Faculty Bibliography

Optogenetics and photopharmacology are powerful approaches to investigating biochemical systems. While the former is based on genetically encoded photoreceptors that utilize abundant chromophores, the latter relies on synthetic photoswitches that are either freely diffusible or covalently attached to specific bioconjugation sites, which are often native or engineered cysteines. The identification of suitable cysteine sites and appropriate linkers for attachment is generally a lengthy and cumbersome process. Herein, we describe an in silico screening approach that is designed to propose a small number of optimal combinations. By applying this computational approach to human carbonic anhydrase and a set of three photochromic tethered ligands, the number of potential site-ligand combinations was narrowed from over 750 down to 6, which we then evaluated experimentally. Two of these six combinations resulted in light-responsive human Carbonic Anhydrases (LihCAs), which were characterized with enzymatic activity assays, mass spectrometry, and X-ray crystallography. Our study also provides insights into the reactivity of cysteines toward maleimides and the hydrolytic stability of the adducts obtained.

A concise route to the azatricyclo[6.2.1.0^4,11]undecane core of (-)-dendrobine and (-)-mubironine C is described wherein an unstabilized azomethine ylide cycloaddition provides the complete carbon framework of the natural products. The cyclization precursor is made in short order from ( R)-carvone through an unconventional high-pressure Ireland-Claisen reaction. Attempts to install a final hydroxyl group through an intramolecular lactonization strategy and the observation of an unexpected and highly complex enal-ene product are also reported.

Neuronal synapse formation and remodeling is essential to central nervous system (CNS) development and is dysfunctional in neurodevelopmental diseases. Innate immune signals regulate tissue remodeling in the periphery, but how this impacts CNS synapses is largely unknown. Here, we show that the IL-1 family cytokine interleukin-33 (IL-33) is produced by developing astrocytes and is developmentally required for normal synapse numbers and neural circuit function in the spinal cord and thalamus. We find that IL-33 signals primarily to microglia under physiologic conditions, that it promotes microglial synapse engulfment, and that it can drive microglial-dependent synapse depletion in vivo. These data reveal a cytokine-mediated mechanism required to maintain synapse homeostasis during CNS development.

Receptor for Advanced Glycation End products (RAGE) has been implicated in amyloid β-peptide (Aβ)-induced perturbation relevant to the pathogenesis of Alzheimer's disease (AD). However, whether and how RAGE regulates Aβ metabolism remains largely unknown. Aβ formation arises from aberrant cleavage of amyloid pre-cursor protein (APP) by β- and γ-secretase. To investigate whether RAGE modulates β- and γ-secretase activity potentiating Aβ formation, we generated mAPP mice with genetic deletion of RAGE (mAPP/RO). These mice displayed reduced cerebral amyloid pathology, inhibited aberrant APP-Aβ metabolism by reducing β- and γ-secretases activity, and attenuated impairment of learning and memory compared with mAPP mice. Similarly, RAGE signal transduction deficient mAPP mice (mAPP/DN-RAGE) exhibited the reduction in Aβ40 and Aβ42 production and decreased β-and γ-secretase activity compared with mAPP mice. Furthermore, RAGE-deficient mAPP brain revealed suppression of activation of p38 MAP kinase and glycogen synthase kinase 3β (GSK3β). Finally, RAGE siRNA-mediated gene silencing or DN-RAGE-mediated signaling deficiency in the enriched human APP neuronal cells demonstrated suppression of activation of GSK3β, accompanied with reduction in Aβ levels and decrease in β- and γ-secretases activity. Our findings highlight that RAGE-dependent signaling pathway regulates β- and γ-secretase cleavage of APP to generate Aβ, at least in part through activation of GSK3β and p38 MAP kinase. RAGE is a potential therapeutic target to limit aberrant APP-Aβ metabolism in halting progression of AD.

Heart failure (HF) has been referred to as the cardiovascular epidemic of our time. Understanding the molecular determinants of HF disease progression and mortality risk is of utmost importance. In this issue of the JCI, Zhang et al. uncover an important link between clinical HF mortality risk and a common variant that regulates SCN5A expression through microRNA-dependent (miR-dependent)mechanisms. They also demonstrate that haploinsufficiency of SCN5A is associated with increased accumulation of reactive oxygen species (ROS) in a genetically engineered murine model. Their data suggest that even modest depression of SCN5A expression may promote pathologic cardiac remodeling and progression of HF.

BACKGROUND:Next-generation sequencing of cell-free DNA (cfDNA) has been shown to be a useful noninvasive test for detecting mutations in solid tumors. METHODS:Targeted gene sequencing was performed with a panel of 263 cancer-related genes for cfDNA and genomic DNA of peripheral blood mononuclear cells (PBMCs) obtained from presurgical specimens of 6 lung cancer patients, and mutation calls in these samples were compared with those of primary tumors and corresponding patient-derived xenografts (PDXs). RESULTS:Approximately 67% of the mutations detected in the tumor samples (primary tumors and/or PDXs) were also detected in genomic DNA from PBMCs as background mutations. These background mutations consisted of germline polymorphisms and a group of mutations with low allele frequencies, mostly <10%. These variants with a low allele frequency were repeatedly detected in all types of samples from the same patients and at similarly low allele frequency levels in PBMCs from different patients; this indicated that their detection might be derived from common causes, such as homologous sequences in the human genome. Allele frequencies of mutations detected in both primary tumors and cfDNA showed 2 patterns: 1) low allele frequencies (approximately 1%-10%) in cfDNA but high allele frequencies (usually >10% or >3-fold increase) in primary tumors and further enrichment in PDXs and 2) similar allele frequencies across samples. CONCLUSIONS:Because only a small fraction of total cfDNA might be derived from tumor cells, only mutations with the first allele frequency pattern may be regarded as tumor-specific mutations in cfDNA. Effective filtering of background mutations will be required to improve the accuracy of mutation calls in cfDNA. Cancer 2018;124:1061-9. © 2017 American Cancer Society.

