increased proliferation, angiogenesis, tumorigenicity and reduced sensitivity to anti-EGFR treatment in xenograft models. Despite low mRNA in GBM, the functional effects of increased ERBB4 activation identify ERBB4 as a potential prognostic and therapeutic target.
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http://dx.doi.org/10.3390/cancers10080243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116191PMC
July 2018

CD52 glycan binds the proinflammatory B box of HMGB1 to engage the Siglec-10 receptor and suppress human T cell function.

Proc Natl Acad Sci U S A 2018 07 11;115(30):7783-7788. Epub 2018 Jul 11.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia;

CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein, is released in a soluble form following T cell activation and binds to the Siglec (sialic acid-binding Ig-like lectin)-10 receptor on T cells to suppress their function. We show that binding of CD52-Fc to Siglec-10 and T cell suppression requires the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). CD52-Fc bound specifically to the proinflammatory Box B domain of HMGB1, and this in turn promoted binding of the CD52 N-linked glycan, in α-2,3 sialic acid linkage with galactose, to Siglec-10. Suppression of T cell function was blocked by anti-HMGB1 antibody or the antiinflammatory Box A domain of HMGB1. CD52-Fc induced tyrosine phosphorylation of Siglec-10 and was recovered from T cells complexed with HMGB1 and Siglec-10 in association with SHP1 phosphatase and the T cell receptor (TCR). Thus, soluble CD52 exerts a concerted immunosuppressive effect by first sequestering HMGB1 to nullify its proinflammatory Box B, followed by binding to the inhibitory Siglec-10 receptor, triggering recruitment of SHP1 to the intracellular immunoreceptor tyrosine-based inhibitory motif of Siglec-10 and its interaction with the TCR. This mechanism may contribute to immune-inflammatory homeostasis in pathophysiologic states and underscores the potential of soluble CD52 as a therapeutic agent.
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http://dx.doi.org/10.1073/pnas.1722056115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065011PMC
July 2018

Differential Sensitivity of Human Hepatocellular Carcinoma Xenografts to an IGF-II Neutralizing Antibody May Involve Activated STAT3.

Transl Oncol 2018 Aug 22;11(4):971-978. Epub 2018 Jun 22.

CSIRO Manufacturing, Parkville, VIC 3052, Australia. Electronic address:

Hepatocellular carcinoma (HCC) is highly refractory to current therapeutics used in the clinic. DX-2647, a recombinant human antibody, potently neutralizes the action of insulin-like growth factor-II (IGF-II), a ligand for three cell-surface receptors (IGF-IR, insulin receptor A and B isoforms, and the cation-independent mannose-6-phosphate receptor) which is overexpressed in primary human HCC. DX-2647 impaired the growth of tumor xenografts of the HCC cell line, Hep3B; however, xenografts of the HCC cell line, HepG2, were largely unresponsive to DX-2647 treatment. Analysis of a number of aspects of the IGF signaling axis in both cell lines did not reveal any significant differences between the two. However, while DX-2647 abolished phospho (p)-IGF-IR, p-IR and p-AKT signaling in both cell lines, HepG2 showed high levels of p-STAT3, which was unaffected by DX-2647 treatment and was absent from the Hep3B cell line. The driver of p-STAT3 was found to be a secreted cytokine, and treatment of HepG2 cells with a pan- JAK kinase inhibitor resulted in a loss of p-STAT3. These findings implicate the activation of STAT3 as one pathway that may mediate resistance to IGF-II-targeted therapy in HCC.
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http://dx.doi.org/10.1016/j.tranon.2018.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020079PMC
August 2018

C6orf106 is a novel inhibitor of the interferon-regulatory factor 3-dependent innate antiviral response.

J Biol Chem 2018 07 25;293(27):10561-10573. Epub 2018 May 25.

