Distinguishing the toxic effects of nanoparticles (NPs) themselves from the well-studied toxic effects of their ions is a critical but challenging measurement for nanotoxicity studies and regulation. This measurement is especially difficult for silver NPs (AgNPs) because in many relevant biological and environmental solutions, dissolved silver forms AgCl NPs or microparticles. Simulations predict that solid AgCl particles form at silver concentrations greater than 0.18 and 0.58 μg/mL in cell culture media and moderately hard reconstituted water (MHRW), respectively. The AgCl NPs are usually not easily separable from AgNPs. Therefore, common existing total silver techniques applied to measure AgNP dissolution, such as inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption, cannot accurately measure the amount of silver remaining in AgNP form, as they cannot distinguish Ag oxidation states. In this work, we introduce a simple localized surface plasmon resonance (LSPR) UV-visible absorbance measurement as a technique to measure the amount of silver remaining in AgNP form for AgNPs with constant agglomeration states. Unlike other existing methods, this absorbance method can be used to measure the amount of silver remaining in AgNP form even in biological and environmental solutions containing chloride because AgCl NPs do not have an associated LSPR absorbance. In addition, no separation step is required to measure the dissolution of the AgNPs. After using ICP-MS to show that the area under the absorbance curve is an accurate measure of silver in AgNP state for unagglomerating AgNPs in non-chloride-containing media, the absorbance is used to measure dissolution rates of AgNPs with different polymer coatings in biological and environmental solutions. We find that the dissolution rate decreases at high AgNP concentrations, 5 kDa polyethylene glycol thiol coatings increase the dissolution rate, and the rate is much higher in cell culture media than in MHRW.

It is widely appreciated that the ends of linear DNA molecules cannot be fully replicated by the conventional replication apparatus. Less well known is that semi-conservative replication of telomeric DNA also presents problems for DNA replication. These problems likely arise from the atypical chromatin structure of telomeres, the GC-richness of telomeric DNA that makes it prone to forming DNA secondary structures, and from RNA-DNA hybrids, formed by transcripts of one or both DNA strands. Given the different aspects of telomeres that complicate their replication, it is not surprising that multiple DNA helicases promote replication of telomeric DNA. This review focuses on one such class of DNA helicases, the Pif1 family of 5′–3′ DNA helicases. In budding and fission yeasts, Pif1 family helicases impact both telomerase-mediated and semi-conservative replication of telomeric DNA as well as recombination-mediated telomere lengthening.

Pif1 family DNA helicases are conserved from bacteria to humans and have critical and diverse functions in vivo that promote genome integrity. Pif1 family helicases share a 23 amino acid region, called the Pif1 signature motif (SM) that is unique to this family. To determine the importance of the SM, we did mutational and functional analysis of the SM from the Saccharomyces cerevisiae Pif1 (ScPif1). The mutations deleted portions of the SM, made one or multiple single amino acid changes in the SM, replaced the SM with its counterpart from a bacterial Pif1 family helicase and substituted an α-helical domain from another helicase for the part of the SM that forms an α helix. Mutants were tested for maintenance of mitochondrial DNA, inhibition of telomerase at telomeres and double strand breaks, and promotion of Okazaki fragment maturation. Although certain single amino acid changes in the SM can be tolerated, the presence and sequence of the ScPif1 SM were essential for all tested in vivo functions. Consistent with the in vivo analyses, in vitro studies showed that the presence and sequence of the ScPif1 SM were critical for ATPase activity but not substrate binding.

Increasing fetal hemoglobin (HbF) provides clinical benefit in patients with sickle cell disease (SCD). We recently identified heme-regulated inhibitor (HRI, EIF2AK1), as a novel HbF regulator. Because HRI is an erythroid-specific protein kinase, it presents a potential target for pharmacologic intervention. We found that maximal HbF induction required >80% to 85% HRI depletion. Because it remains unclear whether this degree of HRI inhibition can be achieved pharmacologically, we explored whether HRI knockdown can be combined with pharmacologic HbF inducers to achieve greater HbF production and minimize potential adverse effects associated with treatments. Strongly cooperative HbF induction was observed when HRI depletion was combined with exposure to pomalidomide or the EHMT1/2 inhibitor UNC0638, but not to hydroxyurea. Mechanistically, reduction in the levels of the HbF repressor BCL11A reflected the cooperativity of HRI loss and pomalidomide treatment, whereas UNC0638 did not modulate BCL11A levels. In conjunction with HRI loss, pomalidomide maintained its HbF-inducing activity at 10-fold lower concentrations, in which condition there were minimal observed detrimental effects on erythroid cell maturation and viability, as well as fewer alterations in the erythroid transcriptome. When tested in cells from patients with SCD, combining HRI depletion with pomalidomide or UNC0638 achieved up to 50% to 60% HbF and 45% to 50% HbF, respectively, as measured by high-performance liquid chromatography, and markedly counteracted cell sickling. In summary, this study provides a foundation for the exploration of combining future small-molecule HRI inhibitors with additional pharmacologic HbF inducers to maximize HbF production and preserve erythroid cell functionality for the treatment of SCD and other hemoglobinopathies.