Faculty Bibliography

In this study, we have evaluated the efficacy of propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) to differentiate between viable and non-viable Ancylostoma caninum ova. The newly developed method was validated using raw wastewater seeded with known numbers of A. caninum ova. Results of this study confirmed that PMA-qPCR has resulted in average of 88 % reduction (P < 0.05) in gene copy numbers for 50 % viable +50 % non-viable when compared with 100 % viable ova. A reduction of 100 % in gene copies was observed for 100 % non-viable ova when compared with 100 % viable ova. Similar reductions (79-80 %) in gene copies were observed for A. caninum ova-seeded raw wastewater samples (n = 18) collected from wastewater treatment plants (WWTPs) A and B. The newly developed PMA-qPCR method was applied to determine the viable ova of different helminths (A. caninum, A. duodenale, Necator americanus and Ascaris lumbricoides) in raw wastewater, human fecal and soil samples. None of the unseeded wastewater samples were positive for the above-mentioned helminths. N. americanus and A. lumbricoides ova were found in unseeded human fecal and soil samples. For the unseeded human fecal samples (1 g), an average gene copy concentration obtained from qPCR and PMA-qPCR was found to be similar (6.8 x 10(5) +/- 6.4 x 10(5) and 6.3 x 10(5) +/- 4.7 x 10(5)) indicating the presence of viable N. americanus ova. Among the 24 unseeded soil samples tested, only one was positive for A. lumbricoides. The mean gene copy concentration in the positively identified soil sample was 1.0 x 10(5) +/- 1.5 x 10(4) (determined by qPCR) compared to 4.9 x 10(4) +/- 3.7 x 10(3) (determined by PMA-qPCR). The newly developed PMA-qPCR methods were able to detect viable helminth ova from wastewater and soil samples and could be adapted for health risk assessment.

BACKGROUND: Gaucher disease (GD) is a genetic disease caused by mutations in the GBA1 gene which result in reduced enzymatic activity of beta-glucocerebrosidase (GCase). This study identified the progranulin (PGRN) gene (GRN) as another gene associated with GD. METHODS: Serum levels of PGRN were measured from 115 GD patients and 99 healthy controls, whole GRN gene from 40 GD patients was sequenced, and the genotyping of 4 SNPs identified in GD patients was performed in 161 GD and 142 healthy control samples. Development of GD in PGRN-deficient mice was characterized, and the therapeutic effect of rPGRN on GD analyzed. FINDINGS: Serum PGRN levels were significantly lower in GD patients (96.65+/-53.45ng/ml) than those in healthy controls of the general population (164.99+/-43.16ng/ml, p<0.0001) and of Ashkenazi Jews (150.64+/-33.99ng/ml, p<0.0001). Four GRN gene SNPs, including rs4792937, rs78403836, rs850713, and rs5848, and three point mutations, were identified in a full-length GRN gene sequencing in 40 GD patients. Large scale SNP genotyping in 161 GD and 142 healthy controls was conducted and the four SNP sites have significantly higher frequency in GD patients. In addition, "aged" and challenged adult PGRN null mice develop GD-like phenotypes, including typical Gaucher-like cells in lung, spleen, and bone marrow. Moreover, lysosomes in PGRN KO mice exhibit a tubular-like appearance. PGRN is required for the lysosomal appearance of GCase and its deficiency leads to GCase accumulation in the cytoplasm. More importantly, recombinant PGRN is therapeutic in various animal models of GD and human fibroblasts from GD patients. INTERPRETATION: Our data demonstrates an unknown association between PGRN and GD and identifies PGRN as an essential factor for GCase's lysosomal localization. These findings not only provide new insight into the pathogenesis of GD, but may also have implications for diagnosis and alternative targeted therapies for GD.

Oral melanoacanthoma (MA) is a rare, benign pigmented lesion that presents as a painless, rapidly growing, brown-black macular lesion that commonly affects the buccal mucosa in areas that are subject to chronic trauma/irritation.1,2 MA is commonly seen in the third and fourth decades of life and primarily affects blacks with a strong female predilection.3,4 Histopathologically, the lesions exhibit proliferation of keratinocytes and dendritic melanocytes.5 This report includes two cases of oral melanoacanthoma and a review of the literature. Case 1: A 43-year-old black female presented with a slowly enlarging pigmented lesion on the right buccal mucosa. The patient did not recall any known trauma to the area or previous infection and reported that the lesion was painless but had a gradually increased in size. Oral examination revealed a 2.0 x 2.0 cm. brown macule on the right buccal mucosa. A punch biopsy was taken of the pigmented area. The tissue was placed in 10% formalin and submitted for microscopic examination. The tissue was stained with hematoxylin and eosin and exhibited acanthotic, stratified squamous epithelium with dendritic melanocytes dispersed throughout the epithelium consistent with a diagnosis of melanoacanthoma. Case 2: A-35 year-old black female presented with a rapidly growing pigmented lesion on the left buccal mucosa. Two years prior to presentation the patient had noted a brown lesion on the buccal mucosa adjacent to a fractured tooth. The lesion remained unchanged and asymptomatic for approximately two years. One week prior to presentation, the patient noted that the lesion was enlarging, but remained painless. Oral examination revealed a 1.5 x 1.5 cm. brown macule surrounded by erythema on the left buccal mucosa adjacent to a fractured tooth. A punch biopsy was taken that included both the pigmented and erythematous areas. The tissue was placed in 10% formalin and submitted for microscopic examination. The tissue was stained with hematoxylin and eosin and exhibited similar histopathologic features to the previous case. Immunohistochemical staining with S-100 and Melan-A dramatically demonstrated the dendritic melanocytes. Review of the literature revealed a total of 50 cases of oral melanoacanthoma. These lesions were reported in black females on the buccal mucosa with subsequent resolution. The cases here demonstrate similar clinical features and age at presentation to previously reported cases. The pathogenesis of oral MA remains unclear, however, most studies suggest this is a reactive process due to chronic irritation.2 Oral MA may regress following biopsy and no surgical intervention is required due to its selfresolving quality.5

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.