From the Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation (CSIRO) Health and Biosecurity, Geelong, Victoria 3220, Australia and

Host recognition of intracellular viral RNA and subsequent induction of cytokine signaling are tightly regulated at the cellular level and are a target for manipulation by viruses and therapeutics alike. Here, we characterize chromosome 6 ORF 106 (C6orf106) as an evolutionarily conserved inhibitor of the innate antiviral response. C6orf106 suppresses the synthesis of interferon (IFN)-α/β and proinflammatory tumor necrosis factor (TNF) α in response to the dsRNA mimic poly(I:C) and to Sendai virus infection. Unlike canonical inhibitors of antiviral signaling, C6orf106 blocks interferon-regulatory factor 3 (IRF3) and, to a lesser extent, NF-κB activity without modulating their activation, nuclear translocation, cellular expression, or degradation. Instead, C6orf106 interacts with IRF3 and inhibits IRF3 recruitment to type I IFN promoter sequences while also reducing the nuclear levels of the coactivator proteins p300 and CREB-binding protein (CBP). In summary, we have defined C6orf106 as a negative regulator of antiviral immunity that blocks IRF3-dependent cytokine production via a noncanonical and poorly defined mechanism. This work presents intriguing implications for antiviral immunity, autoimmune disorders, and cancer.
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http://dx.doi.org/10.1074/jbc.RA117.001491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6036214PMC
July 2018

Electrostatic Interactions between Hendra Virus Matrix Proteins Are Required for Efficient Virus-Like-Particle Assembly.

J Virol 2018 07 13;92(13). Epub 2018 Jun 13.

CSIRO Manufacturing, Parkville, Victoria, Australia.

(HeV) is a zoonotic paramyxovirus belonging to the genus HeV is highly pathogenic, and it can cause severe neurological and respiratory illnesses in both humans and animals, with an extremely high mortality rate of up to 70%. Among the genes that HeV encodes, the matrix (M) protein forms an integral part of the virion structure and plays critical roles in coordinating viral assembly and budding. Nevertheless, the molecular mechanism of this process is not fully elucidated. Here, we determined the crystal structure of HeV M to 2.5-Å resolution. The dimeric structural configuration of HeV M is similar to that of Newcastle disease virus (NDV) M and is fundamental to protein stability and effective virus-like-particle (VLP) formation. Analysis of the crystal packing revealed a notable interface between the α1 and α2 helices of neighboring HeV M dimers, with key residues sharing degrees of sequence conservation among henipavirus M proteins. Structurally, a network of electrostatic interactions dominates the α1-α2 interactions, involving residues Arg57 from the α1 helix and Asp105 and Glu108 from the α2 helix. The disruption of the α1-α2 interactions using engineered charge reversal substitutions (R57E, R57D, and E108R) resulted in significant reduction or abrogation of VLP production. This phenotype was reversible with an R57E E108R mutant that was designed to partly restore salt bridge contacts. Collectively, our results define and validate previously underappreciated regions of henipavirus M proteins that are crucial for productive VLP assembly. Hendra virus is a henipavirus associated with lethal infections in humans. It is classified as a biosafety level 4 (BSL4) agent, and there are currently no preventive or therapeutic treatments available against HeV. Vital to henipavirus pathogenesis, the structural protein M has been implicated in viral assembly and budding, as well as host-virus interactions. However, there is no structural information available for henipavirus M, and the basis of M-driven viral assembly is not fully elucidated. We demonstrate the first three-dimensional structure of a henipavirus M protein. We show the dimeric organization of HeV M as a basic unit for higher-order oligomerization. Additionally, we define key regions/residues of HeV M that are required for productive virus-like-particle formation. These findings provide the first insight into the mechanism of M-driven assembly in henipavirus.
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http://dx.doi.org/10.1128/JVI.00143-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002731PMC
July 2018

Structural characterization of a novel monotreme-specific protein with antimicrobial activity from the milk of the platypus.

Acta Crystallogr F Struct Biol Commun 2018 Jan 1;74(Pt 1):39-45. Epub 2018 Jan 1.

