Cellular Transmembrane Protein Research Articles

Further Characterization of the Antiviral Transmembrane Protein MARCH8.

The cellular transmembrane protein MARCH8 impedes the incorporation of various viral envelope glycoproteins, such as the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G-glycoprotein (VSV-G), into virions by downregulating them from the surface of virus-producing cells. This downregulation significantly reduces the efficiency of virus infection. In this study, we aimed to further characterize this host protein by investigating its species specificity and the domains responsible for its antiviral activity, as well as its ability to inhibit cell-to-cell HIV-1 infection. We found that the antiviral function of MARCH8 is well conserved in the rhesus macaque, mouse, and bovine versions. The RING-CH domains of these versions are functionally important for inhibiting HIV-1 Env and VSV-G-pseudovirus infection, whereas tyrosine motifs are crucial for the former only, consistent with findings in human MARCH8. Through analysis of chimeric proteins between MARCH8 and non-antiviral MARCH3, we determined that both the N-terminal and C-terminal cytoplasmic tails, as well as presumably the N-terminal transmembrane domain, of MARCH8 are critical for its antiviral activity. Notably, we found that MARCH8 is unable to block cell-to-cell HIV-1 infection, likely due to its insufficient downregulation of Env. These findings offer further insights into understanding the biology of this antiviral transmembrane protein.

A viral lncRNA tethers HSV-1 genomes at the nuclear periphery to establish viral latency.

Herpes simplex virus 1 (HSV-1) establishes lifelong latency in neuronal cells. Following a stressor, the virus reactivates from latency, virus is shed at the periphery and recurrent disease can occur. During latency, the viral lncRNA termed the latency-associated transcript (LAT) is known to accumulate to high abundance. The LAT is known to impact many aspects of latency though the molecular events involved are not well understood. Here, we utilized a human neuronal cell line model of HSV latency and reactivation (LUHMES) to identify the molecular-binding partners of the LAT during latency. We found that the LAT binds to both the cellular protein, TMEM43, and HSV-1 genomes in LUHMES cells. Additionally, we find that knockdown of TMEM43 prior to infection results in a decreased ability of HSV-1 to establish latency. This work highlights a potential mechanism for how the LAT facilitates the establishment of HSV-1 latency in human neurons.

Relative resistance of patient-derived envelope sequences to SERINC5-mediated restriction of HIV-1 infectivity.

Pathogenesis of HIV-1 is enhanced through several viral-encoded proteins that counteract a range of host restriction molecules. HIV-1 Nef counteracts the cell membrane protein SERINC5 by downregulating it from the cell surface, thereby enhancing virion infectivity. Some subtype B reference Envelope sequences have shown the ability to bypass SERINC5 infectivity restriction independent of Nef. However, it is not clear if and to what extent circulating HIV-1 strains can exhibit resistance to SERINC5 restriction. Using a panel of Envelope sequences isolated from 50 Tanzanians infected with non-B HIV-1 subtypes, we show that the lentiviral reporters pseudotyped with patient-derived Envelopes have reduced sensitivity to SERINC5 and that this sensitivity differed among viral subtypes. Moreover, we found that SERINC5 sensitivity within patient-derived Envelopes can be modulated by separate regions, highlighting the complexity of viral/host interactions.

SARS-CoV-2 ORF7a potently inhibits the antiviral effect of the host factor SERINC5

Serine Incorporator 5 (SERINC5), a cellular multipass transmembrane protein that is involved in sphingolipid and phosphatydilserine biogenesis, potently restricts a number of retroviruses, including Human Immunodeficiency Virus (HIV). SERINC5 is incorporated in the budding virions leading to the inhibition of virus infectivity. In turn, retroviruses, including HIV, encode factors that counteract the antiviral effect of SERINC5. While SERINC5 has been well studied in retroviruses, little is known about its role in other viral families. Due to the paucity of information regarding host factors targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), we evaluated the effect of SERINC proteins on SARS-CoV-2 infection. Here, we show SERINC5 inhibits SARS-CoV-2 entry by blocking virus-cell fusion, and SARS-CoV-2 ORF7a counteracts the antiviral effect of SERINC5 by blocking the incorporation of over expressed SERINC5 in budding virions.

