Resident Research in Emergency Medicine: An Introduction and Primer.

Training in research methodology represents an important aspect of emergency medicine (EM) resident education, but best methods for design, implementation, and dissemination of resident research remain elusive. Here we describe recommendations and best practices from the existing literature on EM resident research, including helpful tips on how to best implement a resident research program.

Efficacy and safety of simoctocog alfa (Nuwiq®) in patients with severe hemophilia A: a review of clinical trial data from the GENA program.

Simoctocog alfa (human-cl rhFVIII, Nuwiq®) is a 4th generation recombinant FVIII (rFVIII), without chemical modification or fusion with any other protein/fragment. Nuwiq® is produced in a human embryonic kidney cell line (HEK293F), which ensures human-specific post-translational protein processing. Nuwiq® was evaluated in seven prospective clinical studies in 201 adult and pediatric previously treated patients (PTPs) with severe hemophilia A. The NuProtect study in 110 previously untreated patients (PUPs) is ongoing. The mean half-life of Nuwiq® was 15.1-17.1 h in PTP studies with adults and adolescents, and 12.5 h in children aged 2-12 years. Clinical trials in PTPs demonstrated the efficacy and safety of Nuwiq® in the prevention and treatment of bleeds and as surgical prophylaxis. In the NuPreviq study of pharmacokinetic (PK)-guided personalized prophylaxis in 66 adult PTPs, 83% of patients had no spontaneous bleeds during 6 months of personalized prophylaxis and 57% were treated ⩽2 per week. No FVIII inhibitors were detected in PTPs after treatment with 43,267 injections and >80 million IU of Nuwiq®. Interim data for 66 PUPs with ⩾20 exposure days to Nuwiq® in NuProtect demonstrated a low cumulative high-titer inhibitor rate of 12.8% [actual incidence 12.1% (8/66)] and convincing efficacy and safety.

Phase Transition of H2 in Subnanometer Pores Observed at 75 K.

Here we report a phase transition in H2 adsorbed in a locally graphitic Saran carbon with subnanometer pores 0.5-0.65 nm in width, in which two layers of hydrogen can just barely squeeze, provided they pack tightly. The phase transition is observed at 75 K, temperatures far higher than other systems in which an adsorbent is known to increase phase transition temperatures: for instance, H2 melts at 14 K in the bulk, but at 20 K on graphite because the solid H2 is stabilized by the surface structure. Here we observe a transition at 75 K and 77-200 bar: from a low-temperature, low-density phase to a high-temperature, higher density phase. We model the low-density phase as a monolayer commensurate solid composed mostly of para-H2 (the ground nuclear spin state, S = 0) and the high-density phase as an orientationally ordered bilayer commensurate solid composed mostly of ortho-H2 (S = 1). We attribute the increase in density with temperature to the fact that the oblong ortho-H2 can pack more densely. The transition is observed using two experiments. The high-density phase is associated with an increase in neutron backscatter by a factor of 7.0 ± 0.1. Normally, hydrogen produces no backscatter (scattering angle >90°). This backscatter appears along with a discontinuous increase in the excitation mass from 1.2 amu to 21.0 ± 2.3 amu, which we associate with collective nuclear spin excitations in the orientationally ordered phase. Film densities were measured using hydrogen adsorption. No phase transition was observed in H2 adsorbed in control activated carbon materials.

Isotope effect on adsorbed quantum phases: diffusion of H2 and D2 in nanoporous carbon.

Quasielastic neutron scattering of H(2) and D(2) in the same nanoporous carbon at 10-40 K demonstrates extreme quantum sieving, with D(2) diffusing up to 76 times faster. D(2) also shows liquidlike diffusion while H(2) exhibits Chudley-Elliott jump diffusion, evidence of their different relationships with the local lattice of adsorption sites due to quantum effects on intermolecular interactions. The onset of diffusion occurs at 22-25 K for H(2) and 10-13 K for D(2). At these temperatures, H(2) and D(2) have identical thermal de Broglie wavelengths that correlate with the dominant pore size.

Anti-TNFα domain antibody construct CEP-37247: Full antibody functionality at half the size.