Biomedical Manufacturing, CSIRO, 343 Royal Parade, Parkville, VIC 3052, Australia.

Monotreme lactation protein (MLP) is a recently identified protein with antimicrobial activity. It is present in the milk of monotremes and is unique to this lineage. To characterize MLP and to gain insight into the potential role of this protein in the evolution of lactation, the crystal structure of duck-billed platypus (Ornithorhynchus anatinus) MLP was determined at 1.82 Å resolution. This is the first structure to be reported for this novel, mammalian antibacterial protein. MLP was expressed as a FLAG epitope-tagged protein in mammalian cells and crystallized readily, with at least three space groups being observed (P1, C2 and P2). A 1.82 Å resolution native data set was collected from a crystal in space group P1, with unit-cell parameters a = 51.2, b = 59.7, c = 63.1 Å, α = 80.15, β = 82.98, γ = 89.27°. The structure was solved by SAD phasing using a protein crystal derivatized with mercury in space group C2, with unit-cell parameters a = 92.7, b = 73.2, c = 56.5 Å, β = 90.28°. MLP comprises a monomer of 12 helices and two short β-strands, with much of the N-terminus composed of loop regions. The crystal structure of MLP reveals no three-dimensional similarity to any known structures and reveals a heretofore unseen fold, supporting the idea that monotremes may be a rich source for the identification of novel proteins. It is hypothesized that MLP in monotreme milk has evolved to specifically support the unusual lactation strategy of this lineage and may have played a central role in the evolution of these mammals.
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http://dx.doi.org/10.1107/S2053230X17017708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947691PMC
January 2018

CD52 inhibits Toll-like receptor activation of NF-κB and triggers apoptosis to suppress inflammation.

Cell Death Differ 2018 02 15;25(2):392-405. Epub 2017 Dec 15.

The Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.

Soluble CD52 is a small glycoprotein that suppresses T-cell activation, but its effect on innate immune cell function is unknown. Here we demonstrate that soluble CD52 inhibits Toll-like receptor and tumor necrosis factor receptor signaling to limit activation of NF-κB and thereby suppress the production of inflammatory cytokines by macrophages, monocytes and dendritic cells. At higher concentrations, soluble CD52 depletes the short-lived pro-survival protein MCL-1, contributing to activation of the BH3-only proteins BAX and BAK to cause intrinsic apoptotic cell death. In vivo, administration of soluble CD52 suppresses lipopolysaccharide (LPS)-induced cytokine secretion and other features of endotoxic shock, whereas genetic deletion of CD52 exacerbates LPS responses. Thus, soluble CD52 exhibits broad immune suppressive effects that signify its potential as an immunotherapeutic agent.
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http://dx.doi.org/10.1038/cdd.2017.173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762852PMC
February 2018

Development of an anti-ferret CD4 monoclonal antibody for the characterisation of ferret T lymphocytes.

J Immunol Methods 2017 05 16;444:29-35. Epub 2017 Feb 16.

CSIRO Manufacturing, Parkville, Victoria, Australia.

The ferret is an established animal model for a number of human respiratory viral infections, such as influenza virus and more recently, Ebola virus. However, a paucity of immunological reagents has hampered the study of cellular immune responses. Here we describe the development and characterisation of a novel monoclonal antibody (mAb) against the ferret CD4 antigen and the characterisation of ferret CD4 T lymphocytes. Recombinant production and purification of the ferret CD4 ectodomain soluble protein allowed hybridoma generation and the generation of a mAb (FeCD4) showing strong binding to ferret CD4 protein and lymphoid cells by flow cytometry. FeCD4 bound to its cognate antigen post-fixation with paraformaldehyde (PFA) which is routinely used to inactivate highly pathogenic viruses. We have also used FeCD4 in conjunction with other immune cell markers to characterise ferret T cells in both primary and secondary lymphoid organs. In summary, we have developed an important reagent for the study of cellular immunological responses in the ferret model of infectious disease.
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http://dx.doi.org/10.1016/j.jim.2017.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094458PMC
May 2017

New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

Stem Cells 2017 03 19;35(3):626-640. Epub 2017 Jan 19.

