We have demonstrated ultra-high efficiency amplification in Tm-doped fiber with both core- and cladding-pumped configurations using a resonant tandem-pumping approach. These Tm-doped fiber amplifiers are pumped in-band with a 1908 nm Tm-doped fiber laser and operate at 1993 nm with >90% slope efficiency. In a core-pumped configuration, we have achieved 92.1% slope efficiency and 88.4% optical efficiency at 41 W output power. In a cladding-pumped configuration, we have achieved 123.1 W of output power with 90.4% optical efficiency and a 91.6% slope efficiency. We believe these are the highest optical efficiencies achieved in a Tm-doped fiber amplifier operating in the 2-micron spectral region.
The progression of autoimmune diseases is dictated by deviations in the fine balance between pro-inflammatory versus regulatory responses and pathogen recognition receptors (PRRs) play a key role in maintaining the balance. Previously, we have reported that ligation of TLR2 and Dectin 1 on APCs by zymosan results in a regulatory immune response that prevents type 1 diabetes (T1D). Here, we show that TLR2 and Dectin 1 engagement by zymosan promotes regulatory T cell (Treg) responses against the pancreatic ?-cell-specific Ag. Unlike the TLR4 ligand, bacterial lipopolysaccharide, which induced pro-inflammatory cytokines and pathogenic T cells, zymosan induced a mixture of pro- and anti-inflammatory factors and regulatory T cells, both in vitro and in vivo. Ag-specific T cells that are activated using zymosan-exposed dendritic cells (DCs) expressed Foxp3 and produced large amounts of IL-10, TGF-?1 and IL-17. NOD mice that received ?-cell-Ag loaded, zymosan-exposed DCs showed delayed hyperglycemia. Injection of NOD mice at pre-diabetic age and early-hyperglycemic stage with ?-cell-Ag, along with zymosan, results in a superior protection of the NOD mice from diabetes as compared to mice that received zymosan alone. This therapeutic effect was associated with increased frequencies of IL-10-, IL-17-, IL-4-, and Foxp3-positive T cells, especially in the pancreatic lymph nodes. These results show that zymosan can be used as an immune regulatory adjuvant for modulating the T cell response to pancreatic ?-cell-antigen and reversing early stage hyperglycemia in T1D.
Certain aromatic nitriles are well known inhibitors of cysteine proteases. The mode of action of these compounds involves the formation of a reversible or irreversible covalent bond between the nitrile and a thiol group in the active site of the enzyme. However, the reactivity of these aromatic nitrile-substituted heterocycles may lead inadvertently to non-specific interactions with DNA, protein, glutathione and other endogenous components resulting in toxicity and complicating the use of these compounds as therapeutic agents. In the present study, the intrinsic reactivity and associated structure-property relationships of cathepsin K inhibitors featuring substituted pyridazines [6-phenylpyridazine-3-carbonitrile, 6-(4-fluorophenyl)pyridazine-3-carbonitrile, 6-(4-methoxyphenyl)pyridazine-3-carbonitrile, 6-p-tolylpyridazine-3-carbonitrile], pyrimidines [5-p-tolylpyrimidine-2-carbonitrile, 5-(4-fluorophenyl)pyrimidine-2-carbonitrile] and pyridines [5-p-tolylpicolinonitrile and 5-(4-fluorophenyl) picolinonitrile] were evaluated using a combination of computational and analytical approaches to establish correlations between electrophilicity and levels of metabolites that were formed in glutathione- and N-acetylcysteine-supplemented human liver microsomes. Metabolites that were characterized in this study featured substituted thiazolines that were formed following rearrangements of transient glutathione and N-acetylcysteine conjugates. Peptidases including ?-glutamyltranspeptidase were shown to catalyze the formation of these products, which were formed to lesser extents in the presence of the selective ?-glutamyltranspeptidase inhibitor acivicin and the non-specific peptidase inhibitors phenylmethylsulfonyl fluoride and aprotinin. Of the chemical series mentioned above, the pyrimidine series was the most susceptible to metabolism to thiazoline-containing products, followed by the pyridazine and pyridine series in that order. This trend was in keeping with the diminishing electrophilicity across these series as demonstrated by in silico modeling. Hence, mechanistic insights gained from this study could be used to assist a medicinal chemistry campaign to design cysteine protease inhibitors that were less prone to the formation of covalent adducts.
