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The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described, although whether such attenuation is retained for later variants like BA.5 remains controversial. We show that a BA.5 isolate was significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, with BA.5 infections showing increased neuroinvasiveness, resulting in brain infection and mortality, similar to that seen for original ancestral isolates. BA.5 also infected human cortical brain organoids to a greater extent than the BA.1 and original ancestral isolates. In the brains of mice neurons were the main target of infection, and in human organoids neuronal progenitor cells and immature neurons were infected. Evidence for brain infection and brain damage in certain COVID-19 patients is becoming compelling, with the results herein illustrating the increasing intrinsic neuropathogenic potential of evolving omicron variants.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes often debilitating rheumatic disease in tropical Central and South America. There are currently no licensed vaccines or antiviral drugs available for MAYV disease. Here, we generated Mayaro virus-like particles (VLPs) using the scalable baculovirus-insect cell expression system. High-level secretion of MAYV VLPs in the culture fluid of Sf9 insect cells was achieved, and particles with a diameter of 64 to 70 nm were obtained after purification. We characterize a C57BL/6J adult wild-type mouse model of MAYV infection and disease and used this model to compare the immunogenicity of VLPs from insect cells with that of VLPs produced in mammalian cells. Mice received two intramuscular immunizations with 1 μg of nonadjuvanted MAYV VLPs. Potent neutralizing antibody responses were generated against the vaccine strain, BeH407, with comparable activity seen against a contemporary 2018 isolate from Brazil (BR-18), whereas neutralizing activity against chikungunya virus was marginal. Sequencing of BR-18 illustrated that this virus segregates with genotype D isolates, whereas MAYV BeH407 belongs to genotype L. The mammalian cell-derived VLPs induced higher mean neutralizing antibody titers than those produced in insect cells. Both VLP vaccines completely protected adult wild-type mice against viremia, myositis, tendonitis, and joint inflammation after MAYV challenge. IMPORTANCE Mayaro virus (MAYV) is associated with acute rheumatic disease that can be debilitating and can evolve into months of chronic arthralgia. MAYV is believed to have the potential to emerge as a tropical public health threat, especially if it develops the ability to be efficiently transmitted by urban mosquito vectors, such as Aedes aegypti and/or Aedes albopictus. Here, we describe a scalable virus-like particle vaccine against MAYV that induced neutralizing antibodies against a historical and a contemporary isolate of MAYV and protected mice against infection and disease, providing a potential new intervention for MAYV epidemic preparedness.

Branchpoint elements are required for intron removal, and variants at these elements can result in aberrant splicing. We aimed to assess the value of branchpoint annotations generated from recent large-scale studies to select branchpoint-abrogating variants, using hereditary cancer genes as model.We identified branchpoint elements in 119 genes associated with hereditary cancer from 3 genome-wide experimentally-inferred and 2 predicted branchpoint data sets. We then identified variants that occur within branchpoint elements from public databases. We compared conservation, unique variant observations, and population frequencies at different nucleotides within branchpoint motifs. Finally, selected minigene assays were performed to assess the splicing effect of variants at branchpoint elements within mismatch repair genes.There was poor overlap between predicted and experimentally-inferred branchpoints. Our analysis of cancer genes suggested that variants at -2 nucleotide, -1 nucleotide, and branchpoint positions in experimentally-inferred canonical motifs are more likely to be clinically relevant. Minigene assay data showed the -2 nucleotide to be more important to branchpoint motif integrity but also showed fluidity in branchpoint usage.Data from cancer gene analysis suggest that there are few high-risk alleles that severely impact function via branchpoint abrogation. Results of this study inform a general scheme to prioritize branchpoint motif variants for further study.

A frequently repeated premise is that viruses evolve to become less pathogenic. This appears also to be true for SARS-CoV-2, although the increased level of immunity in human populations makes it difficult to distinguish between reduced intrinsic pathogenicity and increasing protective immunity. The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described and appears to be due to reduced utilization of TMPRRS2. That this reduced pathogenicity remains true for omicron BA.5 was recently reported. In sharp contrast, we show that a BA.5 isolate was significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, with BA.5 infection showing increased neurovirulence, encephalitis and mortality, similar to that seen for an original ancestral isolate. BA.5 also infected human cortical brain organoids to a greater extent than a BA.1 and original ancestral isolate. Neurons were the target of infection, with increasing evidence of neuron infection in COVID-19 patients. These results argue that while omicron virus may be associated with reduced respiratory symptoms, BA.5 shows increased neurovirulence compared to earlier omicron sub-variants.