We report preclinical data for CEP-37247, the first human framework domain antibody construct to enter the clinic. At approximately 11 - 13kDa, domain antibodies or dAbs are the smallest antibody domain able to demonstrate the antigen-recognition function of an antibody, e.g. high selectivity and affinity for target antigen. CEP-37247 is a bivalent anti-tumor necrosis factor (TNF)α domain antibody protein construct combining the antigen-recognition function of a dAb with the pharmacological advantages of an antibody Fc region. As a homodimer, with each chain comprising VL dAb, truncated CH1, hinge, CH2 and CH3 domains, CEP-37247 has a molecular mass of approximately 78kDa, which is about half the size of a conventional IgG molecule. Surface plasmon resonance data demonstrate that CEP-37247 possesses high selectivity and affinity for TNFα. CEP-37247 is a potent neutralizer of TNFα activity in vitro in the L929 TNF-mediated cytotoxicity assay. In a human TNFα-over-expressing mouse model of polyarthritis, CEP-37247 prevents development of disease, and is at least as effective as the marketed product etanercept. Fc functionality is intact - CEP-37247 is capable of mediating antibody-dependent cell-mediated cytotoxicity and has a circulating half-life of approximately 4.5 days in cynomolgus macaques. Given the favorable properties outlined above, and its high expression levels (approaching 7 g/L) in a CHOK1 based-expression system, CEP-37247 is progressing into the clinic, where other potential advantages such as enhanced efficacy due to improved tissue distribution, and beneficial immunogenicity profile, will be evaluated.

Comparison of outcomes of permanently closed and periodically harvested coral reef reserves.

In many areas of the developing world, the establishment of permanent marine reserves is inhibited by cultural norms or socioeconomic pressures. Community conserved areas that are periodically harvested are increasingly being implemented as fisheries management tools, but few researchers have empirically compared them with permanently closed reserves. We used a hierarchical control-impact experimental design to compare the abundance and biomass of reef fishes, invertebrates, and substrate composition in periodically harvested and permanent reserves and in openly fished (control sites) of the South Pacific island country of Vanuatu. Fished species had significantly higher biomass in periodically harvested reserves than in adjacent openly fished areas. We did not detect differences in substratum composition between permanent reserves and openly fished areas or between permanent reserves and periodically harvested reserves. Giant clams (tridacnids) and top shells (Trochus niloticus) were vulnerable to periodic harvest, and we suggest that for adequate management of these species, periodically harvested community conservation areas be used in conjunction with other management strategies. Periodic harvest within reserves is an example of adaptive and flexible management that may meet conservation goals and that is suited to the social, economic, and cultural contexts of many coastal communities in the developing world.

Evidence-based medicine audit as a tool for improving emergency ophthalmology.

AIM: To audit the proportion of interventions in emergency ophthalmology that are evidence based and to determine whether the quality of care can be improved. METHODS: Audit of diagnosis-intervention pairs was carried out retrospectively in March 2003. The outcomes were assessed for evidence level reached in the Medline database 1966-2003 and the Cochrane Database of Systematic Reviews. Locally agreed guidelines were issued and the study repeated prospectively in March 2004, when new medical staff were at a similar level of experience. The participants had no prior knowledge of the study to avoid prescribing bias (Hawthorne's phenomenon). RESULTS: In the first part of the audit in 2003, 71% of interventions were evidence based, with 36% derived from systematic reviews, meta-analysis or randomised controlled trials (evidence levels 1-3). After guidelines for care were implemented in 2004, there was an improvement in the number of evidence-based interventions to 82% (P=0.04), and levels 1-3 were reached in 60% (P=0.02). The proportion with no evidence or against evidence dropped from 29 to 18% (P=0.04). An additional benefit was to reduce the number of re-attendances required. CONCLUSION: Evidence-based medicine can be used to improve the quality of care in the acute ophthalmic setting, both in refining the standard of interventions and in reducing the number of hospital visits.

Fascicular multiple ocular motor nerve paresis as first presentation of anaplastic astrocytoma.

