Faculty Bibliography

OBJECTIVE: To study the medical emergencies occurring on a tertiary otolaryngology service identified using a rapid response system (RRS). DESIGN: Retrospective chart review of RRS activations during 21 months. SETTING: Specialised otolaryngology care unit within the University of Pittsburgh Medical Center Presbyterian/Montefiore Hospital, a tertiary, academic, teaching hospital in the USA. INTERVENTION(S): None. RESULTS: 1171 unit admissions. Unit mortality was 5.1/1000 admissions. 53 patients were involved in 67 RRS activations (4/53 deaths). 32 of 67 events were due to respiratory derangements, most commonly pneumonia. 18 of 67 events were due to cardiovascular abnormalities, most commonly hypertension and myocardial infarction. 11 of 67 events were secondary to mental status changes, several of which were related to adverse drug events. 6 of 67 events were secondary to acute bleeding. 23 of 67 events occurred within 24 h of patient transfer/admission, 14 of those after operations. RRS activation was a marker for in-hospital death (RR 42.2, 95% CI 7.9 to 225.2) compared with that in patients not activating the RRS. CONCLUSIONS: Although otolaryngology care units attempt to prevent adverse events, emergencies still occur. RRSs identify deteriorating otolaryngology patients who are at increased risk for mortality. RRSs are an efficient mechanism of intervention during a medical emergency. RRSs provide a convenient method of identifying medical/system errors and educational opportunities.

PURPOSE OF REVIEW: Antifungal drug resistance is a confounding factor that negatively impacts clinical outcome for patients with serious mycoses. Early detection of fungi in blood or other specimens with a rapid assessment of drug susceptibility could improve the survival of patients with invasive disease by accelerating the initiation of appropriate antifungal treatment. Recent years have seen the growth of molecular technology that is ideally suited for fungal identification and assessment of drug resistance mechanisms. RECENT FINDINGS: Elucidation of the genetic mechanisms responsible for triazole and echinocandin resistance in prominent Candida spp. and Aspergillus spp. provides an opportunity to develop molecular diagnostic platforms suitable for rapid detection of primary and secondary drug resistance. Several highly dynamic and robust amplification/detection methodologies are now available that can provide simultaneous species identification and high fidelity discrimination of resistance alleles. SUMMARY: Molecular diagnostic platforms are ideal for rapid detection of fungal pathogens and they provide an opportunity to develop in parallel molecular assays that can evaluate antifungal drug resistance.

An early step in mammalian fertilization is species-restricted binding of sperm to the oocyte's zona pellucida (ZP), a thick extracellular coat. Sperm bind to the ZP of unfertilized oocytes, but not to the ZP of fertilized oocytes. Shortly after binding to the unfertilized oocyte ZP, sperm undergo the acrosome reaction (AR). Three mouse ZP glycoproteins, called mZP1-3, constitute the mouse oocyte's ZP and participate in the process of fertilization. For example, sperm exposed to unfertilized oocyte mZP3 at nanomolar concentrations are inhibited from binding to oocytes and undergo the AR; mZP1 and mZP2 have no effect. mZP3 from fertilized oocytes has no effect on sperm binding and is unable to induce the AR. These properties of mZP3 strongly suggest that it is a receptor for sperm and inducer of the AR. Mapping of the mZP3 combining-site for sperm suggests that it is located near the C-terminus of the polypeptide, in a region encoded by exon-7 of the mZP3 gene. This region of mZP3 is a site of positive Darwinian selection. When mZP3 exon-7 is fused to the Fc fragment of human IgG and sperm are exposed to the chimeric protein, the sperm are inhibited from binding to oocytes, suggesting that the inhibitory activity of mZP3 is dependent on this region of its polypeptide.