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: Reactive oxygen species (ROS) are a major cause of aging in all tissues studied, including reproductive tissues. Recent studies demonstrate extensiveDNA damage repair capacity in oocytes (1), and genetic variation in DNA damage repair pathways is associated with reproductive lifespan in women (2). We hypothesized that MII oocytes are more resistant to oxidative stress than other stages of development. DESIGN: Prospective, randomized study of a biologic intervention on mouse oocytes and embryos. MATERIALS AND METHODS: 80 MII oocytes and 40 cleavage embryos from B6C3F1 mice (Embryotech Laboratories, Inc, USA) were thawed and exposed to oxidative stress induced by Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 750nM), which generates ROS by uncoupling mitochondrial electron transport and disrupting mitochondrial function. Oocytes and embryos were randomized into untreated controls, or 5, 10 or 20 hour exposure to FCCP. DNA damage was determined by immuno- fluorescent staining for gamma-H2AX, or by mean telomere length, measured by single-cell qPCR. Data were analyzed by chi-square test and one-way ANOVA. RESULTS: 20 hours of exposure to FCCP is lethal for all embryos. Embryos exposed to FCCP for 5 and 10 hours of FCCP show increased g- H2AX staining (5 hours- 87.5% vs. 25% positive cells P<0.05; 10 hours- 100% vs 25%, P<0.05). However, these same doses and durations of FCCP do not increase DNA damage in oocytes- there is no significant increase of gamma-H2AX positive MII oocytes compared to controls (30% in the control group, 40% in the 5-hour group and 30% in the 10-hour group, P>0.05). Significantly higher numbers of gamma-H2AX positive MII oocytes are found only in the 20-hour group (100%, P<0.05), a dose which is uniformly lethal to embryos. As previously demonstrated, 45 minutes of 750nM FCCP treatment shortens telomeres in cleavage stage mouse embryos (3). However, MII oocytes exposed to 750nM FCCP for 5, 10, or 20 hours show no statistically significant telomere shortening compared to controls (P>0.05). CONCLUSIONS: MIIoocytes are more resistant to oxidative stress than cleavage embryos

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.

OBJECTIVE: The architecture and structure of the meiotic spindle influence embryo development(1) and risk of aneuploidy in women(2). The factors that disrupt the meiotic spindle remain incompletely understood. Dysfunctional mitochondria produce reactive oxygen species (ROS), which may directly perturb spindles(3). ROS also can disrupt spindles by inducing telomere attrition, since telomeres are essential for spindle formation and are especially susceptible to ROS(4). We studied the impact of ROS, produced by uncoupling mitochondria, on the area and retardance (measure ofmolecular order) ofmeiotic spindles and on mean telomere length of individual human oocytes. DESIGN: Prospective, randomized, paired research laboratory intervention. MATERIALS AND METHODS: 44 germinal vesicle (GV) stage oocytes were accessioned from 16 patients undergoing IVF/ICSI. GVs from each patient were randomly assigned to control or treatment group. Oocytes in the treatment group were cultured in media containing Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 750nM) for one hour to induce mitochondrial stress. Control oocytes were cultured in media without FCCP. GVoocytes from both groups were further cultured to permit meiotic maturation, as indicated by extrusion of the first polar body. Spindles were imaged non-invasively with an orientation independent polarized light microscope (Oosight, Hamilton Thorne, MA, USA). Spindle area and mean retardance were measured with software from the Oosight Imaging System. Mean oocyte telomere length was measured by single cell qPCR. Data were analyzed by Chi-square test and independent t test. RESULTS: Maturation rates of oocytes in the control and FCCP groups were 66.67% and 56.52% respectively. 71.4% of MII oocytes in the control group and 61.5% in the FCCP group had a detectable birefrigent spindle. FCCP decreased the area of the spindles compared to controls (19.84+/-2.11 sq. microns versus 37.77+/-4.79, P=0.011). Mean spindle retardance in the FCCP group also was significantly lower than that of controls (1.45+/-0.11 nm versus 2.19+/-0.16 nm, P=0.003). Telomere length (T/R ratio) did not differ between treatment and control groups (1.11+/-1.56 versus 1.29+/-0.19, P>0.05). Telomere length of oocytes with and without birefringence spindle within the treatment group also did not differ significantly (P>0.05). CONCLUSIONS: Meiotic maturation itself is relatively resistant to ROS, consistent with prior studies showing limited cell cycle check point control during oogenesis. However, ROS produced by acute mitochondrial stress disrupts spindle retardance and size. Reactive oxygen, at least acutely, does not induce telomere attrition in human oocytes

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

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