Effects of ABCG1 knockout on proteomic composition of HDL in mice on a chow diet and a high-fat diet.

ATP-binding cassette transporter G1 (ABCG1) is a cellular transmembrane protein that transports oxysterol efflux from cells to high-density lipoprotein (HDL) particles in the plasma. Previous studies have demonstrated that an ABCG1 deficiency exerts an antiatherosclerotic function through the effects of oxysterol accumulation in cells to enhance apoptosis and regulate inflammatory processes. However, whether the deficiency of ABCG1 and the corresponding changes in the efflux of oxysterols could take a series of impacts on the proteomic composition of HDL remains unclear. Here, plasma HDL of ABCG1(-/-) mice and their wild-type controls on a normal chow diet (NCD) or a high-fat diet (HFD) were isolated by ultracentrifugation. The proportion of 7-ketocholesterol and the proteomic composition of samples were comparatively analyzed by LC-MS/MS. In NCD-fed mice, lipid metabolism-related protein (arachidonate 12-lipoxygenase) and antioxidative protein (pantetheinase) exhibited increased accumulation, and inflammatory response protein (alpha-1-antitrypsin) was decreased in accumulation in ABCG1(-/-) mice HDL. In HFD-fed mice, fewer proteins were detected than that of NCD-fed mice. The ABCG1(-/-) mice HDL exhibited increased accumulation of lipid metabolism-related proteins (e.g., carboxylesterase 1C, apolipoprotein (apo)C-4) and decreased accumulation of alpha-1-antitrypsin, as well as significantly reduced proportion of 7-ketocholesterol. Additionally, positive correlations were found between 7-ketocholesterol and some essential proteins on HDL, such as alpha-1-antitrypsin, apoA-4, apoB-100, and serum amyloid A (SAA). These results suggest a detrimental impact of oxysterols on HDL composition, which might affect the antiatherosclerotic properties of HDL.

Conformational dynamics of the Ebola virus glycoprotein (GP) under conditions relevant for membrane fusion

Ebola virus (EBOV) infects mammalian cells through fusion of the viral and host membranes. This multistep process occurs in cellular low pH endosomal compartments and is mediated by the fusion peptide of the EBOV envelope glycoprotein (GP), however a mechanistic understanding of GP-mediated virus entry remains incomplete. Proteolytic cleavage of GP and interaction with the cellular transmembrane protein Niemann-Pick C1 protein (NPC1) are necessary but not sufficient for fusion. Previously, we applied single-molecule FÃrster resonance energy transfer (smFRET) imaging to investigate the structural dynamics of EBOV GP on the surface of pseudovirions.

Unraveling membrane properties at the organelle-level with LipidDyn

Cellular membranes are formed from different lipids in various amounts and proportions depending on the subcellular localization. The lipid composition of membranes is sensitive to changes in the cellular environment, and its alterations are linked to several diseases. Lipids not only form lipid-lipid interactions but also interact with other biomolecules, including proteins.Molecular dynamics (MD) simulations are a powerful tool to study the properties of cellular membranes and membrane-protein interactions on different timescales and resolutions. Over the last few years, software and hardware for biomolecular simulations have been optimized to routinely run long simulations of large and complex biological systems. On the other hand, high-throughput techniques based on lipidomics provide accurate estimates of the composition of cellular membranes at the level of subcellular compartments. Lipidomic data can be analyzed to design biologically relevant models of membranes for MD simulations. Similar applications easily result in a massive amount of simulation data where the bottleneck becomes the analysis of the data. In this context, we developed LipidDyn, a Python-based pipeline to streamline the analyses of MD simulations of membranes of different compositions. Once the simulations are collected, LipidDyn provides average properties and time series for several membrane properties such as area per lipid, thickness, order parameters, diffusion motions, lipid density, and lipid enrichment/depletion. The calculations exploit parallelization, and the pipeline includes graphical outputs in a publication-ready form. We applied LipidDyn to different case studies to illustrate its potential, including membranes from cellular compartments and transmembrane protein domains. LipidDyn is available free of charge under the GNU General Public License from https://github.com/ELELAB/LipidDyn.