Sexual dysfunction is common among depressed adults. Childhood sexual abuse (CSA) and depressive symptomology are among the risk factors for sexual dysfunction, and these factors may interact to predict adult relationship functioning. Several models have been developed postulating interactions between these variables.
This collaborative project focused on improving self-efficacy and emancipated decision-making skills for nursing learners during an 8-week externship program. The learners attended educational sessions about topics identified as stressful and were paired with a preceptor. The long-term purposes of this pilot program involve nurse recruitment and retention. Job placement and retention will be followed for 2 years. This article will discuss the short-term program outcomes: self-efficacy and emancipated decision making.
?-Glucans are naturally occurring polysaccharides in cereal grains, mushrooms, algae, or microbes, including bacteria, fungi, and yeast. Immune cells recognize these ?-glucans through a cell surface pathogen recognition receptor called Dectin-1. Studies using ?-glucans and other Dectin-1 binding components have demonstrated the potential of these agents in activating the immune cells for cancer treatment and controlling infections. In this study, we show that the ?-glucan from Saccharomyces cerevisiae induces the expression of immune regulatory cytokines (IL-10, TGF-?1, and IL-2) and a tolerogenic enzyme (IDO) in bone marrow-derived dendritic cells as well as spleen cells. These properties can be exploited to modulate autoimmunity in the NOD mouse model of type 1 diabetes (T1D). Treatment of prediabetic NOD mice with low-dose ?-glucan resulted in a profound delay in hyperglycemia, and this protection was associated with increase in the frequencies of Foxp3(+), LAP(+), and GARP(+) T cells. Upon Ag presentation, ?-glucan-exposed dendritic cells induced a significant increase in Foxp3(+) and LAP(+) T cells in in vitro cultures. Furthermore, systemic coadministration of ?-glucan plus pancreatic ? cell Ag resulted in an enhanced protection of NOD mice from T1D as compared with treatment with ?-glucan alone. These observations demonstrate that the innate immune response induced by low-dose ?-glucan is regulatory in nature and can be exploited to modulate T cell response to ? cell Ag for inducing an effective protection from T1D.
Vaccinia virus (VACV) is a large dsDNA virus encoding ~200 proteins, several of which inhibit apoptosis. Here, a comparative study of anti-apoptotic proteins N1, F1, B13 and Golgi anti-apoptotic protein (GAAP) in isolation and during viral infection is presented. VACVs strains engineered to lack each gene separately still blocked apoptosis to some degree because of functional redundancy provided by the other anti-apoptotic proteins. To overcome this redundancy, we inserted each gene separately into a VACV strain (vv811) that lacked all these anti-apoptotic proteins and that induced apoptosis efficiently during infection. Each protein was also expressed in cells using lentivirus vectors. In isolation, each VACV protein showed anti-apoptotic activity in response to specific stimuli, as measured by immunoblotting for cleaved poly(ADP ribose) polymerase-1 and caspase-3 activation. Of the proteins tested, B13 was the most potent inhibitor, blocking both intrinsic and extrinsic stimuli, whilst the activity of the other proteins was largely restricted to inhibition of intrinsic stimuli. In addition, B13 and F1 were effective blockers of apoptosis induced by vv811 infection. Finally, whilst differences in induction of apoptosis were barely detectable during infection with VACV strain Western Reserve compared with derivative viruses lacking individual anti-apoptotic genes, several of these proteins reduced activation of caspase-3 during infection by vv811 strains expressing these proteins. These results illustrated that vv811 was a useful tool to determine the role of VACV proteins during infection and that whilst all of these proteins have some anti-apoptotic activity, B13 was the most potent.