Warmer climatic conditions have been associated with fewer COVID-19 cases. Herein we infected K18-hACE2 mice housed at the standard animal house temperature of ∼22 °C, or at ∼31 °C, which is considered to be thermoneutral for mice. On day 2 post infection, RNA-Seq analyses showed no significant differential gene expression lung in lungs of mice housed at the two temperatures, with almost identical viral loads and type I interferon responses. There was also no significant difference in viral loads in lungs on day 5, but RNA-Seq and histology analyses showed clearly elevated inflammatory signatures and infiltrates. Thermoneutrality thus promoted lung inflammation. On day 2 post infection mice housed at 31 °C showed reduced viral loads in nasal turbinates, consistent with increased mucociliary clearance at the warmer ambient temperature. These mice also had reduced virus levels in the brain, and an ensuing amelioration of weight loss and a delay in mortality. Warmer air temperatures may thus reduce infection of the upper respiratory track and the olfactory epithelium, resulting in reduced brain infection. Potential relevance for anosmia and neurological sequelae in COVID-19 patients is discussed.

Human ACE2 Human angiotensin converting enzyme 2 (hACE2) is the key cell attachment and entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with the original SARS-CoV-2 isolates unable to use mouse ACE2 (mACE2). Herein we describe the emergence of a SARS-CoV-2 strain capable of ACE2-independent infection and the evolution of mouse-adapted (MA) SARS-CoV-2 by in vitro serial passaging of virus in co-cultures of cell lines expressing hACE2 and mACE2. MA viruses evolved with up to five amino acid changes in the spike protein, all of which have been seen in human isolates. MA viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates and caused characteristic lung histopathology. One MA virus also evolved to replicate efficiently in several ACE2-negative cell lines across several species, including clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ACE2 knockout cells. An E484D substitution is likely involved in ACE2-independent entry and has appeared in only ≈0.003 per cent of human isolates globally, suggesting that it provided no significant selection advantage in humans. ACE2-independent entry reveals a SARS-CoV-2 infection mechanism that has potential implications for disease pathogenesis, evolution, tropism, and perhaps also intervention development.

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, withGzma-/-mouse studies having informed our understanding of GZMA’s physiological function. We show herein thatGzma-/-mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereasNntis truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated inGzma-/-mice; however, the presence ofNntand the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6JGzmaS211Amouse provided the first insights into GZMA’s bioactivity free of background issues, with circulating proteolytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain hadNntsequencing reads inconsistent with a C57BL/6J genetic background.Nntand C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

SUMMARYHuman ACE2 (hACE2) is the key cell attachment and entry receptor for SARS-CoV-2, with the original SARS-CoV-2 isolates unable to use mouse ACE2 (mACE2). Herein we describe a new system for generating mouse-adapted SARS-CoV-2 in vitro by serial passaging virus in co-cultures of cell lines expressing hACE2 and mACE2. Mouse-adapted viruses emerged with up to five amino acid changes in the spike protein, all of which have been seen in human isolates. Mouse-adapted viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates, and caused severe lung histopathology. One mouse-adapted virus was also able to replicate efficiently in ACE2-negative cell lines, with ACE2-independent entry by SARS-CoV-2 representing a new biology for SARS-CoV-2 that has potential widespread implications for disease and intervention development.

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma-/- mouse studies having informed our understanding of GZMA’s physiological function. We show herein that Gzma-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J GzmaS211A mouse provided the first insights into GZMA’s bioactivity free of background issues, with circulating proteolytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