Cystic Fibrosis: Advancing Along the Continuum

Cystic fibrosis (CF) is an autosomal recessive genetic disorder resulting from a mutation in the gene which encodes a cellular transmembrane protein channel known as the CF transmembrane conductance regulator. Located systemically on the surface of numerous cells, these altered channels yield multisystem dysfunction. Typical manifestations seen are chronic, progressive, obstructive lung disease, pancreatic insufficiency, CF-related diabetes mellitus, malabsorption and malnutrition, liver disease, and infertility.Once considered a pediatric disorder, through developments in innovative care and therapeutic modalities, CF now spans the life continuum and has established itself as an ageless disease. Facing management of maturing-life issues, advanced practice nurses (APNs) in pediatrics now find themselves needing to collaborate with or facilitate transition of care to other APNs, such as nurse midwives and adult APNs, as well as their counterpart specialists in medicine, all while maintaining open communication with the patient, family and managing CF center.

Persistence of the ABCC6 genes and the emergence of the bony skeleton in vertebrates

The ATP-binding cassette transporter 6 (ABCC6) gene encodes a cellular transmembrane protein transporter (MRP6) that is involved in the regulation of tissue calcification in mammals. Mutations in ABCC6 are associated with human ectopic calcification disorders. To gain insight into its evolution and involvement in tissue calcification we conducted a comparative analysis of the ABCC6 gene and the related gene ABCC1 from invertebrates to vertebrates where a bony endoskeleton first evolved. Taking into consideration the role of ABCC6 in ectopic calcification of human skin we analysed the involvement of both genes in the regeneration of scales, mineralized structures that develop in fish skin. The ABCC6 gene was only found in bony vertebrate genomes and was absent from Elasmobranchs, Agnatha and from invertebrates. In teleost fish the abcc6 gene duplicated but the two genes persisted only in some teleost genomes. Six disease causing amino acid mutations in human MRP6 are a normal feature of abcc6 in fish, suggesting they do not have a deleterious effect on the protein. After scale removal the abcc6 (5 and 10 days) and abcc1 (10 days) gene expression was up-regulated relative to the intact control skin and this coincided with a time of intense scale mineralization.

Abstract 66: CD99 functional analysis in glioblastoma by RNAseq

Abstract CD99 is a membrane protein expressed in a wide variety of normal tissues and implicated in several cellular physiologic processes, including haematopoietic cell differentiation, proliferation, diapedesis, cell-cell adhesion, migration, apoptosis induction, cellular architecture maintenance and transmembrane protein transport. Additionally, aberrant CD99 expression has been associated with numerous malignancies, including astrocytomas. Amongst astrocytomas of different malignant grades, glioblastoma (GBM) is the most aggressive and common of central nervous system tumors. Despite current multimodal therapies, including surgery, chemotherapy and radiotherapy, the median survival duration of patients is 17 months. We have previously demonstrated increased CD99 expression at mRNA and protein levels in astroctyoma of all malignant grades, with GBM showing the highest levels. Knocking down CD99 expression by siRNA efficiently decreased CD99 at mRNA and protein levels and concurrently decreased the migratory property of two GBM cell lines, U87MG and A172. We subsequently analysed the transcriptoma of both cell lines by RNA-seq in Illumina platform after transfection with siRNA for CD99 and compared to non-target control (NTC). CD99 gene and protein silencing was confirmed in both cell lines. A total of 413 and 340 genes for U87MG and A172, respectively, with expression ≤1.3 (p<0.01, Bejamiini-Hochberg test) in knockdown condition in relation to NTC were detected, and they were analysed according to process networks using Metacore. A total of 129 genes had the expression diminished with CD99 in both cell lines, among them genes coding for proteins involved in migration and cytoskeletal remodelling, as expected. Also, we observed a decreased expression of genes involved in molecular mechanisms of translation control and protein folding regulation, as genes encoding for: subunits of the elongation factor-1 complex (EEF1A and EEF1A1), GTP-binding translation elongation factor (EEF2), several ribosomal proteins (PRPL4, RPL8, RPL15, RPL28, RPL32, RPL37A, RPS2, RPS3, RPS4, RPS6, RPS11 and RPS20), tRNA synthetase proteins (VARS and YARS), and heat shock proteins (HSC70, HSP70, HYOU1 and VCP). Furthermore, expression of MTOR, which encodes a common subunit protein of mTORC1 and mTORC2 complexes, is also downregulated with CD99 knockdown. Altogether, these results open insights for new roles of CD99 in GBM in controlling the regulation of translation, a crucial control of cell growth and proliferation. CD99 could thus be targeted to prevent protein translation in cancer cells. Citation Format: Sueli M. Oba-Shinjo, Lais C. Cardoso, Roseli da Silva, Antonio M. Lerario, Miyuki Uno, Suely S.K. Marie. CD99 functional analysis in glioblastoma by RNAseq. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 66. doi:10.1158/1538-7445.AM2015-66

