Differential regulation of miR-21-5p delays wound healing of melanocyte-deprived vitiligo skin by modulating the expression of tumor-suppressors PDCD4 and Maspin.

The loss of melanocytes in vitiligo is associated with architectural, transcriptional, and cellular perturbations of keratinocytes and manifests as a reduced proliferation potential in vitro and delayed re-epithelialization in vivo. To understand the molecular mechanisms underlying this delay, microRNA (miRNA) profiling was performed on split skin biopsies collected on Day 1 (basal level) and Day 14 (wound re-epithelialization) from nonlesional (NL) and lesional (L) skin of five subjects with stable nonsegmental vitiligo and 129 miRNAs were found to be differentially regulated between the NL and L healed epidermis. miR-21-5p, expressed at comparable levels on NL and L Day 1 samples, demonstrated significant upregulation during re-epithelialization. However, the extent of its upregulation was relatively lower in L (10 times compared to Day 1) as compared to NL skin (17 times compared to Day 1). The overexpression of miR-21 in keratinocytes led to a significant increase in the expression of proliferation markers (Ki67 and MCM6 messenger RNA, Ki67 positivity), along with an increase in keratinocyte migration. Using a small interfering RNA mediated knockdown approach, we further demonstrated that miR-21-5p mediates its effects by suppressing the expression of programmed cell death 4 (PDCD4) and mammary serine protease inhibitor (Maspin), both tumor-suppressor genes. Investigation of clinical samples corroborated the lower miR-21 levels and a higher expression of PDCD4 and Maspin in L Day 14 compared to the NL Day 14 epidermis. In conclusion, this study revealed that a relatively lower upregulation of miR-21-5p in L skin leads to significantly higher levels of PDCD4 and Maspin, delaying wound re-epithelialization by reducing the proliferation and migration of keratinocytes.

Transcriptome Analysis of Insulin Signaling-Associated Transcription Factors in C. elegans Reveal Their Genome-Wide Target Genes Specificity and Complexity.

Insulin/IGF-1-like signaling (IIS) plays a crucial, conserved role in development, growth, reproduction, stress tolerance, and longevity. In Caenorhabditis elegans, the enhanced longevity under reduced insulin signaling (rIIS) is primarily regulated by the transcription factors (TFs) DAF-16/FOXO, SKN-1/Nrf-1, and HSF1/HSF-1. The specific and coordinated regulation of gene expression by these TFs under rIIS has not been comprehensively elucidated. Here, using RNA-sequencing analysis, we report a systematic study of the complexity of TF-dependent target gene interactions during rIIS under analogous genetic and experimental conditions. We found that DAF-16 regulates only a fraction of the C. elegans transcriptome but controls a large set of genes under rIIS; SKN-1 and HSF-1 show the opposite trend. Both of the latter TFs function as activators and repressors to a similar extent, while DAF-16 is predominantly an activator. For expression of the genes commonly regulated by TFs under rIIS conditions, DAF-16 is the principal determining factor, dominating over the other two TFs, irrespective of whether they activate or repress these genes. The functional annotations and regulatory networks presented in this study provide novel insights into the complexity of the gene regulatory networks downstream of the IIS pathway that controls diverse phenotypes, including longevity.

Influence of B-ring modifications on proton affinity, transmembrane anion transport and anti-cancer properties of synthetic prodigiosenes.

Prodigiosin is the parent compound of the tripyrrolic natural products known as the prodigiosenes. Some of these natural products and their synthetic analogs show anti-cancer, immunosuppressive and antimicrobial actions, amongst other biological activities. One mechanism put forth to explain their biological activity is that since prodigiosenes are typically protonated at physiological pH they can alter intracellular pH via HCl co-transport (or Cl(-)/OH(-) exchange) across cell membranes. In this study we synthesized a series of prodigiosene analogs with different -O-aryl substituents attached to the B-ring of the tripyrrolic skeleton. NMR studies showed that these analogs can exist as a mixture of two stable α and ß conformers in acidic solution, and that both conformers can bind anions in solution. We found that the electronic nature of the O-aryl substituent on the B-ring influences the rate at which these prodigiosenes catalyze transmembrane anion transport, i.e. the prodigiosenes with the higher pKa had greater Cl(-)/NO3(-) exchange rates. Four of the synthetic prodigiosenes were tested for their in vitro anti-cancer activities in the NCI60 human tumour panel. Despite their promising in vitro anti-cancer activity (GI50 values ranging from 18 to 74 nM), there was no evidence that this activity is influenced by the extent of protonation of these synthetic prodigiosenes.

Synthetic prodigiosenes and the influence of C-ring substitution on DNA cleavage, transmembrane chloride transport and basicity.

Analogues of the tripyrrolic natural product prodigiosin bearing an additional methyl and a carbonyl group at the C-ring were synthesised and evaluated. In vitro anticancer activity screening (NCI) and the study of modes of action (copper-mediated cleavage of double-stranded DNA and transmembrane transport of chloride anions) showed that the presence of the methyl group is not detrimental to activity. Furthermore, although the presence of an ester conjugated to the prodigiosene C-ring seems to decrease both pK(a) and chloride transport efficiency compared to the natural product, these analogues still exhibit a high rate of chloride transport. All analogues exhibit good in vitro anticancer activity and reduced toxicity compared to the natural product: compare an acute systemic toxicity of 100 mg kg(-1) in mice vs. 4 mg kg(-1) for prodigiosin, pointing towards a larger therapeutic window than for the natural product.