BACKGROUND & AIMS: Colorectal cancer is an epigenetically heterogeneous disease, however, the extent and spectrum of the CpG island methylator phenotype (CIMP) is not clear. METHODS: Genome-scale methylation and transcript expression were measured by DNA Methylation and RNA expression microarray in 216 unselected colorectal cancers, and findings were validated using The Cancer Genome Atlas 450K and RNA sequencing data. Mutations in epigenetic regulators were assessed using CIMP-subtyped Cancer Genome Atlas exomes. RESULTS: CIMP-high cancers dichotomized into CIMP-H1 and CIMP-H2 based on methylation profile. KRAS mutation was associated significantly with CIMP-H2 cancers, but not CIMP-H1 cancers. Congruent with increasing methylation, there was a stepwise increase in patient age from 62 years in the CIMP-negative subgroup to 75 years in the CIMP-H1 subgroup (P < .0001). CIMP-H1 predominantly comprised consensus molecular subtype 1 cancers (70%) whereas consensus molecular subtype 3 was over-represented in the CIMP-H2 subgroup (55%). Polycomb Repressive Complex-2 (PRC2)-marked loci were subjected to significant gene body methylation in CIMP cancers (P < 1.6 × 10-78). We identified oncogenes susceptible to gene body methylation and Wnt pathway antagonists resistant to gene body methylation. CIMP cluster-specific mutations were observed in chromatin remodeling genes, such as in the SWItch/Sucrose Non-Fermentable and Chromodomain Helicase DNA-Binding gene families. CONCLUSIONS: There are 5 clinically and molecularly distinct subgroups of colorectal cancer. We show a striking association between CIMP and age, sex, and tumor location, and identify a role for gene body methylation in the progression of serrated neoplasia. These data support our recent findings that CIMP is uncommon in young patients and that BRAF mutant polyps in young patients may have limited potential for malignant progression. ispartof: CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY vol:8 issue:2 pages:269-290 ispartof: location:United States status: published

Granzyme A (GzmA) is a serine protease secreted by cytotoxic lymphocytes, with GzmA-/- mouse studies informing our understanding of GzmA’s physiological function. We show herein that GzmA-/- mice have a mixed C57BL/6J and C57BL/6N background and retain the full length Nicotinamide Nucleotide Transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in GzmA-/- mice; however, the presence of Nnt, rather than loss of GzmA, was responsible for this phenotype by constraining lymphocyte infiltration. A new CRISPR active site mutant C57BL/6J GzmAS211A mouse provided the first insights into GzmA’s bioactivity free of background issues, with circulating proteolytically active GzmA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of Bioprojects listing C57BL/6J as the mouse strain, had Nnt sequencing reads inconsistent with a C57BL/6J background. The Nnt issue has clearly complicated our understanding of GzmA and may similarly have influenced studies across a broad range of fields.

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intranasal hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lungs. RNA-Seq analyses illustrated that the model involves an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. Intranasal hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mice lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.AUTHOR SUMMARYSARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo, with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research.

Poxvirus systems have been extensively used as vaccine vectors. Herein a RNA-Seq analysis of intramuscular injection sites provided detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. The multiple adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1β, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such injection site vaccinology approaches should inform refinements in poxvirus-based vector design., Author summary Poxvirus vector systems have been widely developed for vaccine applications. Despite considerable progress, so far only one recombinant poxvirus vectored vaccine has to date been licensed for human use, with ongoing efforts seeking to enhance immunogenicity whilst minimizing reactogenicity. The latter two characteristics are often determined by early post-vaccination events at the injection site. We therefore undertook an injection site vaccinology approach to analyzing gene expression at the vaccination site after intramuscular inoculation with a recombinant, multiplication defective, vaccinia-based vaccine. This provided detailed insights into inter alia expression of vector-encoded immunoregulatory genes, as well as host innate and adaptive immune responses. We propose that such injection site vaccinology can inform rational vaccine vector design, and we discuss how the information and approach elucidated herein might be used to improve immunogenicity and limit reactogenicity of poxvirus-based vaccine vector systems.

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined, but could be direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory ‘cytokine-storm’, a cocktail of interferon gamma, interleukin 1β and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids and hearts of SARS-CoV-2 infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCO and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the FDA breakthrough designated drug apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage., A combination of phosphoproteomics, drug screening and single-cell sequencing approaches identifies how cytokines elevated in COVID-19 patients drives cardiac dysfunction, with BET inhibitors serving as potential lead candidates decrease ACE2 cardiac expression and infection.