HSV-1 nucleocapsid egress mediated by UL31 in association with UL34 is impeded by cellular transmembrane protein 140

During HSV-1 infection, the viral UL31 protein forms a complex with the UL34 protein at the cellular nuclear membrane, where both proteins play important roles in the envelopment of viral nucleocapsids and their egress into the cytoplasm. To characterize the mechanism of HSV-1 nucleocapsid egress, we screened host proteins to identify proteins that interacted with UL31 via yeast two-hybrid analysis. Transmembrane protein 140 (TMEM140), was identified and confirmed to bind to and co-localize with UL31 during viral infection. Further studies indicated that TMEM140 inhibits HSV-1 proliferation through selectively blocking viral nucleocapsid egress during the viral assembly process. The blockage function of TMEM140 is mediated by impeding the formation of the UL31–UL34 complex due to competitive binding to UL31. Collectively, these data suggest the essentiality of the UL31–UL34 interaction in the viral nucleocapsid egress process and provide a new anti-HSV-1 strategy in viral assembly process of nucleocapsid egress.

Misdirection of membrane trafficking by HIV-1 Vpu and Nef

The HIV-1 accessory protein Nef is well known for its manipulation of host cell endosomal trafficking. By linking transmembrane proteins to endosomal coats, Nef removes them from the surface of infected cells. Modulation of MHC proteins leads to viral evasion of cellular adaptive immunity, whereas modulation of receptors for the HIV envelope glycoprotein, including CD4, enhances viral infectivity. The other HIV-1 accessory proteins, Vif, Vpr and Vpu, share a mechanism of action distinct from Nef in that each interacts with a multi-subunit ubiquitin ligase complex to target cellular proteins for proteosomal degradation. However, newly uncovered functions and mechanistic aspects of Vpu likely involve endosomal trafficking: these include counteraction of the innate antiviral activity of the cellular transmembrane protein BST-2 (tetherin), as well as the removal of the lipid-antigen presenting protein CD1d and the natural killer cell ligand NTB-A from the cell surface. This review focuses on how Nef and Vpu interfere with normal intracellular membrane trafficking to facilitate the spread and virulence of HIV-1.

β-TrCP is dispensable for Vpu's ability to overcome the CD317/Tetherin-imposed restriction to HIV-1 release

BackgroundThe cellular transmembrane protein CD317/BST-2/HM1.24/Tetherin restricts HIV-1 infection by physically tethering mature virions to the surface of infected cells. HIV-1 counteracts this restriction by expressing the accessory protein Vpu, yet the mechanism of this antagonism is incompletely understood. β-TrCP is the substrate recognition domain of an E3 ubiquitin ligase complex that interacts with the di-serine motif S52/S56 in the cytoplasmic tail of Vpu to target the CD4 receptor for proteasomal degradation. Recently, it has been suggested that β-TrCP is also critically involved in Vpu's ability to overcome the CD317-mediated virion release block.ResultsTo test this model, we analyzed the consequences of several experimental strategies to interfere with the Vpu-β-TrCP protein-protein interaction. Under these conditions, we studied effects of Vpu on expression and localization of CD317 and CD4, as well as on its ability to promote HIV-1 release. Our results demonstrate a strict requirement for Vpu's di-serine motif for degradation of CD4 and also CD317, reduction of cell surface exposure of CD317, and HIV-1 release enhancement. We further show a critical role of β-TrCP2, but not of the structurally related β-TrCP1 isoform, for Vpu-mediated degradation of both receptors. Most importantly, Vpu remained active in downregulating CD317 from the cell surface and in overcoming the HIV-1 release restriction in β-TrCP-depleted cells.ConclusionsThese results demonstrate that β-TrCP is not strictly required for Vpu's ability to counteract the CD317-imposed virion release block and support the relevance of cell surface down-modulation of the restriction factor as a central mechanism of Vpu antagonism. Moreover, we propose the existence of a critical, yet to be identified cellular factor that interacts with Vpu via its di-serine motif to alter the trafficking of the restriction factor.

