Faculty Bibliography

Cannabis is one of the most commonly used drugs among adolescents, with initial use beginning between the ages of 12 to 17. Although often perceived as a 'soft drug', both short- and long-term use have been associated with numerous adverse outcomes, including cognitive impairment, increased risk of substance abuse, and heightened risk of psychosis or schizophrenia in individuals with a predisposition. Further, the severity of these impairments is closely linked to initiation of use, i.e. earlier use increases risk. It has been suggested that adolescent vulnerability to the adverse consequences of cannabis use is due to ongoing brain development occurring during this time. Indeed, the adolescent brain continues to be remodeled well into adolescence and early adulthood, particularly in the prefrontal cortex (PFC). The medial prefrontal cortex (mPFC) has been implicated in reward processing and decision-making and alterations in mPFC development due to adolescent cannabis exposure could impair these functions. To model the effects of cannabis on mPFC function, we administered the synthetic cannabinoid WIN 55, 212-2 (WIN) to male and female rats from postnatal day 30-60. Once animals reached adulthood, we used a Probabilistic Reward (PR) choice task to elicit PFC activity and measure how patterns of activity to task-related events were modulated by adolescent WIN-treatment. Adult animals showed subtle effects of WIN-treatment on choice patterns. During task performance, mPFC activity elicited by lever press at the time of choices and reward delivery following choices were reduced in WIN-treated animals. This lasting effect of WIN suggests an impairment of the maturation of excitatory-inhibitory balance of signals in mPFC during adolescence, which may alter executive function into adulthood.

Enterocin (vulgamycin) is a structurally remarkable natural product with significant antibiotic activity. The synthesis of a linear polyketide resembling a biosynthetic precursor was achieved using an unusual acyloin reaction. A diazo group was introduced as a protecting group for an enolizable ketone. We were unable to bring about the envisioned biomimetic aldol addition cascade and gained insights into the feasibility of this process by DFT calculations. As an alternative approach to enterocin, we developed a Cu-catalyzed intramolecular cyclopropanation followed by a MgI₂-induced fragmentation to install the 2-oxabicyclo[3.3.1]nonane core of the natural product.

We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca²⁺) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca²⁺ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca²⁺ imaging in combination with other optogenetic indicators and actuators.

Careful phenotyping of patients to classify those with kidney stones has a long and important history in revealing the chemical basis for stone formation. Advances in our genetic understanding of kidney stones will lead to incredible insights regarding the pathophysiology of this common disorder. At this time, both evaluation of urine chemistry and genotyping of patients are extremely useful in the setting of a university and research-based kidney stone clinic. For much of the world, in a more clinically focused setting, these techniques are neither available nor absolutely necessary. Careful implementation of an empiric prescription based on stone composition would have an important effect to reduce stone recurrence in the world's many stone formers. Increased fluid intake, generic dietary manipulations, and prescription of potassium citrate and thiazides are all appropriate empiric therapies for people with calcium and uric acid kidney stones.

The slow, continuous, devastating march of Alzheimer's disease continues to move across the globe. As a society, we are at a loss for options to treat or reverse the death of neurons-the final, apparently inescapable, hallmark of the disease. A continued focus on these dying neurons has taught us much about the disease but with no knowledge-based effective treatment in sight. A surge of interest in non-neuronal cells, including glia, blood vasculature, and immune cells, has shed new light on how we may better diagnose and treat patients. This may be our best hope to treat the millions patients with cognitive decline and memory loss.-Liddelow, S. A. Modern approaches to investigating non-neuronal aspects of Alzheimer's disease.

The HCN4 gene encodes a subunit of the hyperpolarization-activated cyclic nucleotide-gated channel, type 4 that is essential for the proper generation of pacemaker potentials in the sinoatrial node. The HCN4 gene is often present in targeted genetic testing panels for various cardiac conduction system disorders and there are several reports of HCN4 variants associated with conduction disorders. Here, we report the in vitro functional characterization of four rare variants of uncertain significance (VUS) in HCN4, identified through testing a cohort of 296 sudden unexpected natural deaths. The variants are all missense alterations, leading to single amino acid changes: p.E66Q in the N-terminus, p.D546N in the C-linker domain, and both p.S935Y and p.R1044Q in the C-terminus distal to the CNBD. We also identified a likely benign variant, p. P1063T, which has a high minor allele frequency in the gnomAD, which is utilized here as a negative control. Three of the HCN4 VUS (p.E66Q, p.S935Y, and p.R1044Q) had electrophysiological characteristics similar to the wild-type channel, suggesting that these variants are benign. In contrast, the p.D546N variant in the C-linker domain exhibited a larger current density, slower activation, and was unresponsive to cyclic adenosine monophosphate (cAMP) compared to wild-type. With functional assays, we reclassified three rare HCN4 VUS to likely benign variants, eliminating the necessity for costly and time-consuming further study. Our studies also provide a new lead to investigate how a VUS located in the C-linker connecting the pore to the cAMP binding domain may affect the channel open state probability and cAMP response.