Purpose: Branchpoint elements are required for intron removal, and variations at these elements can result in aberrant splicing. We aimed to assess the value of branchpoint annotations generated from recent large-scale studies to select branchpoint-abrogating variants, using hereditary cancer genes as model.Methods: We identified branchpoint elements in 119 genes associated with hereditary cancer from three genome-wide experimentally-inferred and two predicted branchpoint datasets. We then identified variants from public databases that occur within branchpoint elements. We compared conservation, unique variant observations, and population frequencies at different nucleotides within branchpoint motifs. Finally, selected minigene assays were performed to assess the functional impact of variants in branchpoint elements within mismatch repair genes.Results: There was poor overlap between predicted and experimentally-inferred branchpoints. Our analysis of cancer genes suggested that variants at -2nt, -1nt, and branchpoint positions in experimentally-inferred canonical motifs are more likely to be clinically relevant. Minigene assay data showed the -2nt to be more important to branchpoint motif integrity, but also demonstrated fluidity in branchpoint usage.Conclusion: Data from cancer gene analysis suggests that there will be few high-risk alleles that severely impact function via branchpoint abrogation. Results inform a general scheme to prioritize branchpoint motif variants for further study.

Global microplastic (MP) contamination and the effects on the environment are well described. However, the potential for MP consumption to affect human health remains controversial. Mice consuming ≈80 µg/kg/day of 1 µm polystyrene MPs via their drinking water for a month showed no weight loss, nor were MPs detected in organs. The microbiome was also unchanged. MP consumption did lead to small transcriptional changes in the colon suggesting plasma membrane perturbations and mild inflammation. Mice were challenged with the arthritogenic chikungunya virus, with MP consumption leading to a significantly prolonged arthritic foot swelling that was associated with elevated Th1, NK cell and neutrophil signatures. Immunohistochemistry also showed a significant increase in the ratio of neutrophils to monocyte/macrophages. The picture that emerges is reminiscent of enteropathic arthritis, whereby perturbations in the colon are thought to activate innate lymphoid cells that caninter aliamigrate to joint tissues to promote inflammation.

SUMMARYCardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined, but could be direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory ‘cytokine-storm’, a cocktail of interferon gamma, interleukin 1β and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids and hearts of SARS-CoV-2 infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCO and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the FDA breakthrough designated drug apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.

Poxvirus systems have been extensively used as vaccine vectors. Herein a systems vaccinology analysis of intramuscular injection sites provides detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. Adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1β, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such innate systems vaccinology approaches should inform refinements in poxvirus-based vector design.

Background: WNT activation is a hallmark of colorectal cancer. BRAF mutation is present in 15% of colorectal cancers, and the role of mutations in WNT signaling regulators in this context is unclear. Here, we evaluate the mutational landscape of WNT signaling regulators in BRAF mutant cancers. Methods: we performed exome-sequencing on 24 BRAF mutant colorectal cancers and analyzed these data in combination with 175 publicly available BRAF mutant colorectal cancer exomes. We assessed the somatic mutational landscape of WNT signaling regulators, and performed hotspot and driver mutation analyses to identify potential drivers of WNT signaling. The effects of Apc and Braf mutation were modelled, in vivo, using the Apcmin/+ and BrafV637/Villin-CreERT2/+ mouse, respectively. Results: RNF43 was the most frequently mutated WNT signaling regulator (41%). Mutations in the beta-catenin destruction complex occurred in 48% of cancers. Hotspot analyses identified potential cancer driver genes in the WNT signaling cascade, including MEN1, GNG12 and WNT16. Truncating APC mutation was identified in 20.8% of cancers. Truncating APC mutation was associated with early age at diagnosis (p &lt, 2 × 10−5), advanced stage (p &lt, 0.01), and poor survival (p = 0.026). Apcmin/+/BrafV637 animals had more numerous and larger SI and colonic lesions (p &lt, 0.0001 and p &lt, 0.05, respectively), and a markedly reduced survival (median survival: 3.2 months, p = 8.8 × 10−21), compared to animals with Apc or Braf mutation alone. Conclusions: the WNT signaling axis is frequently mutated in BRAF mutant colorectal cancers. WNT16 and MEN1 may be novel drivers of aberrant WNT signaling in colorectal cancer. Co-mutation of BRAF and APC generates an extremely aggressive neoplastic phenotype that is associated with poor patient outcome.

