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  1. Abstract

    ZF proteins are ubiquitous eukaryotic proteins that play important roles in gene regulation. ZFs contain small domains made up of a combination of four cysteine and histidine residues and are classified on the basis of the identity of these residues and their spacing. One emerging class of ZFs are the Cys3His (or CCCH) class of ZFs. These ZFs play key roles in regulating RNA. In this minireview, an overview of the CCCH class of ZFs, with a focus on tristetraprolin (TTP), is provided. TTP regulates inflammation by controlling cytokine mRNAs, and there is an interest in modulating TTP activity to control inflammation. Two methods to control TTP activity are to target with exogenous metals (a “metals in medicine” approach) or to target with endogenous signaling molecules. Work that has been done to target TTP with Fe, Cu, Cd, and Au as well as with H2S is reviewed. This includes attention to new methods that have been developed to monitor metal exchange with the spectroscopically silent ZnIIincluding native electro‐spray ionization mass spectrometry (ESI‐MS), spin‐filter inductively coupled plasma mass spectrometry (ICP‐MS), and cryo‐electro‐spray mass spectrometry (CSI‐MS); along with fluorescence anisotropy (FA) to follow RNA binding.

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  2. Abstract

    H2S is a gaseous signaling molecule that modifies cysteine residues in proteins to form persulfides (P‐SSH). One family of proteins modified by H2S are zinc finger (ZF) proteins, which contain multiple zinc‐coordinating cysteine residues. Herein, we report the reactivity of H2S with a ZF protein called tristetraprolin (TTP). Rapid persulfidation leading to complete thiol oxidation of TTP mediated by H2S was observed by low‐temperature ESI‐MS and fluorescence spectroscopy. Persulfidation of TTP required O2 , which reacts with H2S to form superoxide, as detected by ESI‐MS, a hydroethidine fluorescence assay, and EPR spin trapping. H2S was observed to inhibit TTP function (binding to TNFα mRNA) by an in vitro fluorescence anisotropy assay and to modulate TNFα in vivo. H2S was unreactive towards TTP when the protein was bound to RNA, thus suggesting a protective effect of RNA.

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  5. Municipal drinking water, regulated by the Environmental Protection Agency via the Safe Drinking Water act, has long been assumed to be contaminant-free. However, crises related to drinking water have emerged, most notably the “Flint Water Crisis” in Flint, MI, where high levels of lead (Pb) were detected in the area’s water. Much of the water-sampling data collected in Flint was obtained by “Citizen Scientists” working closely with a team of researchers at Virginia Tech, who used the analytical technique of Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to quantify metal ions present in the water. Inspired by these efforts, we developed adaptable public water testing outreach efforts, led by students in Baltimore city (Middle School, High School, and College), to test the city’s drinking water. These “student-scientists” read news and scientific articles to understand the public health impact of lead in drinking water and the analytical approaches scientists use to detect metal ions in water. The students then developed a written “water collection protocol” and sought participation from colleagues (other students, faculty, and staff) who collected their home drinking water to be tested. The student scientists prepared and analyzed samples for lead (Pb) as well as copper (Cu), iron (Fe), and zinc (Zn) metal ions commonly found in drinking water, to be tested via ICP-MS. Data were then plotted onto a map of Baltimore City, with the metal levels indicated for each Zip code. This outreach event connects science to real-life news events while teaching analytical methodology and can be tailored to students at various stages of their education. 
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  6. Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric ( Sym CDTb; 3.14 Å) and an asymmetric form ( Asym CDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For Asym CDTb, a Ca 2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile . 
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