OBJECTIVE: Deletion of Akt1 leads to severe atherosclerosis and occlusive coronary artery disease. Vascular smooth muscle cells (VSMCs) are an important component of atherosclerotic plaques, responsible for promoting plaque stability in advanced lesions. Fibrous caps of unstable plaques contain less collagen and ECM components and fewer VSMCs than caps from stable lesions. Here, we investigated the role of Akt1 in VSMC proliferation, migration, and oxidative stress-induced apoptosis. In addition, we also characterized the atherosclerotic plaque morphology and cardiac function in an atherosclerosis-prone mouse model deficient in Akt1. METHODS AND RESULTS: Absence of Akt1 reduces VSMC proliferation and migration. Mechanistically, the proliferation and migratory phenotype found in Akt1-null VSMCs was linked to reduced Rac-1 activity and MMP-2 secretion. Serum starvation and stress-induced apoptosis was enhanced in Akt1 null VSMCs as determined by flow cytometry using Annexin V/PI staining. Immunohistochemical analysis of atherosclerotic plaques from Akt1(-)(/)(-ApoE)(-)(/)(-) mice showed a dramatic increase in plaque vulnerability characteristics such as enlarged necrotic core and reduced fibrous cap and collagen content. Finally, we show evidence of myocardial infarcts and cardiac dysfunction in Akt1(-)(/)(-ApoE)(-)(/)(-) mice analyzed by immunohistochemistry and echocardiography, respectively. CONCLUSIONS: Akt1 is essential for VSMC proliferation, migration, and protection against oxidative stress-induced apoptosis. Absence of Akt1 induces features of plaque vulnerability and cardiac dysfunction in a mouse model of atherosclerosis

Efforts to improve melanoma response rates to temozolomide (TMZ) have thus far been unsuccessful. We screened a library of 2,000 marketed drugs and natural products to identify agents with the potential to sensitize melanoma cells to the effects of TMZ. Celastrol (CEL), a natural compound found in the Thunder of God vine, was identified based on its ability to enhance cell death in TMZ-resistant melanoma cells. A cell proliferation assay was used to compare the growth-inhibitory effects of TMZ alone versus TMZ/CEL combination treatment. Cytotoxic synergy was assessed using combination-index methods. The expression of nuclear factor-kappaB (NF-kappaB), IkappaB, mitogen-activated protein kinase, and ubiquitinated proteins were examined using Western blotting, and the localization of NF-kappaB in CEL-treated melanoma cells was evaluated using immunofluorescence microscopy. The CEL/TMZ combination synergistically inhibited cell proliferation in melanoma cells. CEL treatment increased the levels of ubiquitinated proteins, reduced the levels of tumor necrosis factor-alpha-induced IkappaB phosphorylation, and blocked NF-kappaB translocation to the nucleus. Inhibition of NF-kappaB with small interfering RNA mimicked the ability of CEL to sensitize melanoma cells to TMZ, suggesting that inhibition of NF-kappaB may play a role in TMZ/CEL-induced cytotoxicity. The TMZ/CEL combination induced the phosphorylation of c-Jun NH(2)-terminal kinase, implicating the mitogen-activated protein kinase pathway in the treatment effects. Our data suggest that CEL may be effective in sensitizing resistant melanoma cells to the effects of TMZ. (Mol Cancer Res 2009;7(12):OF1-8)

BACKGROUND: The PEAK PlasmaBlade is a new electrosurgical device that uses pulsed radiofrequency to generate a plasma-mediated discharge along the exposed rim of an insulated blade, creating an effective cutting edge while the blade stays near body temperature. METHODS: Full-thickness incisions were made on the dorsums of pigs with the PlasmaBlade, a conventional electrosurgical device, and a scalpel, and blood loss was quantified. Wounds were harvested at designated time points, tested for wound tensile strength, and examined histologically for scar formation and tissue damage. RESULTS: Bleeding was reduced significantly (59 percent) in PlasmaBlade incisions compared with scalpel incisions, and acute thermal damage from the PlasmaBlade (66 +/- 5 microm) was significantly less than both cut and coagulation mode electrosurgical incisions (456 +/- 35 microm and 615 +/- 22 microm, respectively). Histologic scoring for injury and wound strength was equivalent between the PlasmaBlade and scalpel incisions. By 6 weeks, the healed PlasmaBlade and scalpel incisions were approximately three times stronger, and scar cosmetic appearance was significantly better compared with electrosurgical incisions. CONCLUSIONS: The PlasmaBlade is a promising new surgical instrument that provides atraumatic, scalpel-like cutting precision and electrosurgical-like hemostasis, resulting in minimal bleeding, tissue injury, and scar formation.

