Faculty Bibliography

Ectopic expression of the transcription factors Oct4, Sox2, c-Myc, and Klf4 in fibroblasts generates induced pluripotent stem (iPS) cells. Little is known about the nature and sequence of molecular events accompanying nuclear reprogramming. Using doxycycline-inducible vectors, we have shown that exogenous factors are required for about 10 days, after which cells enter a self-sustaining pluripotent state. We have identified markers that define cell populations prior to and during this transition period. While downregulation of Thy1 and subsequent upregulation of SSEA-1 occur at early time points, reactivation of endogenous Oct4, Sox2, telomerase, and the silent X chromosome mark late events in the reprogramming process. Cell sorting with these markers allows for a significant enrichment of cells with the potential to become iPS cells. Our results suggest that factor-induced reprogramming is a gradual process with defined intermediate cell populations that contain the majority of cells poised to become iPS cells

Neurogenesis, the division, migration, and differentiation of new neurons, occurs throughout life. Brain derived neurotrophic factor (BDNF) has been identified as a potential signaling molecule regulating neurogenesis in the subventricular zone (SVZ), but its functional consequences in vivo have not been well defined. We report marked and unexpected deficits in survival but not proliferation of newly born cells of adult knock-in mice containing a variant form of BDNF [a valine (Val) to methionine (Met) substitution at position 66 in the prodomain of BDNF (Val66Met)], a genetic mutation shown to lead to a selective impairment in activity-dependent BDNF secretion. Utilizing knock-out mouse lines, we identified BDNF and tyrosine receptor kinase B (TrkB) as the critical molecules for the observed impairments in neurogenesis, with p75 knock-out mice showing no effect on cell proliferation or survival. We then localized the activated form of TrkB to a discrete population of cells, type A migrating neuroblasts, and demonstrate a decrease in TrkB phosphorylation in the SVZ of Val66Met mutant mice. With these findings, we identify TrkB signaling, potentially through activity dependent release of BDNF, as a critical step in the survival of migrating neuroblasts. Utilizing a behavioral task shown to be sensitive to disruptions in olfactory bulb neurogenesis, we identified specific impairments in spontaneous olfactory discrimination, but not general olfactory sensitivity or habituation to olfactory stimuli in BDNF mutant mice. Through these observations, we have identified novel links between genetic variant BDNF and adult neurogenesis in vivo, which may contribute to significant impairments in olfactory function

Bacteriophage phi 12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species phi 6, host infection requires binding of a multimeric P3 complex to type IV pili. In species varphi8, phi 12, and phi 13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In phi 8 the protein components are designated P3a and P3b while in species phi 12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T=13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact phi 12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed 'the donut' and appears to inhibit BHT-induced viral envelope fusion

Proteomic analyses of the nucleolus have revealed almost 700 functionally diverse proteins implicated in ribosome biogenesis, nucleolar assembly, and regulation of vital cellular processes. However, this nucleolar inventory has not unveiled a specific consensus motif necessary for nucleolar binding. The ribosomal protein family characterized by their basic nature should exhibit distinct binding sequences that enable interactions with the rRNA precursor molecules facilitating subunit assembly. We succeeded in delineating 2 minimal nucleolar binding sequences of human ribosomal protein S6 by fusing S6 cDNA fragments to the 5' end of the LacZ gene and subsequently detecting the intracellular localization of the beta-galactosidase fusion proteins. Nobis1 (nucleolar binding sequence 1), comprising of 4 highly conserved amino acid clusters separated by glycine or proline, functions independently of the 3 authentic nuclear localization signals (NLSs). Nobis2 consists of 2 conserved peptide clusters and requires the authentic NLS2 in its native context. Similarly, we deduced from previous publications that the single Nobis of ribosomal protein S25 is also highly conserved. The functional protein domain organization of the ribosomal protein S6e family consists of 3 modules: NLS, Nobis, and the C-terminal serine cluster of the phosphorylation sites. This modular structure is evolutionary conserved in vertebrates, invertebrates, and fungi. Remarkably, nucleolar binding sequences of small and large ribosomal proteins reside in peptide clusters conserved over millions of years.