Cystinuria, a genetic disorder of cystine transport, is characterized by excessive excretion of cystine in the urine and recurrent cystine stones in the kidneys and, to a lesser extent, in the bladder. Males generally are more severely affected than females. The disorder may lead to chronic kidney disease in many patients. The cystine transporter (b^0,+) is a heterodimer consisting of the rBAT (encoded by SLC3A1) and b^0,+AT (encoded by SLC7A9) subunits joined by a disulfide bridge. The molecular basis of cystinuria is known in great detail, and this information is now being used to define genotype-phenotype correlations. Current treatments for cystinuria include increased fluid intake to increase cystine solubility and the administration of thiol drugs for more severe cases. These drugs, however, have poor patient compliance due to adverse effects. Thus, there is a need to reduce or eliminate the risks associated with therapy for cystinuria. Four mouse models for cystinuria have been described and these models provide a resource for evaluating the safety and efficacy of new therapies for cystinuria. We are evaluating a new approach for the treatment of cystine stones based on the inhibition of cystine crystal growth by cystine analogs. Our ongoing studies indicate that cystine diamides are effective in preventing cystine stone formation in the Slc3a1 knockout mouse model for cystinuria. In addition to crystal growth, crystal aggregation is required for stone formation. Male and female mice with cystinuria have comparable levels of crystalluria, but very few female mice form stones. The identification of factors that inhibit cystine crystal aggregation in female mice may provide insight into the gender difference in disease severity in patients with cystinuria.

BACKGROUND:Computed tomography (CT) and spirometry are the current standard methods for assessing lung anatomy and pulmonary ventilation, respectively. However, CT provides limited ventilation information and spirometry only provides global measures of lung ventilation. Thus, a method that can enable simultaneous examination of lung anatomy and ventilation is of clinical interest. PURPOSE/OBJECTIVE:To develop and test a 4D respiratory-resolved sparse lung MRI (XD-UTE: eXtra-Dimensional Ultrashort TE imaging) approach for simultaneous evaluation of lung anatomy and pulmonary ventilation. STUDY TYPE/METHODS:Prospective. POPULATION/METHODS:In all, 23 subjects (11 volunteers and 12 patients, mean age = 63.6 ± 8.4). FIELD STRENGTH/SEQUENCE/UNASSIGNED:3T MR; a prototype 3D golden-angle radial UTE sequence, a Cartesian breath-hold volumetric-interpolated examination (BH-VIBE) sequence. ASSESSMENT/RESULTS:All subjects were scanned using the 3D golden-angle radial UTE sequence during normal breathing. Ten subjects underwent an additional scan during alternating normal and deep breathing. Respiratory-motion-resolved sparse reconstruction was performed for all the acquired data to generate dynamic normal-breathing or deep-breathing image series. For comparison, BH-VIBE was performed in 12 subjects. Lung images were visually scored by three experienced chest radiologists and were analyzed by two observers who segmented the left and right lung to derive ventilation parameters in comparison with spirometry. STATISTICAL TESTS/UNASSIGNED:Nonparametric paired two-tailed Wilcoxon signed-rank test; intraclass correlation coefficient, Pearson correlation coefficient. RESULTS:XD-UTE achieved significantly improved image quality compared both with Cartesian BH-VIBE and radial reconstruction without motion compensation (P < 0.05). The global ventilation parameters (a sum of the left and right lung measures) were in good correlation with spirometry in the same subjects (correlation coefficient = 0.724). There were excellent correlations between the results obtained by two observers (intraclass correlation coefficient ranged from 0.8855-0.9995). DATA CONCLUSION/UNASSIGNED:Simultaneous evaluation of lung anatomy and ventilation using XD-UTE is demonstrated, which have shown good potential for improved diagnosis and management of patients with heterogeneous lung diseases. LEVEL OF EVIDENCE/METHODS:2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018.

Objective: Granule cell precursors (GCPs) are a sonic hedgehog (SHH)- dependent progenitor population in the developing cerebellum and the main cell of origin for the SHH subgroup of medulloblastoma (MB). Unlike other subgroups of MB, SHH-MBs occur preferentially in the lateral cerebellum (hemispheres) and have four main driver mutations. We studied whether the timing or type of mutation affects tumor location and identified factors influencing SHH-MB progression.
Method(s): We analyzed the association between type of mutation and tumor location in 38 SHH-MB patient samples. To generate sporadic mouse models of SHH-MB, inducible recombinases were used to express a constitutive activate SMO receptor (SmoM2) or delete Ptch1 in only scattered GCPs. Tumor location, expression profiles and GCP behaviors were analyzed in the models.
Result(s): Our analysis of patient data indicates that adult tumors with SMO mutations form more specifically in the hemispheres than those with PTCH1 mutations. Using sporadic mouse models, we found that regardless of the number of GCPs mutated, timing or type of mutation, tumors developed almost exclusively in the hemispheres with SmoM2-mutants showing a stronger specificity. We further uncovered that GCPs in the hemispheres are more susceptible to high level SHH signaling compared to GCPs in the medial cerebellum (vermis), as more mutant cells in the hemisphere remain undifferentiated and show increased tumorigenicity when transplanted. We also identified location-specific gene expression profiles, and found that deletion of the genes most highly expressed in the hemispheres or vermis showed opposing effects on GCP differentiation.
Conclusion(s): We found that GCPs respond differentially to two driver mutations and a subset of GCPs is more susceptible to high level of SHH signaling as well as tumors formation. We redefined themain cell of origin by showing that GCPs are heterogeneous with molecularly distinct populations based on their location