Conventional adenomas are initiated by APC gene mutation that activates the WNT signal. Serrated neoplasia is commonly initiated by BRAF or KRAS mutation. WNT pathway activation may also occur, however, to what extent this is owing to APC mutation is unknown. We examined aberrant nuclear β-catenin immunolocalization as a surrogate for WNT pathway activation and analyzed the entire APC gene coding sequence in serrated and conventional pathway polyps and cancers. WNT pathway activation was a common event in conventional pathway lesions with aberrant nuclear immunolocalization of β-catenin and truncating APC mutations in 90% and 89% of conventional adenomas and 82% and 70% of BRAF wild-type cancers, respectively. WNT pathway activation was seen to a lesser extent in serrated pathway lesions. It occurred at the transition to dysplasia in serrated polyps with a significant increase in nuclear β-catenin labeling from sessile serrated adenomas (10%) to sessile serrated adenomas with dysplasia (55%) and traditional serrated adenomas (9%) to traditional serrated adenomas with dysplasia (39%) (P=0.0001). However, unlike the conventional pathway, truncating APC mutations were rare in the serrated pathway lesions especially sessile serrated adenomas even when dysplastic (15%) and in the BRAF mutant cancers with microsatellite instability that arise from them (8%). In contrast, APC missense mutations that were rare in conventional pathway adenomas and cancers (3% in BRAF wild-type cancers) were more frequent in BRAF mutant cancers with microsatellite instability (32%). We conclude that increased WNT signaling is important in the transition to malignancy in the serrated pathway but that APC mutation is less common and the spectrum of mutations is different than in conventional colorectal carcinogenesis. Moderate impact APC mutations and non-APC-related causes of increased WNT signaling may have a more important role in serrated neoplasia than the truncating APC mutations common in conventional adenomas.

Colorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The BrafV637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development., Author summary SARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo, with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research.

Serrated pathway colorectal cancers (CRCs) are characterised by a BRAF mutation and half display microsatellite instability (MSI). The Wnt pathway is commonly upregulated in conventional CRC through APC mutation. By contrast, serrated cancers do not mutate APC. We investigated mutation of the ubiquitin ligases RNF43 and ZNRF3 as alternate mechanism of altering the Wnt signal in serrated colorectal neoplasia. RNF43 was mutated in 47/54(87%) BRAF mutant/MSI and 8/33(24%) BRAF mutant/microsatellite stable cancers compared to only 3/79(4%) BRAF wildtype cancers (p

Getah virus (GETV) is a mosquito-transmitted alphavirus primarily associated with disease in horses and pigs in Asia. GETV was also reported to have been isolated from mosquitoes in Australia in 1961, however, retrieval and sequencing of the original isolates (N544 and N554), illustrated that these viruses were virtually identical to the 1955 GETVMM2021 isolate from Malaysia. K-mer mining of the &gt, 40,000 terabases of sequence data in the Sequence Read Archive followed by BLASTn confirmation identified multiple GETV sequences in biosamples from Asia (often as contaminants), but not in biosamples from Australia. In contrast, sequence reads aligning to the Australian Ross River virus (RRV) were readily identified in Australian biosamples. To explore the serological relationship between GETV and other alphaviruses, an adult wild-type mouse model of GETV was established. High levels of cross-reactivity and cross-protection were evident for convalescent sera from mice infected with GETV or RRV, highlighting the difficulties associated with the interpretation of early serosurveys reporting GETV antibodies in Australian cattle and pigs. The evidence that GETV circulates in Australia is thus not compelling.

BACKGROUND:Colorectal cancer is an epigenetically heterogeneous disease, however the extent and spectrum of the CpG Island Methylator Phenotype (CIMP) is not clear.RESULTSAn unselected cohort of 216 colorectal cancers clustered into five clinically and molecularly distinct subgroups using Illumina 450K DNA methylation arrays. CIMP-High cancers were most frequent in the proximal colons of female patients. These dichotomised into CIMP-Hl and CIMP-H2 based on methylation profile which was supported by over representation of BRAF (74%, PKRAS (55%, P78). We identified oncogenes susceptible to gene body methylation and Wnt pathway antagonists resistant to gene body methylation. CIMP cluster specific mutations were observed for genes involved in chromatin remodelling, such as in the SWI/SNF and NuRD complexes, suggesting synthetic lethality.CONCLUSIONThere are five clinically and molecularly distinct subgroups of colorectal cancer based on genome wide epigenetic profiling. These analyses highlighted an unidentified role for gene body methylation in progression of serrated neoplasia. Subgroup-specific mutation of distinct epigenetic regulator genes revealed potentially druggable vulnerabilities for these cancers, which may provide novel precision medicine approaches.

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.

