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1.
Appl Environ Microbiol ; : e0149723, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39012098

RESUMO

The future of the COVID pandemic and its public health and societal impact will be determined by the profile and spread of emerging variants and the timely identification and response to them. Wastewater surveillance of SARS-CoV-2 has been widely adopted in many countries across the globe and has played an important role in tracking infection levels and providing useful epidemiological information that cannot be adequately captured by clinical testing alone. However, novel variants can emerge rapidly, spread globally, and markedly alter the trajectory of the pandemic, as exemplified by the Delta and Omicron variants. Most mutations linked to the emergence of new SARS-CoV-2 variants are found within variable regions of the SARS-CoV-2 Spike protein. We have developed a duplex hemi-nested PCR method that, coupled with short amplicon sequencing, allows simultaneous typing of two of the most highly variable and informative regions of the Spike gene: the N-terminal domain and the receptor binding motif. Using this method in an operationalized public health program, we identified the first known incursion of Omicron BA.1 into Victoria, Australia and demonstrated how sensitive amplicon sequencing methods can be combined with wastewater surveillance as a relatively low-cost solution for early warning of variant incursion and spread.IMPORTANCEThis study offers a rapid, cost-effective, and sensitive approach for monitoring SARS-CoV-2 variants in wastewater. The method's flexibility permits timely modifications, enabling the integration of emerging variants and adaptations to evolving SARS-CoV-2 genetics. Of particular significance for low- and middle-income regions with limited surveillance capabilities, this technique can potentially be utilized to study a range of pathogens or viruses that possess diverse genetic sequences, similar to influenza.

2.
Cell Mol Life Sci ; 78(1): 249-270, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32170339

RESUMO

eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/antagonistas & inibidores , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Morfolinas/farmacologia , Mutagênese Sítio-Dirigida , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
3.
Biochem J ; 477(14): 2735-2754, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32648926

RESUMO

The mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) are serine/threonine protein kinases that are activated by the ERK1/2 (extracellular regulated kinase) and p38α/ß MAPK pathways. The MNKs have previously been implicated in metabolic disease and shown to mediate diet-induced obesity. In particular, knockout of MNK2 in mice protects from the weight gain induced by a high-fat diet. These and other data suggest that MNK2 regulates the expansion of adipose tissue (AT), a stable, long-term energy reserve that plays an important role in regulating whole-body energy homeostasis. Using the well-established mouse 3T3-L1 in vitro model of adipogenesis, the role of the MNKs in adipocyte differentiation and lipid storage was investigated. Inhibition of MNK activity using specific inhibitors failed to impair adipogenesis or lipid accumulation, suggesting that MNK activity is not required for adipocyte differentiation and does not regulate lipid storage. However, small-interfering RNA (siRNA) knock-down of MNK2 did reduce lipid accumulation and regulated the levels of two major lipogenic transcriptional regulators, ChREBP (carbohydrate response element-binding protein) and LPIN1 (Lipin-1). These factors are responsible for controlling the expression of genes for proteins involved in de novo lipogenesis and triglyceride synthesis. The knock-down of MNK2 also increased the expression of hormone-sensitive lipase which catalyses the breakdown of triglyceride. These findings identify MNK2 as a regulator of adipocyte metabolism, independently of its catalytic activity, and reveal some of the mechanisms by which MNK2 drives AT expansion. The development of an MNK2-targeted therapy may, therefore, be a useful intervention for reducing weight caused by excessive nutrient intake.


Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Células 3T3-L1 , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno
4.
Adipocyte ; 9(1): 427-442, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32787498

RESUMO

Given the high and increasing prevalence of obesity and associated disorders, such as type-2 diabetes, it is important to understand the mechanisms that regulate lipid storage and the differentiation of fat cells, a process termed adipogenesis. Using the well-established mouse 3T3-L1 in vitro model of adipogenesis, we refine how the induction of two key adipogenic transcription factors, CCAAT/enhancer-binding proteins (C/EBPs) ß and δ are regulated during early adipogenesis. We identify, in the gene promoters of Cebpb and Cebpd, the DNA response elements responsible for binding transcription factors that are activated by cAMP or glucocorticoids. We also show that mitogen-activated protein kinase (MAPK)-interacting kinase 2 (MNK2; Mknk2), which plays a distinct role in diet-induced obesity, is induced during early adipogenesis and identify the functional DNA response elements responsible for regulating its expression. Mknk2 expression is maintained in differentiated 3T3-L1 adipocytes and is expressed at high levels across a range of mouse adipose tissue depots. Together, these new insights help to clarify the transcriptional programme of early adipogenesis and identify Mknk2 as one of potentially many genes up-regulated during adipogenesis.


Assuntos
Adipogenia/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Elementos de Resposta , Células 3T3-L1 , Animais , Sítios de Ligação , Sequenciamento de Cromatina por Imunoprecipitação , Regulação da Expressão Gênica , Camundongos , Ligação Proteica
5.
Expert Opin Ther Targets ; 23(3): 187-199, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30650992

RESUMO

INTRODUCTION: The mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) are switched on by the oncogenic MAPK (ERK) signalling pathway. They phosphorylate eukaryotic initiation factor (eIF) 4E, a protein which recruits ribosomes to mRNAs and thereby mediates their translation. Importantly, overexpression of eIF4E can transform cells, and its function is controlled by a second oncogenic pathway, mechanistic target of rapamycin complex 1. Areas covered: We have evaluated the literature related to the role of the MNKs in human cancers, including their control by oncogenic signalling pathways; their expression and regulation in cancer cells and preclinical cancer models; and their roles in the proliferation, survival and migration/invasion of cancer cells. We also discuss progress towards generating specific and potent inhibitors of the MNKs and data obtained using such compounds. Expert opinion: The available data indicate that MNKs and/or eIF4E phosphorylation play a role in oncogenic transformation, the progression of at least some tumours and especially in processes related to tumour metastasis. MNKs are clearly druggable targets and, as they are not essential, significant 'side effects' of inhibiting the MNKs are likely to be limited. Further work is required to assess the efficacy of MNK inhibition in tackling tumour development, progression and metastasis.


Assuntos
Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Animais , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos
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