All of the available ‘universal’ primers introduce a degree of bias in the amplification of fungal DNA, which could lead to over- or under-representations of certain OTUs. We have utilized three primer sets targeting ITS and LSU regions of the ribosomal genes that were used in other mycobiome studies. However, these primers sets are known to be not equally efficient in the amplification of ascomycetes, basidiomycetes and EDFL. Additional difficulties in the use of cloned libraries come from their dependence on the alignments of database nucleotide sequences to determine OTUs. The assignment of ITS sequences is less problematic as there is a comprehensive nucleotide database for the species level identification of many fungi. Higher taxonomic assignments especially of unknown fungi are more problematic as they rely on LSU sequences that are not as numerous in the databases. A key question surrounding Cl is whether anti-atrophic properties contributed to enhanced recovery rates produced by the Harefield Protocol. Findings of similar myocyte size in both explanted and non-recovered patients treated by this protocol, suggest Cl’s pro-recovery effects were unrelated to myocardial size. This present study reinforces this prospect. We show Cl to be ineffective in preventing myocardial ARRY-142886 atrophy associated with prolonged unloading, and this agrees with previous work in non-failing rat hearts undergoing 2 weeks unloading. In contrast, we previously showed Cl to limit regression of rodent HF myocyte hypertrophy during short-term unloading . However, such a brief period of MU, during which atrophic remodelling is regarded as being submaximal was considered inadequate, and a poor representation of prolonged clinical LVAD support. As we have previously shown, Met prevented myocardial atrophy, but this effect was lost during combined MetCl therapy with actual worsening of atrophy. The latter observation is unexpected, if compared with the effects of the combined therapy in normally-loaded hearts; this can be due to additional detrimental consequences of mechanical unloading and require to be further studied. Atrophic remodelling is complex and the multiple pathways involved poorly defined. The ubiquitin proteosome, calpain, lysosomal proteolysis and authophagy systems, and mTOR IGF-1/PI3K/AKT and ERK-1 pathways are all altered during MU. These growth regulatory pathways, along with the TGF-b, CAMKII and calcineurin/ NFAT hypertrophic signalling pathways, known components under b-AR influence, may represent Met’s route of action, and augmentation of myocyte number via regenerative mechanisms is another possibility. This finding is particularly important as, to date, no pharmacotherapy has proven effective in attenuating myocardial atrophy, with success via haemodynamic loading strategies alone, re-emphasising the critical importance of load in regulation of cardiac mass. Such loading, potentially brought about by HR reduction and subsequent augmentation of LV filling, may have driven Met’s anti-atrophic actions; but the lack of effect during combined MetCl therapy, despite an equivalent reduction in HR, makes this mechanism unlikely. The current treatment methods for breast cancer include chemotherapy, radiotherapy.