Formalin fixation and embedding of clinical tissue samples in paraffin is a common method for archiving biological material. These samples are often well annotated and provide an invaluable resource for research. However, this process of fixation and storage of tissue leads to DNA damage and fragmentation. The use of DNA from formalin fixed, paraffin-embedded (FFPE) tissue to interrogate methylation levels on a genome-wide scale can pose challenges. We compared fresh and matched FFPE tissue DNA samples using the Illumina Infinium HD Human Methylation 450K BeadChip platform with a companion application for repair and "restoration" of DNA from FFPE tissue. Our results showed good correlation between fresh and FFPE sample data. FFPE DNA captured 99% of the CpG sites on the array on average. Significant cancer subgroups based on the CpG island methylator phenotype (CIMP) were clearly distinguished for both fresh and FFPE sample sets with cluster and scaling analysis. The DNA methylation status for the five standard CIMP panel genes which was evaluated for all samples by the MethyLight assay was correctly assigned in both fresh and FFPE samples by the array data. We conclude that the "restoration" method followed by assay on the Infinium HD Human Methylation 450K microarray can produce good quality data for DNA from FFPE samples.

BACKGROUND AND AIMS: Colorectal cancer is an epigenetically heterogeneous disease, however the extent and spectrum of the CpG Island Methylator Phenotype (CIMP) is not clear.METHODS: Genome scale methylation and transcript expression were measured using the Illumina HM450 DNA methylation and HT12 V3 expression microarrays in 216 unselected colorectal cancers. Mutations in epigenetic regulators were assessed using CIMP-classified Cancer Genome Atlas exomes.RESULTS: CIMP-High cancers dichotomised into CIMP-H1 and CIMP-H2 based on methylation profile, which was supported by over-representation of BRAF (74%, P Citation Format: Vicki L. Whitehall, Lochlan Fennell, Troy Dumenil, Gunter Hartel, Katia Nones, Catherine Bond, Diane McKeone, Ann-Marie Patch, Stephen Kazakoff, John Pearson, Nicola Waddell, Pratyaksha Wirapati, Paul Lochead, Shuji Ogino, Sabine Tejpar, Barbara Leggett. BRAF and KRAS mutation define distinct subtypes of the CpG island methylator phenotype in colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 479.

Background The length polymorphism repeat in the promoter region of the serotonin transporter gene (5HTTLPR) is one of the most studied polymorphisms for association with a range of psychiatric and personality phenotypes. However, the original 5HTTLPR assay is prone to bias toward short allele calling. Methods We designed new assays for the 5HTTLPR suitable for large-scale genotyping projects and we genotyped 13 single nucleotide polymorphisms (SNPs) in a 38-kilobase region around the 5HTTLPR, including SNP rs25531, a polymorphism of the 5HTTLPR long allele. Association analysis was conducted for major depression and/or anxiety disorder in unrelated cases ( n = 1161) and control subjects ( n = 1051) identified through psychiatric interviews administered to a large population sample of Australian twin families. Participants had been scored for personality traits several years earlier ( n ≥ 2643 unrelated individuals). Results We identified a two-SNP haplotype proxy for 5HTTLPR; the CA haplotype of SNPs rs4251417 and rs2020934 is coupled with the short allele of 5HTTLPR ( r 2 = .72). We found evidence for association ( p = .0062, after accounting for multiple testing) for SLC6A4 SNPs rs6354 and rs2020936 (positioned in a different linkage disequilibrium [LD] block about 15.5 kb from 5HTTLPR) with anxiety and/or depression and neuroticism, with the strongest association for recurrent depression with onset in young adulthood (odds ratio=1.55, 95% confidence interval=1.16–2.06). Conclusions The associated SNPs are in the same LD block as the variable number of tandem repeats serotonin transporter intron 2 marker, for which association has previously been reported.

Somatic mutations of the BRAF gene are common in melanomas and nevi but the contribution of polymorphisms in this gene to melanoma or nevus susceptibility remains unclear. An Australian melanoma case-control sample was typed for 16 single nucleotide polymorphisms (SNP) within the BRAF gene, and five SNP in three neighboring genes. The sample comprised 755 melanoma cases from 740 families stratified by family history of melanoma and controls from 635 unselected twin families (2,239 individuals). Ancestry of the cases and controls was recorded, and the twins had undergone skin examination to assess total body nevus count, degree of freckling, and pigmentation phenotype. Genotyping was carried out via primer extension followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. SNP in the BRAF gene were found to be weakly associated with melanoma status but not with development of nevi or freckles. The estimated proportion of attributable risk of melanoma due to variants in BRAF is 1.6%. This study shows that BRAF polymorphisms predispose to melanoma but the causal variant has yet to be determined. The burden of disease associated with this variant is greater than that associated with the major melanoma susceptibility locus CDKN2A, which has an estimated attributable risk of 0.2%.

