Faculty Bibliography

As a result of its importance in key RNA metabolic processes, the ribonucleolytic RNA exosome complex has been the focus of intense study for almost two decades. Research on exosome subunit assembly, cofactor and substrate interaction, enzymatic catalysis and structure have largely been conducted using complexes produced in the yeast Saccharomyces cerevisiae or in bacteria. Here, we examine different populations of endogenous exosomes from human embryonic kidney (HEK) 293 cells and test their enzymatic activity and structural integrity. We describe methods to prepare EXOSC10-containing, enzymatically active endogenous human exosomes at suitable yield and purity for in vitro biochemistry and negative stain transmission electron microscopy. This opens the door for assays designed to test the in vitro effects of putative cofactors on human exosome activity and will enable structural studies of preparations from endogenous sources.

OBJECTIVE: The chronic inflammation associated with atherosclerosis is caused by lipid deposition followed by leukocyte recruitment to the arterial wall. We previously showed that the hematopoietic cell-specific adaptor protein Cas- and Hef1-associated signal transducer hematopoietic isoform (Chat-H)/SHEP1 regulated lymphocyte adhesion and migration. In this study, we analyzed the role of Chat-H in atherosclerosis development. APPROACH AND RESULTS: Using Chat-H-deficient bone marrow transplantation in low-density lipoprotein receptor-deficient mice, we found that Chat-H regulated atherosclerotic plaque formation. Chat-H deficiency in hematopoietic cells associated with lower plaque complexity and fewer leukocytes in the lesions, whereas myeloid-specific deletion of Chat-H was sufficient for conferring atheroprotection. Chat-H deficiency resulted in reduced recruitment of classical Ly6chigh and nonclassical Ly6clow monocytes to the plaques, which was accompanied by increased numbers of both monocyte subsets in the blood. This was associated with defective adhesion of Chat-H-deficient Ly6chigh and Ly6clow monocytes to vascular cell adhesion molecule-1 in vitro and impaired infiltration of fluorescent bead-loaded monocytes to atherosclerotic plaques. In contrast, Chat-H was dispensable for CX3CL1 and CCR1/CCR5-dependent migration of monocytes. CONCLUSIONS: Our findings highlight Chat-H as a key protein that regulates atherosclerosis development by controlling monocyte adhesion and recruitment to the plaques and identify a novel target that may be exploited for treating atherosclerosis.

BACKGROUND: Patients with atopic dermatitis (AD) are prone to skin infections, with microbes such as Staphylococcus aureus suspected of contributing to pathogenesis. Bleach baths might improve AD by reducing skin microbial burden. OBJECTIVE: We sought to characterize the microbiota of lesional and nonlesional skin in young children with AD and control subjects and compare changes after treatment with a topical corticosteroid (TCS) alone or TCS + dilute bleach bath. METHODS: In a randomized, placebo-controlled, single-blinded clinical trial in 21 children with AD and 14 healthy children, lesional and nonlesional AD skin was examined at baseline and after 4-week treatment with TCS alone or TCS plus bleach bath. Microbial DNA was extracted for quantitative polymerase chain reaction of predominant genera and 16S rRNA sequencing. RESULTS: At baseline, densities of total bacteria and Staphylococcus, including Staphylococcus aureus, were significantly higher at the worst AD lesional site than nonlesional (P = .001) or control (P < .001) skin; bacterial communities on lesional and nonlesional AD skin significantly differed from each other (P = .04) and from control (P < .001). After TCS + bleach bath or TCS alone, bacterial compositions on lesional skin normalized (P < .0001), resembling nonlesional skin, with microbial diversity restored to control skin levels. LIMITATIONS: The 4-week time period and/or the twice-weekly baths may not have been sufficient for additional impact on the cutaneous microbiome. More detailed sequencing may allow better characterization of the distinguishing taxa with bleach bath treatment. CONCLUSIONS: Treatment with a TCS cream suffices to normalize the cutaneous microbiota on lesional AD; after treatment, bacterial communities on lesional skin resemble nonlesional skin but remain distinct from control.