Clayton and Parkville, CSIRO Manufacturing, Victoria, Australia.

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640.
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http://dx.doi.org/10.1002/stem.2558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412944PMC
March 2017

Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection.

PLoS Pathog 2016 Mar 24;12(3):e1005478. Epub 2016 Mar 24.

CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, Victoria, Australia.

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.
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http://dx.doi.org/10.1371/journal.ppat.1005478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806981PMC
March 2016

Incomplete target neutralization by the anti-cancer antibody rilotumumab.

MAbs 2016 ;8(2):246-52

a Oncogenic Signalling Laboratory and Brain Cancer Discovery Collaborative, Centre for Cancer Research, Hudson Institute of Medical Research , 27-31 Wright Street, Clayton , VIC 3168 , Australia.

The antibody rilotumumab, which has been tested in multiple Phase 2 and Phase 3 trials, has been reported to neutralize hepatocyte growth factor (HGF), the ligand for the oncogene MET. However, we report that rilotumumab does not prevent HGF from directly binding to MET on conventional and primary patient-derived human gliomasphere lines, a trait driven by the HGF α-chain, which remains free to engage cell-surface glycosaminoglycans and the receptor MET. This binding induces MET phosphorylation, initiates robust AKT and ERK signaling and potentiates biological effects such as cell scattering. This partial antagonism was highly exacerbated in the presence of activated epidermal growth factor receptor, which is common in several cancers. Hence, we confirm that rilotumumab is only a partial antagonist of HGF activity, a finding that has considerable implications for the therapeutic use of rilotumumab.
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http://dx.doi.org/10.1080/19420862.2015.1122149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966628PMC
December 2016

Structural and functional characterisation of ferret interleukin-2.

Dev Comp Immunol 2016 Feb 22;55:32-8. Epub 2015 Oct 22.

CSIRO Manufacturing, Parkville, VIC 3052, Australia. Electronic address:

While the ferret is a valuable animal model for a number of human viral infections, such as influenza, Hendra and Nipah, evaluating the cellular immune response following infection has been hampered by the lack of a number of species-specific immunological reagents. Interleukin 2 (IL-2) is one such key cytokine. Ferret recombinant IL-2 incorporating a C-terminal histidine tag was expressed and purified and the three-dimensional structure solved and refined at 1.89 Å by X-ray crystallography, which represents the highest resolution and first non-human IL-2 structure. While ferret IL-2 displays the classic cytokine fold of the four-helix bundle structure, conformational flexibility was observed at the second helix and its neighbouring region in the bundle, which may result in the disruption of the spatial arrangement of residues involved in receptor binding interactions, implicating subtle differences between ferret and human IL-2 when initiating biological functions. Ferret recombinant IL-2 stimulated the proliferation of ferret lymph node cells and induced the expression of mRNA for IFN-γ and Granzyme A.
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http://dx.doi.org/10.1016/j.dci.2015.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7102629PMC
February 2016

Structural and biochemical analyses of a Clostridium perfringens sortase D transpeptidase.

Acta Crystallogr D Biol Crystallogr 2015 Jul 30;71(Pt 7):1505-13. Epub 2015 Jun 30.

Manufacturing Flagship, Commonwealth Scientific and Industrial Research Organisation, 343 Royal Parade, Parkville, Victoria 3052, Australia.

The assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria is mediated by the sortase family of enzymes. These cysteine transpeptidases catalyze a unique sorting signal motif located at the C-terminus of their target substrate and promote the covalent attachment of these proteins onto an amino nucleophile located on another protein or on the bacterial cell wall. Each of the six distinct classes of sortases displays a unique biological role, with sequential activation of multiple sortases often observed in many Gram-positive bacteria to decorate their peptidoglycans. Less is known about the members of the class D family of sortases (SrtD), but they have a suggested role in spore formation in an oxygen-limiting environment. Here, the crystal structure of the SrtD enzyme from Clostridium perfringens was determined at 1.99 Å resolution. Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded β-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro, with a significant preference for the LPQTGS sorting motif. Additionally, the catalytic activity of SrtD is most efficient at 316 K and can be further improved in the presence of magnesium cations. Since C. perfringens spores are heat-resistant and lead to foodborne illnesses, characterization of the spore-promoting sortase SrtD may lead to the development of new antimicrobial agents.
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http://dx.doi.org/10.1107/S1399004715009219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498605PMC
July 2015

Blood-based protein biomarker panel for the detection of colorectal cancer.

PLoS One 2015 20;10(3):e0120425. Epub 2015 Mar 20.

CSIRO Preventative Health National Research Flagship, Adelaide, South Australia, Australia.

Background: The majority of colorectal cancer (CRC) cases are preventable by early detection and removal of precancerous polyps. Even though CRC is the second most common internal cancer in Australia, only 30 per cent of the population considered to have risk factors participate in stool-based test screening programs. Evidence indicates a robust, blood-based, diagnostic assay would increase screening compliance. A number of potential diagnostic blood-based protein biomarkers for CRC have been reported, but all lack sensitivity or specificity for use as a stand-alone diagnostic. The aim of this study was to identify and validate a panel of protein-based biomarkers in independent cohorts that could be translated to a reliable, non-invasive blood-based screening test.

Principal Findings: In two independent cohorts (n = 145 and n = 197), we evaluated seven single biomarkers in serum of CRC patients and age/gender matched controls that showed a significant difference between controls and CRC, but individually lack the sensitivity for diagnostic application. Using logistic regression strategies, we identified a panel of three biomarkers that discriminated between controls and CRC with 73% sensitivity at 95% specificity, when applied to either of the two cohorts. This panel comprised of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2).

Conclusions: Due to the heterogeneous nature of CRC, a single biomarker is unlikely to have sufficient sensitivity or specificity for use as a stand-alone diagnostic screening test and a panel of markers may be more effective. We have identified a 3 biomarker panel that has higher sensitivity and specificity for early stage (Stage I and -II) disease than the faecal occult blood test, raising the possibility for its use as a non-invasive blood diagnostic or screening test.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120425PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368610PMC
February 2016

LRIG1 extracellular domain: structure and function analysis.

J Mol Biol 2015 May 9;427(10):1934-48. Epub 2015 Mar 9.

Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia; Department of Surgery, RMH, University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

We have expressed and purified three soluble fragments of the human LRIG1-ECD (extracellular domain): the LRIG1-LRR (leucine-rich repeat) domain, the LRIG1-3Ig (immunoglobulin-like) domain, and the LRIG1-LRR-1Ig fragment using baculovirus vectors in insect cells. The two LRIG1 domains crystallised so that we have been able to determine the three-dimensional structures at 2.3Å resolution. We developed a three-dimensional structure for the LRIG1-ECD using homology modelling based on the LINGO-1 structure. The LRIG1-LRR domain and the LRIG1-LRR-1Ig fragment are monomers in solution, whereas the LRIG1-3Ig domain appears to be dimeric. We could not detect any binding of the LRIG1 domains or the LRIG1-LRR-1Ig fragment to the EGF receptor (EGFR), either in solution using biosensor analysis or when the EGFR was expressed on the cell surface. The FLAG-tagged LRIG1-LRR-1Ig fragment binds weakly to colon cancer cells regardless of the presence of EGFRs. Similarly, neither the soluble LRIG1-LRR nor the LRIG1-3Ig domains nor the full-length LRIG1 co-expressed in HEK293 cells inhibited ligand-stimulated activation of cell-surface EGFR.
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http://dx.doi.org/10.1016/j.jmb.2015.03.001DOI Listing
May 2015

Notch ligand delta-like1: X-ray crystal structure and binding affinity.

