Advances in myotonic dystrophy type 1 drug discovery through design of novel ligands and mechanism establishment

Myotonic dystrophy type 1 (DM1) is caused by an expanded CUG repeat (CUGexp) that sequesters muscleblind-like 1 protein (MBNL1), a protein that regulates alternative splicing. CUGexp RNA is a validated drug target for this currently untreatable disease. Herein, we describe the development of a bioactive small molecule (Chapter 2) and a small library of dimeric ligands (Chapter 3) leading to an optimized bivalent ligand. These novel ligands target CUGexp RNA and are able to inhibit the CUGexp⋅MBNL1 interaction in cells that model DM1. In a DM1 cell model these ligands were found to disperse CUGexp ribonuclear foci, release MBNL1, and partially reverse the mis-splicing of the insulin receptor pre-mRNA. Direct evidence for ribonuclear foci dispersion by this ligand was obtained in a live DM1 cell model using time-lapse confocal microscopy. In Chapter 4, We report a single-molecule approach to study the binding of MBNL1 to (CUG)n=4,6 and the effect of small molecules on this interaction. MBNL1 is able to bind to the (CUG)n･inhibitor complex indicating that the inhibition is not a straight forward competitive process. A simple bivalent ligand, shows a binding to (CUG)n almost 50-fold more tightly than the corresponding monomeric ligand and is more effective in destabilizing MBNL1･(CUG)4. The single-molecule method and the analysis framework might be extended to the study of other biomolecular interactions. Chapter 5 includes a preliminary effort to solve the mystery of CUGexp unfolding/folding upon interaction with MBNL1. To approach this unanswered yet key structural question about how MBNL1 binds CUGexp, preliminary bulk FRET (Fluorescence Resonance Energy Transfer) studies, as well as single-molecule FRET studies are describe

Ectopic “Ectopic” Gastric Mucosa

Meckel’s diverticulum is a developmental GI anomaly. It is a remnant of the omphalomesenteric duct (vitelline duct) and the most common congenital anomaly found in the small intestine. It contains ectopic/heterotopic gastric mucosa in half of the cases. Imaging investigations for diagnosing Meckel’s diverticulum may include a plain radiography; however, this has a very limited diagnostic value. A blind-ending fluid-filled structure can sometimes be seen with sonography, but again, this technique’s diagnostic value is limited due to multiple factors. A CT scan may be helpful in localizing the bleeding diverticulum, which can be better visualized with CT enterography. Diverticula containing gastric mucosa can be diagnosed with a higher sensitivity with Tc-99 scintigraphy. The typical location of Meckel’s diverticulum is within two feet of the ileocecal valve; thus, ectopic gastric mucosal uptake is typically seen in the lower right quadrant in scintigraphy. We present a rare case of Tc-99 pertechnetate scintigraphy showing ectopic gastric mucosa in the upper mid abdomen, which was surgically proven to be at the mid ileum. To our knowledge, there is no ectopic Meckel’s diverticulum case published in the literature. Familiarity with this atypical imaging presentation of relatively common ectopic gastric mucosa may help the radiologists in the timely diagnosis and management of the patient

Variant allele fraction of genomic alterations in circulating tumor DNA (%ctDNA) correlates with SUV

The relationship between higher variant allele fraction (VAF) of genomic alterations in circulating tumor DNA (%ctDNA), an indicator of poor outcome, and maximum standardized uptake value (SU

Comparison of immunohistochemical expression of CD10 in keratocystic odontogenic tumor and ameloblastoma