Surface roughness is one of the most important qualities in haptic perception. Roughness is a major identifier for judgments of material composition, comfort and friction, and is tied closely to manual dexterity. Some attention has been given to the study of roughness perception in the past, but it has typically focused on non-controllable natural materials or on a narrow range of artificial materials. The advent of high-resolution 3D printing technology provides the ability to fabricate arbitrary 3D textures with precise surface geometry to be used in tactile studies. We used parametric modeling and 3D printing to manufacture a set of textured plates with defined element spacing, shape, and arrangement. Using active touch and two-alternative forced choice protocols, we investigated the contributions of these surface parameters to roughness perception in human subjects. Results indicate that large spatial periods produce higher estimations of roughness (with Weber fraction = 0.19), small texture elements are perceived as rougher than large texture elements of the same wavelength, perceptual differences exist between textures with the same spacing but different arrangements, and roughness equivalencies exist between textures differing along different parameters. We posit that papillary ridges serve as tactile processing units, and neural ensembles encode the spatial profiles of the texture contact area to produce roughness estimates. The stimuli and the manufacturing process may be used in further studies of tactile roughness perception and in related neurophysiological applications.

OBJECTIVE:Blunted tachycardia during hypotension is a characteristic feature of patients with autonomic failure, but the range has not been defined. This study reports the range of orthostatic heart rate (HR) changes in patients with autonomic failure caused by neurodegenerative synucleinopathies. METHODS:Patients evaluated at sites of the U.S. Autonomic Consortium (NCT01799915) underwent standardized autonomic function tests and full neurological evaluation. RESULTS:We identified 402 patients with orthostatic hypotension (OH) who had normal sinus rhythm. Of these, 378 had impaired sympathetic activation, i.e., neurogenic OH, and based on their neurological examination were diagnosed with Parkinson disease, dementia with Lewy bodies, pure autonomic failure or multiple system atrophy. The remaining 24 patients had preserved sympathetic activation and their OH was classified as non-neurogenic, due to volume depletion, anemia or polypharmacy. Patients with neurogenic OH had twice the fall in systolic blood pressure (SBP) [-44±25 vs. -21±14 mmHg (mean±SD), p<0.0001] but only one third of the increase in HR than those with non-neurogenic OH (8±8 vs. 25±11 bpm, p<0.0001). A ΔHR/ΔSBP ratio of 0.492 bpm/mmHg had excellent sensitivity (91.3%) and specificity (88.4%) to distinguish between patients with neurogenic vs. non-neurogenic OH (AUC=0.96, p<0.0001). Within patients with neurogenic OH, HR increased more in those with multiple system atrophy (p=0.0003), but there was considerable overlap with patients with Lewy body disorders. INTERPRETATION/CONCLUSIONS:A blunted HR increase during hypotension suggests a neurogenic cause. A ΔHR/ΔSBP ratio lower than 0.5 bpm/mmHg is diagnostic of neurogenic OH.

Purpose: To demonstrate the appearance of osseous pathologies other than traumatic bone marrow edema using DECT with virtual noncalcium application for bone marrow edema and color overlay in patients presenting acutely to the emergency department with atraumatic musculoskeletal pain. Materials and Methods: This study was IRB approved and informed consent was waived. 166 consecutive patients presenting to the emergency department from 2/1/2017 - 7/1/2017 who underwent DECT (Somatom Force, Siemens) for musculoskeletal indications were retrospectively identified. CTs performed for the indication of trauma (n=113) were excluded. Post-processing was performed offline using a virtual noncalcium algorithm with color overlay (syngo.via; Siemans). Demographics were extracted from the electronic medical record. Descriptive statistics were performed. Results: In the study period, 20 females and 31 males, average age 59 years (range 20-92) underwent 53 CTs. Indications for imaging were infection (n=28), postoperative pain (n=2), and atraumatic pain (n=23). 34 (64%) had only soft tissue findings or were negative. 19 (36%) demonstrated atraumatic osseous etiologies of pain including metastasis, primary bone tumor, osteomyelitis, and inflammatory or infectious arthropathy. The appearance of these etiologies with color overlay is illustrated. 15 (28%) underwent subsequent imaging with MRI, bone scan, or PET with concordant results and these correlates are shown. Conclusion: DECT has emerged as a technology for detecting traumatic bone marrow edema. Bone marrow edema related to other, atraumatic etiologies including inflammatory arthropathy, tumor, and infection are also visually highlighted by this technique. In the emergent setting, DECT with virtual noncalcium subtraction and color overlay may be a useful adjunct to provide a visual aid for the detection or exclusion of marrow edema or amarrow infiltrating process in patients presenting with atraumatic musculoskeletal pain