Psoriatic arthritis is a complex, chronic inflammatory disease with both skin and joint involvement. Clinical presentation varies considerably among patients and during the course of the disease. Assessment of patients for psoriatic arthritis requires careful attention to patient history, a focused physical examination, and inspection for characteristic radiographic changes. Although this disease was once thought to be a rare and mild form of arthritis, recent studies have shown that patients with psoriatic arthritis may develop significant disability, with up to 20% of cases demonstrating a rapidly progressive, debilitating clinical course. Orthopedic manifestations of the disease can be severe and can cause significant physical disability. Although surgical intervention for psoriatic arthritis is relatively uncommon, having an understanding of the assessment, available treatment options, and surgical considerations allows for improved outcome in the management of this complex patient population

Constrained acetabular liners can fail leading to recurrent dislocation. Failure can occur at any of the five possible interfaces: bone-acetabular shell, acetabular shell-constrained liner insert, constrained liner insert-bipolar head, bipolar head- femoral head and femoral head-trunion. We report a patient who presented with dissociation of the cemented Osteonics acetabular constrained liner (Stryker-Howmedica-Osteonics, Rutherford, New Jersey). The failure interface was at the factory pre-assembled constrained liner insert-bipolar head without any locking ring failure; instead there was deformation of the constrained liner insert's polyethylene rim, which facilitated dissociation. To our knowledge, there are no previous reports of such a failure mode pertaining to this type of constrained liner. Constrained acetabular liners are indicated during primary or revision total hip arthroplasty for patients who are at high risk for dislocations or who have had recurrent dislocations. Failure rates (typically recurrent dislocation) range from 4% to 29% at mid-term follow-up. The first report on the Osteonics acetabular constrained liner was published in 1994. Failures have been reported previously to occur at surgically controllable interfaces, such as the acetabular shell from the bony surface and the constrained liner insert from the acetabular metal shell, and have been attributable to excessive constraint or improper technique. All dissociations pertaining to factory-preassembled component interfaces have been attributed to breakage of the locking ring. This article presents the first case of disengagement of the tripolar constrained liner without disruption of the locking ring

Human exposure to air pollutants, including ambient particulate matter, has been proposed as a mechanism for the rise in allergic disorders. Diesel exhaust particles, a major component of ambient particulate matter, induce sensitization to neoallergens, but the mechanisms by which sensitization occur remain unclear. We show that diesel exhaust particles upregulate thymic stromal lymphopoietin in human bronchial epithelial cells in an oxidant-dependent manner. Thymic stromal lymphopoietin induced by diesel exhaust particles was associated with maturation of myeloid dendritic cells, which was blocked by anti-thymic stromal lymphopoietin antibodies or silencing epithelial cell-derived thymic stromal lymphopoietin. Dendritic cells exposed to diesel exhaust particle-treated human bronchial epithelial cells induced Th2 polarization in a thymic stromal lymphopoietin-dependent manner. These findings provide new insight into the mechanisms by which diesel exhaust particles modify human lung mucosal immunity

Macrophage migration inhibitory factor (MIF), a proinflammatory mediator, has been shown to be elevated following heart surgery in adults and may be associated with several postoperative complications, including cardiac and pulmonary dysfunction. In this study, we aimed to measure perioperative plasma MIF, interleukin (IL)-8, and free T4 in 20 children age <4 years undergoing surgical repair of congenital heart lesions with left ventricular volume overload, and to determine whether the response of these mediators determined postoperative outcomes. Plasma samples were obtained preoperatively, immediately on arrival in the pediatric intensive care unit (PICU), and at 12, 24, and 48 h. Patients were continuously monitored in the PICU, and data were recorded daily for therapeutic and monitoring procedures that reflected the invasiveness, intensity, and complexity of care rendered (therapeutic interventional scoring system, TISS). Preoperative plasma MIF, IL-8, and free T4 were not different from age-matched healthy children. However, plasma MIF and IL-8 increased significantly 2 h after completion of cardiopulmonary bypass, and then normalized within 24 h. Peak postoperative levels of MIF (48 +/- 24 ng/mL) and IL-8 (79 +/- 57 pg/mL) correlated significantly with duration of cardiopulmonary bypass. The magnitude of the postoperative increase in plasma MIF was associated with increased number of days required for mechanical ventilation (r = 0.553; P = 0.012), and peak plasma IL-8 correlated significantly with the fraction of inhaled oxygen (FiO(2)) required immediately after surgery (r = 0.510; P = 0.02). Higher circulating MIF levels correlated significantly with increased inotropic support requirements on the second postoperative day, whereas higher postoperative IL-8 levels were associated with higher TISS scores, suggesting increased need for postoperative medical care. These data suggest a potential negative effect of high circulating levels of MIF in the immediate postoperative period on respiratory and cardiovascular functions, and support the development of therapeutic strategies targeting MIF function in this clinical setting.

The expansion of gene therapy applications from inherited disorders to acquired conditions has been mirrored by an exponential rise in both experimental work and clinical trials. This review highlights current plastic surgical delivery systems and clinical applications for targeted gene therapy. We revisit some of the vectors used both experimentally and in clinical gene therapy trials, with an emphasis on developments in plastic surgical delivery systems resulting in improved targeting of therapeutic genes. In addition, we discuss a novel technique for the delivery of gene therapy using the ex vivo transduction of free flaps, developed in our laboratory. This delivery system achieves targeted high-level transgene expression with minimal demonstrable systemic toxicity. Advances in delivery systems are essential for translating basic research into clinical therapeutics