The WNT and MAPK signaling pathways are central to colorectal homeostasis. Mutation of the APC tumor suppressor gene activates the WNT pathway to initiate most conventional adenomas. By contrast, oncogenic BRAF mutation initiates development of sessile serrated adenomas (SSA) via activation of the MAPK pathway. The purpose of this study was to investigate the frequency, timing and mechanism of WNT pathway activation in serrated colorectal neoplasia. Methods: The WNT pathway was examined in a range of colorectal polyp and cancer subtypes. Aberrant nuclear immuno-localization of β-catenin was used as a surrogate for WNT pathway activation, compared to the normal membranous staining pattern. The entire coding region of the APC gene was sequenced using a custom enrichment panel that included 242 primer pairs (GeneRead DNAseq Targeted Panel, Qiagen) and the constructed library was sequenced using Illumina's HT TruSeq Kit. As another mechanism of activating WNT, mutations were also assessed in the E3 ubiquitin ligase genes RNF43 and ZNRF3 using Sanger sequencing. To test the interaction of the WNT and MAPK pathways in vivo, a conditional BRAF mutant mouse model was crossed with an inducible Cre driven by the murine Villin gene promoter, to restrict Cre expression and induction of the BRAF mutation to the intestine at 2 weeks. The chemical carcinogen azoxymethane was administered weekly for 6 weeks to activate the WNT pathway from 3 weeks of age. The potential therapeutic implication of targeting these pathways was tested in vitro using the porcupine inhibitor LGK974 and MEK inhibitor PD0325901. Results: The majority of conventional pathway polyps and cancers had nuclear localization of β-catenin (22/28, 79% adenomas and 36/42, 86% BRAF wildtype cancers) and mutations in APC (17/20, 85% adenomas and 19/30, 63% BRAF wild type cancers). Sessile serrated adenomas infrequently showed nuclear staining of β-catenin and 0/20 SSA had mutations. By contrast, the WNT signal was increased in the dysplastic component of SSA with a focus of dysplasia (76/137, 55%), however APC mutations were uncommon (3/20, 15%). Similarly, BRAF mutant cancers had nuclear β-catenin in 36/92 (39%) cancers compared to APC mutation in only 9/80 (11%) samples. This did not significantly differ when samples were stratified by microsatellite instability status. RNF43 and ZNRF3 were commonly mutated in BRAF mutant / microsatellite unstable cancers (47/54, 87% and 16/54, 30%, respectively) as well as BRAF mutant / microsatellite stable cancers (8/33, 24% and 5/33, 15%) compared to only 3/79 (4%) RNF43 mutations and 0/27 ZNRF3 mutations in BRAF wildtype cancers. This was accompanied by lower transcript expression for both RNF43 and ZNRF3 in BRAF mutant compared to BRAF wildtype cancers (P Conclusions: The WNT pathway is commonly activated in serrated neoplasia at the transition to dysplasia. This is not due APC mutation but rather mutation of the upstream RNF43 and/or ZNRF43 genes or other WNT pathway targets. The combination of WNT and MAPK pathway inhibition offers potential for improving therapy for patients with serrated pathway cancers. This abstract is also being presented as Poster A21. Citation Format: Vicki Whitehall, Jennifer Borowsky, Catherine Bond, Mark Bettington, Sally-Ann Pearson, Murugan Kalimutho, John Liu, Troy Dumenil, Diane McKeone, Saara Jamieson, Winnie Fernando, Lochlan Fennell, Andrew Clouston, Christophe Rosty, Ian Brown, Neal Walker, Barbara Leggett. Wnt and MAPK pathway activation in conventional and serrated colorectal neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr PR04.

The length polymorphism repeat (5HTTLPR) in the promoter region of the serotonin transporter gene (SLC6A4, also known as 5HTT) is extensively studied in the context of psychiatric phenotypes, particularly in major depressive disorder. However, investigation of this polymorphism in the context of the current generation of large-scale genome-wide association studies is precluded, as the genotyping technology is limited to single-nucleotide polymorphisms (SNPs). Using genome-wide and 5HTTLPR genotype data from a total of 2823 unrelated individuals, we show that no single SNP is in high linkage disequilibrium (LD) with 5HTTLPR but some two-SNP haplotypes provide reasonable predictors. Hence, two-SNP haplotypes can be used as proxies for 5HTTLPR in genome-wide association studies. Analyses are repeated for sets of SNPs that are included in different genome-wide SNP platforms.