A case of spontaneous, painless partial III (pupil-sparing) and IV fascicular nerve paresis as the first presentation of anaplastic astrocytoma is reported. The other ocular, neurological and systemic examination was within normal limits. The literature and possible anatomical location of this atypical presentation is reviewed.

Analysis of the structure and function of the transcriptional coregulator HOP.

Homeodomain-only protein (HOP) is an 8-kDa transcriptional corepressor that is essential for the normal development of the mammalian heart. Previous studies have shown that HOP, which consists entirely of a putative homeodomain, acts downstream of Nkx2.5 and associates with the serum response factor (SRF), repressing transcription from SRF-responsive genes. HOP is also able to recruit histone deacetylase (HDAC) activity, consistent with its ability to repress transcription. Unlike other classic homeodomain proteins, HOP does not appear to interact with DNA, although it has been unclear if this is because of an overall divergent structure or because of specific amino acid differences between HOP and other homeodomains. To work toward an understanding of HOP function, we have determined the 3D structure of full-length HOP and used a range of biochemical assays to define the parts of the protein that are functionally important for its repression activity. We show that HOP forms a classical homeodomain fold but that it cannot recognize double stranded DNA, a result that emphasizes the importance of caution in predicting protein function from sequence homology alone. We also demonstrate that two distinct regions on the surface of HOP are required for its ability to repress an SRF-driven reporter gene, and it is likely that these motifs direct interactions between HOP and partner proteins such as SRF- and HDAC-containing complexes. Our results demonstrate that the homeodomain fold has been co-opted during evolution for functions other than sequence-specific DNA binding and suggest that HOP functions as an adaptor protein to mediate transcriptional repression.

Molecular analysis of the interaction between the hematopoietic master transcription factors GATA-1 and PU.1.

GATA-1 and PU.1 are transcription factors that control erythroid and myeloid development, respectively. The two proteins have been shown to function in an antagonistic fashion, with GATA-1 repressing PU.1 activity during erythropoiesis and PU.1 repressing GATA-1 function during myelopoiesis. It has also become clear that this functional antagonism involves direct interactions between the two proteins. However, the molecular basis for these interactions is not known, and a number of inconsistencies exist in the literature. We have used a range of biophysical methods to define the molecular details of the GATA-1-PU.1 interaction. A combination of NMR titration data and extensive mutagenesis revealed that the PU.1-Ets domain and the GATA-1 C-terminal zinc finger (CF) form a low affinity interaction in which specific regions of each protein are implicated. Surprisingly, the interaction cannot be disrupted by single alanine substitution mutations, suggesting that binding is distributed over an extended interface. The C-terminal basic tail region of CF appears to be sufficient to mediate an interaction with PU.1-Ets, and neither acetylation nor phosphorylation of a peptide corresponding to this region disrupts binding, indicating that the interaction is not dominated by electrostatic interactions. The CF basic tail shares significant sequence homology with the PU.1 interacting motif from c-Jun, suggesting that GATA-1 and c-Jun might compete to bind PU.1. Taken together, our data provide a molecular perspective on the GATA-1-PU.1 interaction, resolving several issues in the existing data and providing insight into the mechanisms through which these two proteins combine to regulate blood development.

Zinc fingers as protein recognition motifs: structural basis for the GATA-1/friend of GATA interaction.

GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.

A classic zinc finger from friend of GATA mediates an interaction with the coiled-coil of transforming acidic coiled-coil 3.

Classic zinc finger domains (cZFs) consist of a beta-hairpin followed by an alpha-helix. They are among the most abundant of all protein domains and are often found in tandem arrays in DNA-binding proteins, with each finger contributing an alpha-helix to effect sequence-specific DNA recognition. Lone cZFs, not found in tandem arrays, have been postulated to function in protein interactions. We have studied the transcriptional co-regulator Friend of GATA (FOG), which contains nine zinc fingers. We have discovered that the third cZF of FOG contacts a coiled-coil domain in the centrosomal protein transforming acidic coiled-coil 3 (TACC3). Although FOG-ZF3 exhibited low solubility, we have used a combination of mutational mapping and protein engineering to generate a derivative that was suitable for in vitro and structural analysis. We report that the alpha-helix of FOG-ZF3 recognizes a C-terminal portion of the TACC3 coiled-coil. Remarkably, the alpha-helical surface utilized by FOG-ZF3 is the same surface responsible for the well established sequence-specific DNA-binding properties of many other cZFs. Our data demonstrate the versatility of cZFs and have implications for the analysis of many as yet uncharacterized cZF proteins.

