Faculty Bibliography

Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. Here, we report the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix-fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, we found that Osk-C binds the 3'UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3'UTRs, and provide structural insights into a novel protein-RNA interaction motif involving a hydrolase-related domain.

AIMS: Progress in tissue preservation (high-pressure freezing), data acquisition (tomographic electron microscopy; TEM) and analysis (image segmentation and quantification) have greatly improved the level of information extracted from ultrastructural images. Here, we combined these methods and developed analytical tools to provide an in-depth morphometric description of the intercalated disc (ID) in adult murine ventricle. As a point of comparison, we characterized the ultrastructure of the ID in mice heterozygous-null for the desmosomal gene plakophilin-2 (PKP2; mice dubbed PKP2-Hz). METHODS AND RESULTS: Tomographic EM images of thin sections of adult mouse ventricular tissue were processed by image segmentation analysis. Novel morphometric routines allowed us to generate the first quantitative description of the ID intercellular space based on three-dimensional data. We show that complex invaginations of the cell membrane increased total ID surface area by two orders of magnitude. In addition, PKP2-Hz samples showed increased average intercellular spacing, intercalated disc surface area and membrane tortuosity, as well as reduced number and length of mechanical junctions when compared to control. Finally, we observed membranous structures reminiscent of junctional sarcoplasmic reticulum at the ID, which were significantly more abundant in PKP2-Hz hearts. CONCLUSIONS: We have developed a systematic method to characterize the ultrastructure of the intercellular space in the adult murine ventricle and have provided a quantitative description of the structure of the intercellular membranes and of the intercellular space. We further show that PKP2 deficiency associates with ultrastructural defects. The possible importance of the intercellular space in cardiac behavior is discussed.

Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca(2+) homeostasis, but correcting lysosomal Ca(2+) deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

Cleft palate is a common birth defect in humans. Therefore, understanding the molecular genetics of palate development is important from both scientific and medical perspectives.Lhx6 and Lhx8 encode LIM homeodomain transcription factors, and inactivation of both genes in mice resulted in profound craniofacial defects including cleft secondary palate. The initial outgrowth of the palate was severely impaired in the mutant embryos, due to decreased cell proliferation. Through genome-wide transcriptional profiling, we discovered that p57Kip2 (Cdkn1c), encoding a cell cycle inhibitor, was up-regulated in the prospective palate of Lhx6-/-;Lhx8-/- mutants. p57Kip2 has been linked to Beckwith-Wiedemann syndrome and IMAGe syndrome in humans, which are developmental disorders with increased incidents of palate defects among the patients. To determine the molecular mechanism underlying the regulation of p57Kip2 by the Lhx genes, we combined chromatin immunoprecipitation, in silico search for transcription factor binding motifs, and in vitro reporter assays with putative cis-regulatory elements. The results of these experiments indicated that LHX6 and LHX8 regulated p57Kip2 via both direct and indirect mechanisms, with the latter mediated by Forkhead box (FOX) family transcription factors.Together, our findings uncovered a novel connection between the initiation of palate development and a cell cycle inhibitor via LHX. We propose a model in which Lhx6 and Lhx8 negatively regulate p57Kip2 expression in the prospective palate area to allow adequate levels of cell proliferation, and thereby promote normal palate development. This is the first report elucidating a molecular genetic pathway downstream of Lhx in palate development.

Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes, however their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a/b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a/b regulates endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a/b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Altogether, our work identifies a novel mechanism by which miRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM/miR-199a/b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking.

The widespread use of the mouse as a model system to study brain development has created the need for noninvasive neuroimaging methods that can be applied to early postnatal mice. The goal of this study was to optimize in vivo three- (3D) and four-dimensional (4D) manganese (Mn)-enhanced MRI (MEMRI) approaches for acquiring and analyzing data from the developing mouse brain. The combination of custom, stage-dependent holders and self-gated (motion-correcting) 3D MRI sequences enabled the acquisition of high-resolution (100-mum isotropic), motion artifact-free brain images with a high level of contrast due to Mn-enhancement of numerous brain regions and nuclei. We acquired high-quality longitudinal brain images from two groups of FVB/N strain mice, six mice per group, each mouse imaged on alternate odd or even days (6 3D MEMRI images at each day) covering the developmental stages between postnatal days 1 to 11. The effects of Mn-exposure, anesthesia and MRI were assessed, showing small but significant transient effects on body weight and brain volume, which recovered with time and did not result in significant morphological differences when compared to controls. Metrics derived from deformation-based morphometry (DBM) were used for quantitative analysis of changes in volume, position and signal intensity of a number of brain regions. The cerebellum, a brain region undergoing significant changes in size and patterning at early postnatal stages, was analyzed in detail to demonstrate the spatiotemporal characterization made possible by this new atlas of mouse brain development. These results show that MEMRI is a powerful tool for quantitative analysis of mouse brain development, with great potential for in vivo phenotype analysis in mouse models of neurodevelopmental diseases.