Biochem J 2015 May;468(1):159-66

*Department of Structural Biology, Walter and Eliza Hall Institute of Medical Research and Department of Medical Biology, University of Melbourne, Parkville, 3052 Australia.

The Notch pathway is a fundamental signalling system in most multicellular animals. We have determined the X-ray crystal structure of the extracellular domain of the Notch ligand delta-like ligand-1 (Dll-1). The structure incorporates the N-terminal C2 domain, receptor-binding DSL domain and the first six (of eight) EGF (epidermal growth factor)-like repeats, which form a highly extended conformation, confirmed by analytical ultracentrifugation. Comparison of our structure with a fragment of Jagged1 ligand allows us to dissect the similarities and differences between the ligand families. Differences in the C2 domains of Dll-1 and Jagged1 suggest their lipid-binding properties are likely to differ. A conserved hydrophobic patch on the surface of both Dll-1 and Jagged1 provides a likely receptor-interaction site that is common to both ligands. We also explore the binding affinity of Dll-1 for a fragment of Notch1 using different techniques. Apparent binding affinities vary when different techniques are used, explaining discrepancies in the literature. Using analytical ultracentrifugation, we perform for the first time binding analyses where both receptor and ligand are in solution, which confirms a Kd of 10 μM for this interaction.
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http://dx.doi.org/10.1042/BJ20150010DOI Listing
May 2015

The structure of vanin 1: a key enzyme linking metabolic disease and inflammation.

Acta Crystallogr D Biol Crystallogr 2014 Dec 28;70(Pt 12):3320-9. Epub 2014 Nov 28.

CSIRO Biosciences Program, 343 Royal Parade, Parkville, VIC 3052, Australia.

Although part of the coenzyme A pathway, vanin 1 (also known as pantetheinase) sits on the cell surface of many cell types as an ectoenzyme, catalyzing the breakdown of pantetheine to pantothenic acid (vitamin B5) and cysteamine, a strong reducing agent. Vanin 1 was initially discovered as a protein involved in the homing of leukocytes to the thymus. Numerous studies have shown that vanin 1 is involved in inflammation, and more recent studies have shown a key role in metabolic disease. Here, the X-ray crystal structure of human vanin 1 at 2.25 Å resolution is presented, which is the first reported structure from the vanin family, as well as a crystal structure of vanin 1 bound to a specific inhibitor. These structures illuminate how vanin 1 can mediate its biological roles by way of both enzymatic activity and protein-protein interactions. Furthermore, it sheds light on how the enzymatic activity is regulated by a novel allosteric mechanism at a domain interface.
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http://dx.doi.org/10.1107/S1399004714022767DOI Listing
December 2014

Comparison of alternative nucleophiles for Sortase A-mediated bioconjugation and application in neuronal cell labelling.

Org Biomol Chem 2014 May;12(17):2675-85

CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052, Australia.