Background: Odontogenic keratocyst (OKC), also called keratocystic odontogenic tumor (KCOT), is a developmental lesion which should be carefully monitored and it exhibits development mechanisms and biologic behaviors different from those of other more common lesions such as dentigerous and radicular cysts. CD10 antigen is a cell surface metalloendopeptidase, which inactivates various peptides that are physiologically active. Studies have shown that increase in the expression of CD10 in the stromal cells helps the progression of the tumor. Ameloblastoma (AB) is a local invasive tumor and given the role of supporting connective tissue stroma in the aggression and progression. The aim of the present study was to comparatively evaluate the expression of CD10 in the connective tissue stroma of AB and OKC as a KCOT. Materials and Methods: In this retrospective, cross-sectional study, 14 paraffin blocks of KCOT and 9 of AB (7 multicystic and 2 unicystic) were evaluated with CD10 immunohistochemical expression in the connective tissue stroma of AB and the connective tissue wall of KCOT. The data were analyzed with Fisher′s exact test (P < 0.05). Results: In 8 samples of 9 AB and in 13 samples of 14 KOT lesions, expression of CD10 was shown. Fisher′s exact test did not reveal any significant differences between these two lesions in the expression of CD10 (P = 0.64). Conclusion: The results of this study propose that high expression rate of CD10 might be one of the reasons for the aggressive behavior of AB and high recurrence rate of OKC and reinforce the classification of OKC as an odontogenic tumor

Antigenic structure of the hemagglutinin of H9N2 influenza viruses.

The hemagglutinins (HAs) of H9 influenza viruses isolated from birds and mammals of different species were antigenically and genetically analyzed. Antigenic variants were selected from A/swine/Hong Kong/10/98 (H9N2) and A/duck/Hokkaido/13/00 (H9N2) in the presence of monoclonal antibodies (MAbs), respectively. Based on the reactivity patterns of these mutants with a panel of MAbs, at least 5 non-overlapping antigenic sites were defined using 8 MAbs which recognized 7 distinct epitopes on the H9 HA molecule. Based on the reactivity patterns with the panel of monoclonal antibodies, 21 H9N2 virus strains isolated from birds and mammals were divided into 7 antigenically distinct groups. The present findings indicate that it is important to monitor the antigenic variation in H9 influenza viruses. The panel of MAbs in the present study, thus, should be useful for detailed antigenic analysis of the H9 HAs for epidemiological studies, the selection of vaccine strains, and diagnosis

Fluorous-Soluble Metal Chelate for Sensitive Fluorine-19 Magnetic Resonance Imaging Nanoemulsion Probes.

Fluorine-19 MRI is an emerging cellular imaging approach, enabling lucid, quantitative "hot-spot" imaging with no background signal. The utility of 19F-MRI to detect inflammation and cell therapy products in vivo could be expanded by improving the intrinsic sensitivity of the probe by molecular design. We describe a metal chelate based on a salicylidene-tris(aminomethyl)ethane core, with solubility in perfluorocarbon (PFC) oils, and a potent accelerator of the 19F longitudinal relaxation time ( T1). Shortening T1 can increase the 19F image sensitivity per time and decrease the minimum number of detectable cells. We used the condensation between the tripodal ligand tris-1,1,1-(aminomethyl)ethane and salicylaldehyde to form the salicylidene-tris(aminomethyl)ethane chelating agent (SALTAME). We purified four isomers of SALTAME, elucidated structures using X-ray scattering and NMR, and identified a single isomer with high PFC solubility. Mn4+, Fe3+, Co3+, and Ga3+ cations formed stable and separable chelates with SALTAME, but only Fe3+ yielded superior T1 shortening with modest line broadening at 3 and 9.4 T. We mixed Fe3+ chelate with perfluorooctyl bromide (PFOB) to formulate a stable paramagnetic nanoemulsion imaging probe and assessed its biocompatibility in macrophages in vitro using proliferation, cytotoxicity, and phenotypic cell assays. Signal-to-noise modeling of paramagnetic PFOB shows that sensitivity enhancement of nearly 4-fold is feasible at clinical magnetic field strengths using a 19F spin-density-weighted gradient-echo pulse sequence. We demonstrate the utility of this paramagnetic nanoemulsion as an in vivo MRI probe for detecting inflammation macrophages in mice. Overall, these paramagnetic PFC compounds represent a platform for the development of sensitive 19F probes