MUC1 aptamer-tethered H40-TEPA-PEG nanoconjugates for targeted siRNA-delivery and gene silencing in breast cancer cells.

With a prevalence of 12.5% of all new cancer cases annually, breast cancer stands as the most common form of cancer worldwide. The current therapies utilized for breast cancer are constrained and ineffective in addressing the condition. siRNA-based gene silencing is a promising method for treating breast cancer. We have developed an aptamer-conjugated dendritic multilayered nanoconjugate to treat breast cancer. Initially, we transformed the hydroxyl groups of the hyperbranched bis-MPA polyester dendrimer into carboxylic groups. Subsequently, we linked these carboxylic groups to tetraethylenepentamine to form a positively charged dendrimer. In addition, the mucin-1 (MUC1) aptamer was attached to the dendrimer using a heterobifunctional polyethylene glycol. Characterizing dendrimers involved 1H NMR and dynamic light scattering techniques at every production stage. A gel retardation experiment was conducted to evaluate the successful binding of siRNA with targeted and non-targeted dendrimers. The targeted dendrimers exhibited no harmful effects on the NIH-3T3 fibroblast cells and RBCs, indicating their biocompatible characteristics. Confocal microscopy demonstrated significant higher uptake of targeted dendrimers than non-targeted dendrimers in MCF-7 breast cancer cells. The real-time PCR results demonstrated that the targeted dendrimers exhibited the most pronounced inhibition of the target gene expression compared to the non-targeted dendrimers and lipofectamine-2000. The caspase activation study confirmed the functional effect of survivin silencing by dendrimer, which led to the induction of apoptosis in breast cancer cells. The findings indicated that Mucin-1 targeted hyperbranched bis-MPA polyester dendrimer carrying siRNA could successfully suppress the expression of the target gene in breast cancer cells.

In silico identification and characterization of sensory motifs in the transcriptional regulators of the ArsR-SmtB family.

The ArsR-SmtB family of proteins displays the greatest diversity among the bacterial metal-binding transcriptional regulators with regard to the variety of metal ions that they can sense. In the presence of increased levels of toxic heavy metals, these proteins dissociate from their cognate DNA upon the direct binding of metal ions to the appropriate sites, designated motifs on the proteins, either at the interface of the dimers or at the intra-subunit locations. In addition to the metal-mediated regulation, some proteins were also found to control transcription via redox reactions. In the present work, we have identified several new sequence motifs and expanded the knowledge base of metal binding sites in the ArsR-SmtB family of transcriptional repressors, and characterized them in terms of the ligands to the metal, distribution among different phyla of bacteria and archaea, amino acid propensities, protein length distributions and evolutionary interrelationships. We built structural models of the motifs to show the importance of specific residues in an individual motif. The wide abundance of these motifs in sequences of bacteria and archaea indicates the importance of these regulators in combating metal-toxicity within and outside of the hosts. We also show that by using residue composition, one can distinguish the ArsR-SmtB proteins from other metalloregulatory families. In addition, we show the importance of horizontal gene transfer in microorganisms, residing in similar habitats, on the evolution of the structural motifs in the family. Knowledge of the diverse metalloregulatory systems in microorganisms could enable us to manipulate specific genes that may result in a toxic metal-free environment.

Metal homeostasis in bacteria: the role of ArsR-SmtB family of transcriptional repressors in combating varying metal concentrations in the environment.

Bacterial infections cause severe medical problems worldwide, resulting in considerable death and loss of capital. With the ever-increasing rise of antibiotic-resistant bacteria and the lack of development of new antibiotics, research on metal-based antimicrobial therapy has now gained pace. Metal ions are essential for survival, but can be highly toxic to organisms if their concentrations are not strictly controlled. Through evolution, bacteria have acquired complex metal-management systems that allow them to acquire metals that they need for survival in different challenging environments while evading metal toxicity. Metalloproteins that controls these elaborate systems in the cell, and linked to key virulence factors, are promising targets for the anti-bacterial drug development. Among several metal-sensory transcriptional regulators, the ArsR-SmtB family displays greatest diversity with several distinct metal-binding and nonmetal-binding motifs that have been characterized. These prokaryotic metolloregulatory transcriptional repressors represses the expression of operons linked to stress-inducing concentrations of metal ions by directly binding to the regulatory regions of DNA, while derepression results from direct binding of metal ions by these homodimeric proteins. Many bacteria, e.g., Mycobacterium tuberculosis, Bacillus anthracis, etc., have evolved to acquire multiple metal-sensory motifs which clearly demonstrate the importance of regulating concentrations of multiple metal ions. Here, we discussed the mechanisms of how ArsR-SmtB family regulates the intracellular bioavailability of metal ions both inside and outside of the host. Knowledge of the metal-challenges faced by bacterial pathogens and their survival strategies will enable us to develop the next generation drugs.