Solitary fibrous tumors (SFTs) are a relatively rare group of mesenchymal neoplasms. Klemperer and Rabin first described a case in the pleura in 1931, but SFTs have also been reported in extrapleural sites, including the oral cavity. SFTs of the oral cavity most commonly affect the buccal mucosa and tongue of female patients in their sixth decade of life. To date, only seven cases of oral SFTs located in the palate (three soft palate, four hard palate) have been documented in the literature. We present a case of a solitary fibrous tumor of the hard palate with review of the literature. A 26-year-old female with a past medical history significant for tuberous sclerosis presented to NYU College of Dentistry reporting a several year history of a painless mass of the hard palate. The mass was biopsied, initially diagnosed as cellular angiofibroma, and referred to the Department of Oral & Maxillofacial Surgery at Bellevue Hospital Center for further management. Examination revealed a 3x3 cm exophytic lesion on the right hard palate extending past the midline. The mass was non-tender to palpation and the mucosa overlying the lesion was intact without evidence of ulceration or necrosis. A CTA of the lesion showed mild prominence of vasculature along the right lateral soft and hard palate, possibly demonstrating supply from the ascending palatine artery. Postoperative surgical histopathology demonstrated a well-circumscribed, non-encapsulated lesion composed of spindle cells with admixed background slit-and-staghorn vessels in a patternless pattern. Immunohistochemical staining was diffusely positive for CD34 and Bcl-2 while negative for SMA, CD31, AE1/AE3, CAM5.2, S-100, EMA and demonstrated low KI-67 immunolabeling. SFTs constitute a heterogeneous group of rare spindlecell tumors that include benign and malignant neoplasms. Their cell of origin remains uncertain since CD34-positive spindle cells are also found in other mesenchymal neoplasms, such as giant cell angiofibromas and hemangiopericytoma, and share similar microscopic, immunohistochemical and biologic features. SFTs are usually well-demarcated and partially encapsulated neoplasms. Microscopically, SFTs show a wide range of morphological characteristics from predominantly fibrous lesions containing alternating fibrous areas and hyalinized thick-walled vessels to more cellular fibrous neoplasms with a "patternless pattern" and thinwalled branching vessels. Immunohistochemically, SFTs usually demonstrate CD34 and CD99, and vimentin positivity with variable Bcl-2, EMA and SMA positivity and are usually negative for CD68, desmin, pan-cytokeratins, and S-100 protein immunoreactivity. Malignant SFTs tend to demonstrate nuclear atypia, hypercellularity, loss of margin integrity, high mitotic rate (>4 per 10 high power fields) and lose CD34 immunoreactivity while overexpressing S-100 and p53. Our patient's lesion demonstrated positivity for only CD34 and Bcl-2, which, along with its aforementioned histological characteristics, was more consistent with the diagnosis of benign SFT than for the original diagnosis of cellular angiofibroma. CT imaging typically demonstrates SFTs as well-circumscribed, hypervascular masses with varying degrees of enhancement, necrosis or cystic change, and may show occasional internal calcification. Our patient's lesion demonstrated mild prominence of vasculature associated with the lesion with no invasion into the adjacent hard palate, consistent with a benign tumor. Due to its rare entity, SFTs are seldom considered in the differential diagnosis for submucosal masses of the oral cavity. However, reports suggest that SFTs may possess malignant characteristics and, thus, should be considered when evaluating well-circumscribed, solid masses in the oral cavity

Selective serotonin reuptake inhibitors (SSRIs) are a class of antidepressants that are utilized to treatmajor depressive disorders and anxiety disorders; SSRIs are the most widely used antidepressants worldwide.3 Past literature on SSRIs have documented that use of SSRIs reduce bone formation thus increasing the risk of bone fracture. Inhibiting the reuptake of 5-HTs results in increased osteoclast differentiation and inhibition of osteoblast proliferation, leading to an overall decrease in bone mass and bone mineral density. 1Recent studies have suggested that use of SSRIs is associated with increased risk of dental implant failure, although other studies involving bone loss and remodeling have reported conflicting results. In addition to SSRIs, proton- proton inhibitors and anti-epileptic drugs have also been implicated in impaired bone remodeling. We conducted a retrospective study on patients who completed dental implant therapy from December 2007 to January 2016 at a single institution. A total of 510 dental implants (167 implants placed in 29 patients using SSRIs) placed in 108 male patients were used to assess the risk associated with the use of SSRI. The data was analyzed with a multivariate analysis and linear regression model. Osseointegration is defined as a direct structural and functional connection between ordered living bone and the surface of a load-carrying implant, that is critical for implant stability.2 In order for implants to be considered successful, they must be fully osseointegrated with the bone and provide function. Implant failure is defined as presenting with one or more of the following: clinical signs of mobility, pain, infection, total loss of implant, radiographic bone loss and/or peri-apical pathology. The results of our study show that in patients taking an SSRI at the time of dental implant therapy, 10 implants failed and 157 were successful (5.99% failure rate), while in those who were not taking an SSRI, 20 implants failed and 323 were successful (5.83% failure rate). We found no association between SSRI and dental implant failure risk (RR = 1.03; 95% confidence interval, 0.4918-2.1443; p = 0.9436). A secondary outcome that was associated with increased failure rate was smoking habits (p = 0.03), which is in agreement with previous studies. Additionally, we did not find a dose-dependent risk of dental implant failure in patients who were taking a low-moderate vs. moderate- high dose of SSRIs (RR = 1.91, 95% confidence interval, 0.4201 -8.6981; p = 0.40) The study assessed the use of proton- pump inhibitors and anti-epileptic drugs in the above patients, demonstrating that there was no associated risk of implant failure (p =0.93; p = 0.84, respectively). Our results conclude that treatment with SSRIs is not associated with an increased failure rate of osseointegration of dental implants, which is not in agreement with previously reported studies. 4