With the advent of the microvascular fibula free flap (MVFFF), maxillofacial reconstruction following ablative surgery has been a viable solution for patients with large maxillary or mandibular defects. Furthermore, total maxillofacial reconstruction in a two-stage process, where the fibula is harvested and dental implants placed (Stage I) followed by ablative surgery, inset and immediate loading with a dental prosthesis (Stage 2) has been well documented.1 This procedure, however, requires two separate surgical procedures and a delay of at least 10 weeks between each stage where the patient is often left partially or completely edentulous. The incorporation of computer-aided surgical simulation (CASS) and computer-aided design/computer-aided manufacturing (CAD/CAM) has made it possible to not only complete total maxillofacial reconstruction from tumor ablationto immediate insertion of an implant-retained dental prosthesis in a single OR procedure, butithas also increased the predictability and accuracy of maxillofacial reconstruction and decreased intraoperative time.2 Patients requiring more complex maxillofacial reconstruction heavily benefit from increased precision of the final surgical outcome as the accuracy of each osteotomy influences subsequent steps. The aim of our study is to assess the predictability and accuracy of virtually planned, single-stage total maxillofacial reconstruction, also known as 'Jaw in a Day'.³ We conducted a retrospective chart review of all patients who underwent maxillofacial tumor ablation, MVFFF reconstruction, implant placement and immediate implant loading with a dental prosthesis in a single OR procedure. These procedures were completed at Bellevue Hospital Center and NYU Langone Medical Center from January 2011 to January 2015. All cases were virtually planned with Medical Modeling (Golden, CO), and stereolithographic models, osteotomy guides, implant guides, and dental prosthesis were fabricated via CAD/CAM technology. To determine the precision and accuracy of the post-surgical outcomes, we compared the final positions of the implants and fibula on postoperative CT imaging with the planned positions of the implants and fibula based on preoperative virtual planning with Medical Modeling. A total of 8 patients underwent tumor ablation, MVFFF reconstruction, implant placement and immediateimplant loading with a dental prosthesis in a single OR procedure. All patients were diagnosed with benign mandibular (7) and maxillary (1) tumors, including ameloblastoma (6), odontogenic myxoma (1), and AVmalformation (1).Atotal of 35implants were placed with satisfactory primary stability at the time of surgery. On average, the final positions of the implants placed were within 2mm of the virtually treatment planned positions within the fibula. To date, there have been no flap failures and only one implant has failed osseointegration into the MVFFF. Total maxillofacial reconstruction via CASS and CAD/CAM technology has made it possible for surgeons to complete these procedures with high precision and accuracy while minimizing intraoperative time. Additionally, immediate dental rehabilitation is possible at the time of ablation, eliminating the period of edentulism for these patients. Given the highly predictable and accurate postoperative outcomes and low complications rates of virtually planned total maxillofacial reconstruction with a MVFFF and immediate dental rehabilitation, this technique is quickly becoming the standard of care for patients requiring complex maxillofacial reconstruction

BACKGROUND: Patients undergoing cardiovascular procedures remain at increased risk for myocardial infarction, stroke, and cardiovascular death. Risk factor control in this patient population remains suboptimal and would likely benefit from strategies targeting education, lifestyle, and healthy behaviors. DESIGN: The IMPACT trial is a 400-subject prospective randomized trial designed to compare different cardiovascular prevention strategies in subjects following a cardiovascular intervention. The trial began enrollment in the Spring of 2012 and is randomizing subjects in a 1:1:1 manner to usual care, a one-time cardiovascular prevention consult, or a one-time cardiovascular prevention consult plus behavioral intervention program (telephone-based motivational interviewing and tailored text messages) over a 6-month period. The primary end point is non-high-density lipoprotein cholesterol. Secondary end points include other plasma lipid values, metabolic risk, smoking cessation, physical activity, dietary intake, medication use and adherence, and quality of life. CONCLUSIONS: The IMPACT trial provides data on different management strategies for risk factor optimization in subjects following cardiovascular procedures. The results will provide a platform for the continued development of novel multidisciplinary interventions in this high-risk population.

The LTBPs (or latent transforming growth factor beta binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, -2, -3, and -4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here. The LTBPs were first identified as forming latent complexes with TGFbeta by covalently binding the TGFbeta propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFbeta precursor in the trans-golgi network but LAP and TGFbeta remain strongly bound through non-covalent interactions. LAP, TGFbeta, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFbeta latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFbeta activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFbeta regulation, TGFbeta-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly.

Children having cerebral palsy will incur life-long disabilities, which require high costs of medical and nursing care. This imposes a tremendous burden on the families of the affected children, whether financially or emotionally. It is understandable for the affected families to initiate court litigation in order to alleviate the financial burden and at the same time to overcome the emotional pain associated with the permanent and lifetime implications which cerebral palsy entails. However, suing for such injuries in court and identification of medical malpractice is not an easy task for the families. Further, court litigation tends to be tedious, lengthy and unpleasant. The hazards of litigation have prompted several countries to find an available alternative to court litigation, such as the implementation of a no-fault compensation system, to settle these types of claims. Thus, it is much applauded that the Japan Obstetric Compensation System for Cerebral Palsy was established in January 2009, with the aim of helping children with such disabilities to improve their quality of life and to provide monetary compensation in order to lessen the economic burden on the family. The system features two vital pillars; that is, compensation and causal analysis prevention. The system aims at improving the quality of maternity care and analyzing the causes of accidents in order to prevent similar cases from happening in the future. Overall, the system clearly depicts social solidarity in encouraging collective responsibility for the mishaps suffered by the community.