During pathogenesis, Mycobacterium tuberculosis (Mtb) colonizes environments, such as the macrophage or necrotic granuloma, that are acidic and rich in cholesterol and fatty acids. The goal of this study was to examine how acidic pH and available carbon sources interact to regulate Mtb physiology. Here we report that Mtb growth at acidic pH requires host-associated carbon sources that function at the intersection of glycolysis and the TCA cycle, such as pyruvate, acetate, oxaloacetate and cholesterol. In contrast, in other tested carbon sources, Mtb fully arrests its growth at acidic pH and establishes a state of non-replicating persistence. Growth-arrested Mtb is resuscitated by the addition of pyruvate suggesting that growth arrest is due to a pH-dependent checkpoint on metabolism. Additionally, we demonstrate that the phoPR two-component regulatory system is required to slow Mtb growth at acidic pH and functions to maintain redox homeostasis. Transcriptional profiling and functional metabolic studies demonstrate that signals from acidic pH and carbon source are integrated to remodel pathways associated with anaplerotic central metabolism, lipid anabolism and the regeneration of oxidized cofactors. Because phoPR is required for Mtb virulence in animals, we suggest that pH-driven adaptation may be critical to Mtb pathogenesis.
Cholesterol-dependent cytolysins (CDCs) constitute a family of pore forming toxins secreted by Gram-positive bacteria. These toxins form transmembrane pores by inserting a large ?-barrel into cholesterol-containing membrane bilayers. Binding of water-soluble CDCs to the membrane triggers the formation of oligomers containing 35-50 monomers. The coordinated insertion of more than seventy ?-hairpins into the membrane requires multiple structural conformational changes. Perfringolysin O (PFO), secreted by Clostridium perfringens, has become the prototype for the CDCs. In this chapter, we will describe current knowledge on the mechanism of PFO cytolysis, with special focus on cholesterol recognition, oligomerization, and the conformational changes involved in pore formation.
The proximal femoral locking compression plate is a fixed angled anatomically contoured stainless steel plate used to treat pertrochanteric fractures of the proximal femur. Recent reports quote a high failure rate associated with this implant. We aimed to identify the common methods of failure and determine the elements of surgical techniques that could be altered to potentially improve outcomes should this implant be used for the treatment of unstable pertrochanteric fractures.
Microbubbles offer unique properties as combined carriers of therapeutic payloads and diagnostic agents. Here we report on the development of novel microbubble architectures that in addition to the usual lipid shell have an actin cytoskeletal cortex assembled on their exterior. We show, using atomic force microscopy that this biomimetic coating creates a thin mesh that allows tuning of the mechanical properties of microbubbles and that the nature of actin assembly is determined by the fluidity of the lipid layer. Further, we show that it is possible to attach payloads and targeting-ligands to the actin scaffold. Resistance to gas permeation showed that the additional actin layer reduces gas diffusion across the shell and thus increases bubble lifetime. This study demonstrates a one step method to creating more complex microbubble architectures, which would be capable of further modification and tuning through the inclusion of actin binding proteins.
The biphenyl derivatives 2 and 3 are prototypes of a novel class of NS5A replication complex inhibitors that demonstrate high inhibitory potency toward a panel of clinically relevant HCV strains encompassing genotypes 1-6. However, these compounds exhibit poor systemic exposure in rat pharmacokinetic studies after oral dosing. The structure-activity relationship investigations that improved the exposure properties of the parent bis-phenylimidazole chemotype, culminating in the identification of the highly potent NS5A replication complex inhibitor daclatasvir (33) are described. An element critical to success was the realization that the arylglycine cap of 2 could be replaced with an alkylglycine derivative and still maintain the high inhibitory potency of the series if accompanied with a stereoinversion, a finding that enabled a rapid optimization of exposure properties. Compound 33 had EC50 values of 50 and 9 pM toward genotype-1a and -1b replicons, respectively, and oral bioavailabilities of 38-108% in preclinical species. Compound 33 provided clinical proof-of-concept for the NS5A replication complex inhibitor class, and regulatory approval to market it with the NS3/4A protease inhibitor asunaprevir for the treatment of HCV genotype-1b infection has recently been sought in Japan.
We have demonstrated what we believe is the highest slope efficiency reported for a Tm-doped fiber laser operating in the 2-micron spectral region. Using a 1908 nm Tm-doped fiber laser as an in-band pump source, we generated 1.43 W of output power at 2005 nm with 81.25% optical efficiency and 90.2% slope efficiency, with respect to launched pump power. This resonant-pumping approach allowed us to examine the bleaching effects in Tm-doped fiber under resonant pumping. We also analytically show that this pumping method can scale to high power levels while maintaining high efficiency.
