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\section{Materials and methods}   Please provide a detailed account of the methods and materials used for the study in order to allow replication by other researchers.  If you have used a standard protocol that is published elsewhere (e.g. Nature Protocols, Current Protocols), a reference may be used. Please include \begin{itemize}  \item  the source of all samples, reagents, antibodies etc.,\item  \end{itemize}how samples were selected; what exclusions were made, if any; what was being measured; for processed data, any software used to process the data and, where possible, the source code should be made openly available.  For laboratory animal studies, we recommend compliance with the 'Animal Research: Reporting In Vivo Experiments' (ARRIVE) guidelines, developed by NC3Rs to improve standards of reporting to ensure that the data from animal experiments can be fully scrutinized and utilized.  Allowances made, if any, for controlling bias or unwanted sources of variability.