CCHX zinc finger derivatives retain the ability to bind Zn(II) and mediate protein-DNA interactions.

Classical (CCHH) zinc fingers are among the most common protein domains found in eukaryotes. They function as molecular recognition elements that mediate specific contact with DNA, RNA, or other proteins and are composed of a betabetaalpha fold surrounding a single zinc ion that is ligated by two cysteine and two histidine residues. In a number of variant zinc fingers, the final histidine is not conserved, and in other unrelated zinc binding domains, residues such as aspartate can function as zinc ligands. To test whether the final histidine is required for normal folding and the DNA-binding function of classical zinc fingers, we focused on finger 3 of basic Krüppel-like factor. The structure of this domain was determined using NMR spectroscopy and found to constitute a typical classical zinc finger. We generated a panel of substitution mutants at the final histidine in this finger and found that several of the mutants retained some ability to fold in the presence of zinc. Consistent with this result, we showed that mutation of the final histidine had only a modest effect on DNA binding in the context of the full three-finger DNA-binding domain of basic Krüppel-like factor. Further, the zinc binding ability of one of the point mutants was tested and found to be indistinguishable from the wild-type domain. These results suggest that the final zinc chelating histidine is not an essential feature of classical zinc fingers and have implications for zinc finger evolution, regulation, and the design of experiments testing the functional roles of these domains.

Decreased visual acuity in patients with cytomegalovirus retinitis and AIDS.

PURPOSE: To investigate the causes of decreased visual acuity in patients with cytomegalovirus (CMV) retinitis in the acquired immunodeficiency syndrome (AIDS). METHODS: All human immunodeficiency virus (HIV)-positive patients seen in two ophthalmology units over a 15 month period from September 1996 were included in this retrospective study. A detailed ophthalmic examination was performed on all patients and in addition those with CMV retinitis underwent serial fundus photography. Decreased visual acuity was defined as a best corrected visual acuity < or = 6/12. CMV and retroviral treatment, CD4+ count and HIV viral load were also documented for each patient. RESULTS: Of 110 patients seen over the 15 month period, 26 (41 eyes) had a diagnosis of CMV retinitis. Twelve patients (16 eyes) with CMV retinitis had decreased visual acuity. The decreased visual acuity in 7 eyes was initially due to the CMV retinitis involving the macula and the optic nerve. Retinal detachment was responsible in 2 eyes and optic nerve atrophy in 1 eye. In 6 eyes (4 patients) the decreased visual acuity was due to a maculopathy--cystoid macular oedema and/or an epiretinal membrane in the presence of an inactive zone 2 or 3 CMV retinitis--with all these patients exhibiting a vitritis of varying grade. The decreased visual acuity in the maculopathy subgroup was irreversible in all except 1 eye, and 2 eyes in this category later developed a cataract. CONCLUSION: In this series, CMV-retinitis-'related' maculopathy was a major (38%) cause of decreased visual acuity, occurring in the absence of zone 1 retinitis and despite inactive peripheral CMV retinitis. A varying degree of vitritis was an associated feature in all these patients. This study therefore highlights maculopathy as an important and previously unrecognised significant cause of visual morbidity in CMV retinitis.

Ocular complications of intravenous cidofovir for cytomegalovirus retinitis in patients with AIDS.