Total talar dislocation is a rare injury that is usually open. We report a case of closed anterolateral dislocation in a 19 year old, following a fall from a bicycle. He was treated with prompt closed reduction. A magnetic resonance imaging scan 6 months later showed no signs of avascular necrosis. At 2 years follow-up, the patient had a full, pain free, range of motion at the ankle and subtalar joints.Levels of Evidence: Therapeutic, Level IV: Case Study.
The non-obese diabetic (NOD) mice spontaneously develop T1D, progression of which is similar to that in humans and therefore, are widely used as a model for understanding the immunological basis of this disease. The incidence of T1D in NOD mice is influenced by the degree of cleanliness of the mouse colony and the gut microflora. In this report, we show that the T1D incidence and rate of disease progression are profoundly influenced by the pH of drinking water, which also affects the composition and diversity of commensal bacteria in the gut. Female NOD mice that were maintained on acidic pH water (AW) developed insulitis and hyperglycemia rapidly as compared to those on neutral pH water (NW). Interestingly, forced dysbiosis by segmented filamentous bacteria (SFB)-positive fecal transfer (FT) significantly suppressed the insulitis and T1D incidence in mice that were on AW, but not on NW. The 16S rDNA-targeted pyrosequencing revealed a significant change in the composition and diversity of gut flora, when the pH of drinking water was altered. Importantly, autoantigen specific T cell frequencies in the periphery and pro-inflammatory cytokine response in the intestinal mucosa are significantly higher in AW recipient mice compared to their NW counterparts. These observations suggest that pH of drinking water affects the composition of gut microflora, leading to an altered autoimmune response and T1D incidence in NOD mice.
Identified over a dozen years ago in the brain and pancreatic islet, ?IV-spectrin is critical for the local organization of protein complexes throughout the nervous system. ?IV-Spectrin targets ion channels and adapter proteins to axon initial segments and nodes of Ranvier in neurons, and ?IV-spectrin dysfunction underlies ataxia and early death in mice. Despite advances in ?IV-spectrin research in the nervous system, its role in pancreatic islet biology is unknown. Here, we report that ?IV-spectrin serves as a multifunctional structural and signaling platform in the pancreatic islet. We report that ?IV-spectrin directly associates with and targets the calcium/calmodulin-dependent protein kinase II (CaMKII) in pancreatic islets. In parallel, ?IV-spectrin targets ankyrin-B and the ATP-sensitive potassium channel. Consistent with these findings, ?IV-spectrin mutant mice lacking CaMKII- or ankyrin-binding motifs display selective loss of expression and targeting of key protein components, including CaMKII?. ?IV-Spectrin-targeted CaMKII directly phosphorylates the inwardly-rectifying potassium channel, Kir6.2 (alpha subunit of KATP channel complex), and we identify the specific residue, Kir6.2 T224, responsible for CaMKII-dependent regulation of KATP channel function. CaMKII-dependent phosphorylation alters channel regulation resulting in KATP channel inhibition, a cellular phenotype consistent with aberrant insulin regulation. Finally, we demonstrate aberrant KATP channel phosphorylation in ?IV-spectrin mutant mice. In summary, our findings establish a broader role for ?IV-spectrin in regulation of cell membrane excitability in the pancreatic islet, define the pathway for CaMKII local control in pancreatic beta cells, and identify the mechanism for CaMKII-dependent regulation of KATP channels.
Golgi antiapoptotic proteins (GAAPs) are highly conserved Golgi membrane proteins that inhibit apoptosis and promote Ca(2+) release from intracellular stores. Given the role of Ca(2+) in controlling cell adhesion and motility, we hypothesized that human GAAP (hGAAP) might influence these events. In this paper, we present evidence that hGAAP increased cell adhesion, spreading, and migration in a manner that depended on the C-terminal domain of hGAAP. We show that hGAAP increased store-operated Ca(2+) entry and thereby the activity of calpain at newly forming protrusions. These hGAAP-dependent effects regulated focal adhesion dynamics and cell migration. Indeed, inhibition or knockdown of calpain 2 abrogated the effects of hGAAP on cell spreading and migration. Our data reveal that hGAAP is a novel regulator of focal adhesion dynamics, cell adhesion, and migration by controlling localized Ca(2+)-dependent activation of calpain.