Abstract 2968: Aberrant expression of polycystin-1 in renal cell tumors

Abstract Polycystin-1 (PC1) is a cellular transmembrane protein encoded by the polycystic kidney disease gene (PKD1) prevalently expressed in developing/mature kidney and in autosomal polycystic kidney disease (ADPKD). Limited data are available concerning the PC1 involvement in renal tumorigenesis. Polycystin-1 expression was evaluated by immunohistochemistry (IHC) in 8 clear cell renal cell carcinomas (RCCs), 7 tubulopapilary cell type tumors, 3 solid RCCs developed in patients with von Hippel-Lindau disease (VHL), one RCC developed in a patient on chronic haemodyalisis and one angyomyolipoma in a patient with Tuberous sclerosis (TS). In parallel, the BIZ cell line derived from a high grade clear cell RCC and the WUR cell line derived from a tubulopapillary type RCC, were used to measure the level of PC1 expression by both immunolabeling and immunoblotting analyses. Five cases of clear cell type RCCs, and two tubulopapillary cell type RCCs consistently expressed PC1 with a percentage of labeled neoplastic cells ranging between 15% and 65%. Un the VHL disease-associated renal cell carcinoma, both the tumor cells and cystic tissues areas weakly expressed the PC1 protein. In TS-associated angiomyolipoma, the vascular component was PC1 positive, while the tumor cells were scarcely stained. High level of expression of PC1 was confirmed by both IHC and immunoblotting in WUR RCC cells (80% of labeled cells), while BIZ RCC cells exhibited a moderate PC1 staining (30% of labeled cells). Comparatively, in the normal kidney, consistent level of polycystin 1 was detected in distal tubules, collecting duct, glomerular podocytes and vascular smooth muscle cells. The strongest immunoreactivity against PC1 was observed in epithelial cells lining the cystic components in all ADPKD tissues. In conclusion, the presented data indicate consistent level of expression of the polycystin-1 protein in a number renal cell carcinomas and suggest that PC1 expression is linked to the tumor cell type, being more frequent event in clear cell type RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2968.

Aberrant Expression of Polycystin-1 in Renal Cell Tumors

Polycystin-1 (PC1) is a cellular transmembrane protein coded by the polycystic kidney disease (PKD1) gene, prevalently expressed in developing/mature kidney and in autosomal polycystic kidney disease (ADPKD). Limited data are available concerning the PC1 involvement in renal tumorigenenesis. Polycystin-1 expression was evaluated in 8 clear cell renal cell carcinomas (RCCs), 7 tubulopapillary cell type tumors, 3 solid RCCs developed in patients with von Hippel-Lindau disease (VHLD), one RCC developed in a patient on chronic haemodialysis and one angiomyolipoma in a patient with Tuberous sclerosis (TS). In the normal kidney, consistent level of polycystin-1 was detected in distal tubules, collecting duct, glomerular podocytes and vascular smooth muscle cells. The strongest immunoreactivity against polycystin-1 was observed in epithelial cells lining the cystic components in all ADPKD tissues. Five cases of clear cell type RCCs and two-tubulopapillary cell type RCCs consistently expressed PC1. In the VHL disease associated renal carcinomas, both the neoplastic cells and cystic tissue areas weakly expressed PC1. In TS-associated angiomyolipoma, the vascular component was PC1 positive, while the tumoral cells were scarcely stained. The present report indicates consistent expression of the PKD1 gene product polycystin-1, in normal kidney, ADPKD tissues, and renal cell carcinomas. The findings suggest that the level of PC1 expression is linked to tumor cell type, being a more frequent event in clear cell RCC.

