Brandon Ritzo

Single Molecule Investigation of Ag+ Interactions with Single Cytosine-, Methylcytosine- and Hydroxymethylcytosine-Cytosine Mismatches in a Nanopore

Both cytosine-Ag-cytosine interactions and cytosine modifications in a DNA duplex have attracted great interest for research. Cytosine (C) modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) are associated with tumorigenesis. However, a method for directly discriminating C, mC and hmC bases without labeling, modification and amplification is still missing. Additionally, the nature of coordination of Ag+ with cytosine-cytosine (C-C) mismatches is not clearly understood. Utilizing the alpha-hemolysin nanopore, we show that in the presence of Ag+, duplex stability is most increased for the cytosine-cytosine (C-C) pair, followed by the cytosine-methylcytosine (C-mC) pair, and the cytosine-hydroxymethylcytosine (C-hmC) pair, which has no observable Ag+ induced stabilization. Molecular dynamics simulations reveal that the hydrogen-bond-mediated paring of a C-C mismatch results in a binding site for Ag+. Cytosine modifications (such as mC and hmC) disrupted the hydrogen bond, resulting in disruption of the Ag+ binding site. Our experimental method provides a novel platform to study the metal ion-DNA interactions and could also serve as a direct detection method for nucleobase modifications.

Incidence and type of meniscal injury and associated long-term clinical outcomes in dogs treated surgically for cranial cruciate ligament disease.

OBJECTIVE To evaluate factors related to meniscal pathology and their effect on clinical outcome in dogs treated for cranial cruciate ligament (CCL) disease. STUDY DESIGN Prospective cross-sectional study. ANIMALS Dogs (n = 163) with CCL disease (n = 223 stifles). METHODS CCL disease was treated by (1) arthroscopy and TightRope (TR) stabilization; (2) arthroscopy and tibial plateau leveling osteotomy (TPLO); or (3) open arthrotomy and TPLO. Incidences of concurrent and subsequent meniscal tears, meniscal treatments, mid-(6 months) and long-(>1 year) term outcomes by owner assessment were compared among surgical treatment groups. RESULTS Concurrent meniscal tears were diagnosed in 83% of stifles assessed by arthroscopy and 44% of stifles assessed by arthrotomy, with concurrent tear diagnosis being 1.9 times more likely by arthroscopy than arthrotomy (P < .001). Incidence of diagnosis of subsequent meniscal tears was 6.7% with median time to diagnosis of 5.8 months. Differences in proportion of subsequent meniscal tears among treatment groups were not significant (P = .69). Subsequent meniscal tears were diagnosed in 21% of cases without concurrent meniscal tears, but only 1.3% of cases with concurrent meniscal tears (P < .001). Cases treated with meniscal release did not have subsequent meniscal tears, whereas dogs not treated with meniscal release had a subsequent meniscal tear rate of 11% (P = .0013). Cases diagnosed and treated for concurrent meniscal tears were 1.3 times more likely to have a successful long-term outcome (P = .007). CONCLUSIONS CCL surgical technique did not affect subsequent meniscal tear rate or mid-term or long-term functional outcomes, whereas diagnosis and treatment of concurrent meniscal tears did significantly affect both.

Silver(I) ions modulate the stability of DNA duplexes containing cytosine, methylcytosine and hydroxymethylcytosine at different salt concentrations

Silver(I) ions can stabilize cytosine-cytosine, cytosine (C)-methylcytosine (5mC) and cytosine-hydroxymethylcytosine (5hmC) mismatched-base pairs. While cytosine modifications regulate DNA stability to regulate cellular functions, silver ions can modulate the stability of C-C, C-5mC and C-5hmC containing DNA duplexes in a salt concentration dependent manner.

Biosensing: When less is more in a nanopore

Chemically modifying solid-state nanopores with a single nitrilotriacetic acid receptor allows the reversible detection of single proteins.

Tuning the tetraethylammonium sensitivity of potassium channel Kcv by subunit combination

Tetraethylammonium (TEA) is a potassium (K+) channel inhibitor that has been extensively used as a molecular probe to explore the structure of channels’ ion pathway. In this study, we identified that Leu70 of the virus-encoded potassium channel Kcv is a key amino acid that plays an important role in regulating the channel’s TEA sensitivity. Site-directed mutagenesis of Leu70 can change the TEA sensitivity by 1,000-fold from ∼100 µM to ∼100 mM. Because no compelling trends exist to explain this amino acid’s specific interaction with TEA, the role of Leu70 at the binding site is likely to ensure an optimal conformation of the extracellular mouth that confers high TEA affinity. We further assembled the subunits of mutant and wt-Kcv into a series of heterotetramers. The differences in these heterochannels suggest that all of the four subunits in a Kcv channel additively participate in the TEA binding, and each of the four residues at the binding site independently contributes an equal binding energy. We therefore can present a series of mutant/wild-type tetramer combinations that can probe TEA over three orders of magnitude in concentration. This study may give insight into the mechanism for the interaction between the potassium channel and its inhibitor.

When less is more in a nanopore.

Chemically modifying solid-state nanopores with a single nitrilotriacetic acid receptor allows the reversible detection of single proteins.

When less is more in a nanopore: Biosensing

Chemically modifying solid-state nanopores with a single nitrilotriacetic acid receptor allows the reversible detection of single proteins.