A common single nucleotide polymorphism (SNP) in the 5′ untranslated region (5′UTR) of the epidermal growth factor ( EGF ) gene modulates the level of transcription of this gene and hence is associated with serum levels of EGF. This variant may be associated with melanoma risk, but conflicting findings have been reported. An Australian melanoma case–control sample was typed for the EGF+61A>G transversion (rs4444903). The sample comprised 753 melanoma cases from 738 families stratified by family history of melanoma and 2387 controls from 645 unselected twin families. Ancestry of the cases and controls was recorded, and the twins had undergone skin examination to assess total body nevus count, degree of freckling and pigmentation phenotype. SNP genotyping was carried out via primer extension followed by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The EGF+61 SNP was not found to be significantly associated with melanoma status or with development of nevi or freckles. Among melanoma cases, however, G homozygotes had thicker tumors (p=0.05), in keeping with two previous studies. The EGF polymorphism does not appear to predispose to melanoma or nevus development, but its significant association with tumor thickness implies that it may be a useful marker of prognosis.

Association of the valine/methionine variant at codon 66 (Val66Met) of brain derived neurotrophic factor (BDNF) has been reported inconsistently across a spectrum of psychiatric disorders. Haplotypes of six tagging single nucleotide polymorphisms (SNPs) of a 37-kb region of dystrobrevin-binding protein 1 (DTNBP1) were found to be associated with schizophrenia. These haplotypes have not been studied extensively for other psychiatric disorders but are plausible candidates for anxiety and depression disorders. Here, association between variants of BDNF and DTNBP1, and multiple anxiety and depression phenotypes is explored.Study participants were selected as sibling pairs that were either concordant or discordant for extreme neuroticism scores from a total sample of 18 742 Australian twin individuals and their siblings. All participants completed detailed Composite International Diagnostic Interview from which diagnoses of Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV depression and anxiety disorders were determined. Six hundred and seventy-four participants had a diagnosis of anxiety and/or depression from 492 families. The BDNF Val66Met and six DTNBP1 (rs3213207, rs1011313, rs2619528, rs760761, rs1018381, rs2619538) SNPs were genotyped on samples from study participants (n=2045 from 987 families) and, where possible, their parents (n=787). Family-based association tests were conducted between the individual SNPs and the DTNBP1 six SNP haplotypes and anxiety, depression, and neuroticism.We found no convincing evidence for association between any of the variants studied and anxiety, depression, or neuroticism.This study sample is relatively large but our results do not support an association between BDNF Val66Met and anxiety, depression, or neuroticisim. DTNBP1 haplotypes, which have been found to be risk factors for schizophrenia, are unlikely to be risk factors for anxiety and depression.

The Val158Met polymorphism of the gene encoding catechol-O-methyltransferase (COMT) is one of the most widely tested variants for association with psychiatric disorders, but replication has been inconsistent including both sex limitation and heterogeneity of the associated allele. In this study we investigate the association between three SNPs from COMT and anxiety and depression disorders and neuroticism. all measured within the same study sample. Participants were selected as sibling pairs (or multiples) that were either concordant or discordant for extreme neuroticism. scores from a total sample of 18,742 Australian twin individuals and their siblings. All participants completed the Composite International Diagnostic Interview (CIDI) from which diagnoses of DSM-IV depression and anxiety disorders were determined. Of the participants, 674 had a diagnosis of anxiety and/or depression from 492 families. Study participants (n = 2,045 from 987 families) plus, where possible, their parents were genotyped for rs737865, rs4680 (Val158Met), and rs165599. Using family based tests we looked for association between these variants and neuroticism, depression, anxiety, panic disorder and agarophobia (PDAG) and obsessive compulsive disorder. We found no convincing evidence for association either in allelic or genotypic tests for the total sample or when the sample was stratified by sex. Haplotype T-G-G showed weak association (P=0.042) with PDAG before correction for multiple testing, association between this haplotype and schizophrenia has been previously reported in an Australian sample. (C) 2008 Wiley-Liss, Inc.