The Sortase A (SrtA) enzyme from Staphylococcus aureus catalyses covalent attachment of protein substrates to pentaglycine cross-bridges in the Gram positive bacterial cell wall. In vitro SrtA-mediated protein ligation is now an important protein engineering tool for conjugation of substrates containing the LPXTGX peptide recognition sequence to oligo-glycine nucleophiles. In order to explore the use of alternative nucleophiles in this system, five different rhodamine-labelled compounds, with N-terminal nucleophilic amino acids, triglycine, glycine, and lysine, or N-terminal non-amino acid nucleophiles ethylenediamine and cadaverine, were synthesized. These compounds were tested for their relative abilities to function as nucleophiles in SrtA-mediated bioconjugation reactions. N-Terminal triglycine, glycine and ethylenediamine were all efficient in labelling a range of LPETGG containing recombinant antibody and scaffold proteins and peptides, while reduced activity was observed for the other nucleophiles across the range of proteins and peptides studied. Expansion of the range of available nucleophiles which can be utilised in SrtA-mediated bioconjugation expands the range of potential applications for this technology. As a demonstration of the utility of this system, SrtA coupling was used to conjugate the triglycine rhodamine-labelled nucleophile to the C-terminus of an Im7 scaffold protein displaying Aβ, a neurologically important peptide implicated in Alzheimer's disease. Purified, labelled protein showed Aβ-specific targeting to mammalian neuronal cells. Demonstration of targeting neuronal cells with a chimeric protein illustrates the power of this system, and suggests that SrtA-mediated direct cell-surface labelling and visualisation is an achievable goal.
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http://dx.doi.org/10.1039/c3ob42325eDOI Listing
May 2014

Colorectal cancer biomarkers: to be or not to be? Cautionary tales from a road well travelled.

World J Gastroenterol 2014 Jan;20(4):888-98

Kim YC Fung, Ilka Priebe, Damien Belobrajdic, Leanne Purins, Bruce Tabor, Celine Pompeia, Trevor Lockett, Leah Cosgrove, CSIRO, Preventative Health National Research Flagship, Adelaide, SA 5000, Australia.

Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide and places a major economic burden on the global health care system. The time frame for development from premalignant to malignant disease typically spans 10-15 years, and this latent period provides an ideal opportunity for early detection and intervention to improve patient outcomes. Currently, early diagnosis of CRC is hampered by a lack of suitable non-invasive biomarkers that are clinically or economically acceptable for population-based screening. New blood-based protein biomarkers for early detection of CRC are therefore urgently required. The success of clinical biomarker discovery and validation studies is critically dependent on understanding and adjusting for potential experimental, analytical, and biological factors that can interfere with the robust interpretation of results. In this review we outline some important considerations for research groups undertaking biomarker research with exemplars from our studies. Implementation of experimental strategies to minimise the potential effects of these problems will facilitate the identification of panels of biomarkers with the sensitivity and specificity required for the development of successful tests for the early detection and surveillance of CRC.
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http://dx.doi.org/10.3748/wjg.v20.i4.888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3921542PMC
January 2014

Megakaryocytes co-localise with hemopoietic stem cells and release cytokines that up-regulate stem cell proliferation.

Stem Cell Res 2013 Sep 28;11(2):782-92. Epub 2013 May 28.

Materials Science and Engineering, Commonwealth Scientific and Industrial Research Organization, Melbourne, Australia.

We report transplanted hemopoietic stem cells (HSC) preferentially lodge within two cells of mature megakaryocytes (MM). With both populations comprising ~0.2% of bone marrow cells, this strongly suggests a key functional interaction. HSC isolated from the endosteum (eLSKSLAM) showed significantly increased hemopoietic cell proliferation while in co-culture with MM. Furthermore, eLSKSLAM progeny retained HSC potential, maintaining long-term multi-lineage reconstitution capacity in lethally ablated recipients. Increased hemopoietic cell proliferation was not MM contact dependent and could be recapitulated with media supplemented with two factors identified in MM-conditioned media: insulin-like growth factor binding protein-3 (IGFBP-3) and insulin-like growth factor-1 (IGF-1). We demonstrate that HSC express the receptor for IGF-1 and that IGF-1/IGFBP-3 induced increased hemopoietic cell proliferation can be blocked by an anti-IGF-1 neutralising antibody. However, co-cultures of 8N, 16N or 32N MM with eLSKSLAM showed that MM of individual ploidy did not significantly increase hemopoietic cell proliferation. Our data suggests that MM are an important component of the HSC niche and regulate hemopoietic cell proliferation through cytokine release.
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http://dx.doi.org/10.1016/j.scr.2013.05.007DOI Listing
September 2013