Iron plays an especially important role in human physiological functions and pathological impairments. The superior properties of carbon nanotubes (CNTs) and their modification with bismuth and magnetic nanoparticles as developed in this work have led to an extraordinary and novel material to facilitate ultrasensitive detection in the nanomolar range. Here, we present the development of an electrochemical sensor for detection of ferrous (Fe²⁺) and ferric (Fe³⁺) iron by means of CNTs modified with bismuth and magnetic nanoparticles for higher sensitivity of detection. The sensor fabrication includes microfabrication methodologies, soft lithography, and electrodeposition. Cyclic voltammetry and differential pulse voltammetry are used for the electroanalytical studies and detection of the ions in samples. The sensor has a dynamic range of detection from 0.01 nm to 10 mm. The performance of the sensor with modified CNTs was explored for sensitivity and specificity. CNTs, modified with bismuth and magnetic nanoparticles by means of electrodeposition, enhanced the detection limit significantly down to 0.01 nm.

OBJECTIVE: Telomere attrition may mediate some of the effects of aging on reproductive function in women. Mice null or haploinsufficient for telomerase phenocopy the profile of reproductive aging in women, with progressive infertility caused by numerous defects in their reproductive cells. Telomeropathies, such as Dyskeratosis Congenita, provide a natural experiment to test the Telomere Theory of Reproductive Aging in women. This study attempts to extensively characterize the reproductive function in women with telomeropathies for the first time. DESIGN: Blood samples, cumulus cells, and arrested embryos were collected following the cycle of A 30 year old woman with a precocious aging syndrome and aplastic anemia, attributed to a telomeropathy (AMH=0.3 and AFC=8). She underwent, controlled ovarian stimulation with E2 prime protocol and 600 IU/day of gonadotropin, using mixed protocol and GnRH antagonist for 18 days. MATERIALS AND METHODS: Monochrome multiplex quantitative polymerase chain reaction (qPCR) assay (Cawthon 2009) measured telomere length in leukocytes extracted from whole blood as well as, cumulus cells stripped from retrieved follicles. Telomere (T) amplification was normalized to a single copy gene (S), resulting in a T/S ratio proportional to average telomere length in the population. Single-Cell Amplification of Telomere Repeats (SCATR) PCR (Wang 2013) was used to measure telomere length in discarded embryo blastomeres. Telomere (T) amplification was normalized a reference gene (R), producing a T/R ratio . One-Way ANOVA test was used to determine statistical significance. RESULTS: Hyperstimulation resulted in only 7 oocytes and 1 euploid blastocyst. Over the treatment course, leukocyte telomere length increased from T/S ratio= 0.192+/-.0157 to 0.234+/-.0306 and there was a statistically significant (p= .0256) linear increase during the treatment. Further, telomere length in a retrieved parthenogenetic, 2-cell embryo was (T/R average= .169+/-.021) and that in cumulus cells (T/S= 0.586+/-.0147). Telomere lengths in all assayed cell types were shorter than those from age matched controls. CONCLUSIONS: Young woman with reduced ovarian reserve, poor response to ovarian stimulation and a high percentage of arrested embryos and aneuploid embryos was still able to generate one euploid blastocyst with high dose controlled ovarian stimulation, demonstrating the promise of ART for fertility preservation in women with telomeropathies. Intriguingly, controlled ovarian hyperstimulation increased her leukocyte telomere length. Presumably, the supraphysiologic levels of estrogen activated telomerase activity, consistent with prior studies reporting an estrogen response element in the TERT gene. Future studies should examine whether women with telomeropathies may benefit from estrogen supplementation

This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.

The structure of the infectious prion protein (PrPSc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrPSc replicates by converting the non-infectious, cellular prion protein (PrPC) into the misfolded, infectious conformer through an unknown mechanism. PrPSc and its N-terminally truncated variant, PrP 27-30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27-30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 A. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 A3, predicted the height of each PrP 27-30 molecule as ~17.7 A. Together, the data indicate a four-rung beta-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.

Neurofilaments are uniquely complex among classes of intermediate filaments in being composed of four subunits (NFL, NFM, NFH and alpha-internexin in the CNS) that differ in structure, regulation, and function. Although neurofilaments have been traditionally viewed as axonal structural components, recent evidence has revealed that distinctive assemblies of neurofilament subunits are integral components of synapses, especially at postsynaptic sites. Within the synaptic compartment, the individual subunits differentially modulate neurotransmission and behavior through interactions with specific neurotransmitter receptors. These newly uncovered functions suggest that alterations of neurofilament proteins not only underlie axonopathy in various neurological disorders but also may play vital roles in cognition and neuropsychiatric diseases. Here, we review evidence that synaptic neurofilament proteins are a sizable population in the CNS and we advance the concept that changes in the levels or post-translational modification of individual NF subunits contribute to synaptic and behavioral dysfunction in certain neuropsychiatric conditions.