Murine colonic mucosa hyperproliferation. II. PKC-beta activation and cPKC-mediated cellular CFTR overexpression.

In the companion article (Umar S, Scott J, Sellin JH, Dubinsky WP, and Morris AP, Am J Physiol Gastrointest Liver Physiol 278: 753-764, 2000), we have shown that transmissible murine colonic hyperplasia (TMCH) increased cellular cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein expression, relocalized CFTR within colonocytes, and enhanced mucosal cAMP-dependent Cl(-) secretion. We show here that these changes were dependent on elevated cellular levels of membrane-bound Ca(2+)- and diacylglycerol-sensitive protein kinase C (PKC) activity (12-fold), induced by selective (3- to 4-fold) rises in conventional PKC (cPKC) isoform expression and membrane translocation. Three cPKC isoforms were detected in isolated crypts: alpha, beta1, and beta2. cPKC-beta1 rises preceded and those of cPKC-alpha and cPKC-beta2 paralleled cellular hyperproliferation and its effects on CFTR expression and cAMP-dependent Cl(-) current secretion. Only cPKC-beta1 and cPKC-beta2 were membrane translocated during TMCH. Furthermore, only cPKC-beta1 trafficked to the nucleus, whereas cPKC-beta2 remained partitioned among cytosolic, membrane, and cytoskeletal subcellular fractions. Modest increases in novel PKC-epsilon (nPKC-epsilon) expression and subcellular membrane partitioning were recorded during TMCH, but no changes were seen for PKC-delta or -eta. No nPKC isoform nuclear partitioning was detected. The orally bioactive cPKC inhibitor Ro-32-0432 reversed both TMCH and elevated cellular CFTR mRNA levels, whereas a pharmacologically inert analog (Ro-31-6045) failed to inhibit either response. On the basis of these facts, we present a new hypothesis whereby PKC-dependent cellular proliferation promotes endogenous cellular CFTR levels. PKC-beta1 was identified as a candidate regulatory PKC isoform.

The 21-kDa polypeptide (VAP21) in the rabies virion is a CD99-related host cell protein.

In our monoclonal antibody (MAb) stocks prepared against the BHK-21 cell antigens, two (#11875 and 28276) recognized a 21-kDa polypeptide (referred to as VAP21) which is efficiently incorporated into the rabies virion. By using these MAbs, we isolated the cDNA clones that encoded a polypeptide of 144 amino acids from our BHK-21 cell cDNA library. Based on the following evidence, the cDNA was assumed to encode a full-length sequence of VAP21 antigen: i) expression of the cDNA in animal cells resulted in the production of a polypeptide recognized by the two MAbs, and its electrophoretic mobility was the same as that of authentic VAP21 antigen; and ii) immunization with the products from the cDNA-transformed E. coli cells raised specific antibodies in rabbits that recognized a 21-kDa polypeptide in the virion. From the deduced amino acid sequence, it is suggested that the VAP21 antigen has a molecular structure of type-I transmembrane protein containing characteristic proline-rich and glycine-rich regions in its ectodomain. Homology searches resulted in finding homologous sequences (totally about 40% homology) in the human MIC2 gene product (CD99; 32-kDa) of T lymphocytes. These results suggest that the VAP21 antigen in the rabies virion is a cellular CD99-related transmembrane protein.

CD30 ligand is expressed on resting normal and malignant human B lymphocytes.

CD30, a member of the tumour necrosis receptor superfamily, is physiologically expressed on a subpopulation of T helper cells in normal individuals but is also expressed on several malignant and virally transformed cells. Its ligand (CD30L) is a pleiotropic cellular transmembrane protein that can induce cell death in several CD30+ cell lines. CD30L expression has been reported on activated human peripheral blood T lymphocytes and macrophages but not on B cells. Here we show that the CD30L is expressed on resting normal and on malignant B cells in addition to both CD4+ and CD8+ subsets of activated T cells, making it the second tumour necrosis family member, in addition to the CD27 ligand, that can be expressed on both T and B cells. These findings raise the possibility that the CD30L has a role in B-cell/T-cell communication and that B and T cells are likely to be involved in the growth regulation of CD30+ tumours.