PURPOSE: To describe the frequency of anterior uveitis and ocular hypotony in cidofovir-treated patients with acquired immune deficiency syndrome (AIDS)-related cytomegalovirus (CMV) retinitis. METHODS: A retrospective review was performed of all patients with AIDS-related CMV retinitis during a 12-month period. The CMV retinitis activity, concurrent illnesses and medications, and CD4+ lymphocyte count were recorded in addition to the degree of anterior chamber inflammation and intraocular pressure at each visit. The frequency of uveitis and ocular hypotony in cidofovir-treated patients was determined and the possible influence of other ocular and systemic factors considered. RESULTS: Eight of 9 patients on cidofovir developed anterior uveitis. The cellular anterior chamber activity resolved with topical corticosteroid administration in all eyes with uveitis but significant flare persisted despite topical steroids in 3 patients. Posterior synechiae responded poorly to topical mydriatic therapy, resulting in inadequate mydriasis which significantly limited the fundal view. One patient developed a visually significant unilateral hypotonous maculopathy. CONCLUSIONS: Patients treated with intravenous cidofovir for AIDS-related CMV retinitis are at significant risk of ocular adverse effects. Prompt treatment with topical corticosteroids and mydriatics may control uveitis and in some cases cidofovir treatment may be cautiously continued. In the event of ocular hypotony cidofovir should be discontinued in favour of an alternative anti-cytomegaloviral agent.

Neuronal apoptosis induced by HIV-1 Tat protein and TNF-alpha: potentiation of neurotoxicity mediated by oxidative stress and implications for HIV-1 dementia.

Apoptosis of neurons and non-neuronal cells has been demonstrated in the brain of AIDS patients with dementia. Previous studies suggest that the apoptotic stimuli are likely to be soluble factors. Several candidates for the soluble factors that lead to neuronal apoptosis in HIV-1 infection have been proposed, including the HIV-1 Tat protein and TNF-alpha. The mechanisms that lead to neuronal apoptosis in the brain of AIDS patients in vivo, may involve the combined effects of more than one pro-apoptotic factor. In this study, we examine whether exposure of primary human neurons to the combination of HIV-1 Tat and TNF-alpha can potentiate the induction of neuronal apoptosis compared with exposure to either factor alone. TNF-alpha was shown to potentiate the induction of neuronal apoptosis by HIV-1 Tat via a mechanism that involves increased oxidative stress. Antioxidants inhibited, but did not completely abolish the induction of neuronal apoptosis by Tat, suggesting that other mechanisms are also likely to be involved. These findings suggest that soluble HIV-1 Tat and TNF-alpha may play a role in neuronal apoptosis induced by HIV-1 infection of the CNS, particularly when present in combination. Our findings further suggest that one mechanism whereby combinations of pro-apoptotic factors may potentiate the induction of neuronal apoptosis in the brain of AIDS patients is by increasing oxidative stress. Understanding the role of oxidative stress and other mechanisms that lead to apoptosis in HIV-1 infection of the CNS may advance the development of new therapeutic strategies to prevent neuronal cell death and improve neurologic function in AIDS patients.

A comparison of Wright card with Teller card for detection of amblyopia.

PURPOSE: Teller acuity cards are used to assess visual acuity in infants but can underestimate amblyopia. In order to improve amblyopia detection, a new luminance balanced checkered card of 4.8 cycles/cm frequency was developed by Wright and Vistech (Dayton, Ohio). The Wright card was compared with a corresponding Teller card (of 4.8 cycles/cm) for detection of amblyopia. METHODS: A prospective masked study of 44 children was carried out. Each was assessed for amblyopia using both the Wright and Teller cards in addition to the Snellen or Allen card (optotype). The results were analyzed statistically using repeated measures of ordinal data. RESULTS: Thirteen eyes were found to be amblyopic with optotype testing, two with the Teller card and 17 with the Wright card. With optotype acuity as the standard, the sensitivity and specificity of the Teller card was 15.3% and 100%, while that of the Wright card was 100% and 94.6%. DISCUSSION: The Teller card is associated with high false negative test rate because it assesses grating acuity and allows spurious resolution possibly due to an edge artifact. This artifact is less pronounced with the Wright card. Further, the stripes on the Teller card require resolution only in horizontal axis, while the checkers requires uniform resolution in all axes. Different pathways for neural processing may also contribute to the disparate results with these methods. CONCLUSIONS: The Wright card was more sensitive than the Teller card for the detection of amblyopia in this study population and has potential value in preverbal children.