The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1-induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA-binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus nondifferentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1-bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that polycomb repressive complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1-repressed genes. These data provide insights into GATA-1-mediated gene regulation in vivo.

Although mature neurotrophins are well described trophic factors that elicit retrograde survival signaling, the precursor forms of neurotrophins (i.e., proneurotrophins) can function as high-affinity apoptotic ligands for selected neural populations. An outstanding question is whether target-derived proneurotrophins might affect neuronal survival/death decisions through a retrograde transport mechanism. Since neurotrophin-3 (NT-3) is highly expressed in non-neural tissues that receive peripheral innervation, we investigated the localized actions of its precursor (proNT-3) on sympathetic neurons in the present study. Pharmacological inhibition of intracellular furin proteinase activity in 293T cells resulted in proNT-3 release instead of mature NT-3, whereas membrane depolarization in cerebellar granule neurons stimulated endogenous proNT-3 secretion, suggesting that proNT-3 is an inducible bona fide ligand in the nervous system. Our data also indicate that recombinant proNT-3 induced sympathetic neuron death that is p75(NTR)- and sortilin-dependent, with hallmark features of apoptosis including JNK (c-Jun N-terminal kinase) activation and nuclear fragmentation. Using compartmentalized culture systems that segregate neuronal cell bodies from axons, proNT-3, acting within the distal axon compartment, elicited sympathetic neuron death and overrode the survival-promoting actions of NGF. Together, these results raise the intriguing possibility that dysregulation of proneurotrophin processing/release by innervated targets can be deleterious to the neurons projecting to these sites

The retrotrapezoid nucleus (RTN) is a group of neurons in the rostral medulla, defined here as Phox2b-, Vglut2-, neurokinin1 receptor-, and Atoh1-expressing cells in the parafacial region, which have been proposed to function both as generators of respiratory rhythm and as central respiratory chemoreceptors. The present study was undertaken to assess these two putative functions using genetic tools. We generated two conditional Phox2b mutations, which target different subsets of Phox2b-expressing cells, but have in common a massive depletion of RTN neurons. In both conditional mutants as well as in the previously described Phox2b(27Ala) mutants, in which the RTN is also compromised, the respiratory-like rhythmic activity normally seen in the parafacial region of fetal brainstem preparations was completely abrogated. Rhythmic motor bursts were recorded from the phrenic nerve roots in the mutants, but their frequency was markedly reduced. Both the rhythmic activity in the RTN region and the phrenic nerve discharges responded to a low pH challenge in control, but not in the mutant embryos. Together, our results provide genetic evidence for the essential role of the Phox2b-expressing RTN neurons both in establishing a normal respiratory rhythm before birth and in providing chemosensory drive.

Receptor protein tyrosine phosphatases (RPTPs) control many aspects of nervous system development. At the Drosophila neuromuscular junction (NMJ), regulation of synapse growth and maturation by the RPTP LAR depends on catalytic phosphatase activity and on the extracellular ligands Syndecan and Dally-like. We show here that the function of LAR in controlling R7 photoreceptor axon targeting in the visual system differs in several respects. The extracellular domain of LAR important for this process is distinct from the domains known to bind Syndecan and Dally-like, suggesting the involvement of a different ligand. R7 targeting does not require LAR phosphatase activity, but instead depends on the phosphatase activity of another RPTP, PTP69D. In addition, a mutation that prevents dimerization of the intracellular domain of LAR interferes with its ability to promote R7 targeting, although it does not disrupt phosphatase activity or neuromuscular synapse growth. We propose that LAR function in R7 is independent of its phosphatase activity, but requires structural features that allow dimerization and may promote the assembly of downstream effectors