We report on the use of supported lipid bilayers to reveal dynamics of actin polymerization from a nonpolymerizing subphase via cationic phospholipids. Using varying fractions of charged lipid, lipid mobility, and buffer conditions, we show that dynamics at the nanoscale can be used to control the self-assembly of these structures. In the case of fluid-phase lipid bilayers, the actin adsorbs to form a uniform two-dimensional layer with complete surface coverage whereas gel-phase bilayers induce a network of randomly oriented actin filaments, of lower coverage. Reducing the pH increased the polymerization rate, the number of nucleation events, and the total coverage of actin. A model of the adsorption/diffusion process is developed to provide a description of the experimental data and shows that, in the case of fluid-phase bilayers, polymerization arises equally due to the adsorption and diffusion of surface-bound monomers and the addition of monomers directly from the solution phase. In contrast, in the case of gel-phase bilayers, polymerization is dominated by the addition of monomers from solution. In both cases, the filaments are stable for long times even when the G-actin is removed from the supernatant-making this a practical approach for creating stable lipid-actin systems via self-assembly.
The present study describes a novel methodology for the detection of reactive compounds using in vitro peptide-trapping and liquid chromatography-high-resolution accurate mass spectrometry (LC-HRMS). Compounds that contain electrophilic groups can covalently bind to nucleophilic moieties in proteins and form adducts. Such adducts are thought to be associated with drug-mediated toxicity and therefore represent potential liabilities in drug discovery programs. In addition, reactive compounds identified in biological screening can be associated with data that can be misinterpreted if the reactive nature of the compound is not appreciated. In this work, to facilitate the triage of hits from high-throughput screening (HTS), a novel assay was developed to monitor the formation of covalent peptide adducts by compounds suspected to be chemically reactive. The assay consists of in vitro incubations of test compounds (under conditions of physiological pH) with synthetically prepared peptides presenting a variety of nucleophilic moieties such as cysteine, lysine, histidine, arginine, serine, and tyrosine. Reaction mixtures were analyzed using full-scan LC-HRMS, the data were interrogated using postacquisition data mining, and modified amino acids were identified by subsequent LC-HRMS/mass spectrometry. The study demonstrated that in vitro nucleophilic peptide trapping followed by LC-HRMS analysis is a useful approach for screening of intrinsically reactive compounds identified from HTS exercises, which are then removed from follow-up processes, thus obviating the generation of data from biochemical activity assays.
Golgi anti-apoptotic proteins (GAAPs) are hydrophobic proteins resident in membranes of the Golgi complex. They protect cells from a range of apoptotic stimuli, reduce the Ca(2+) content of intracellular stores, and regulate Ca(2+) fluxes. GAAP was discovered in camelpox virus, but it is highly conserved throughout evolution and encoded by all eukaryote genomes examined. GAAPs are part of the transmembrane Bax inhibitor-containing motif (TMBIM) family that also includes other anti-apoptotic and Ca(2+)-modulating membrane proteins. Most TMBIM members show multiple bands when analyzed by SDS-PAGE, suggesting that they may be oligomeric. However, the molecular mechanisms of oligomerization, the native state of GAAPs in living cells and the functional significance of oligomerization have not been addressed. TMBIM members are thought to have evolved from an ancestral GAAP. Two different GAAPs, human (h) and viral (v)GAAP were therefore selected as models to examine oligomerization of TMBIM family members. We show that both hGAAP and vGAAP in their native states form oligomers and that oligomerization is pH-dependent. Surprisingly, hGAAP and vGAAP do not share the same oligomerization mechanism. Oligomerization of hGAAP is independent of cysteines, but oligomerization of vGAAP depends on cysteines 9 and 60. A mutant vGAAP that is unable to oligomerize revealed that monomeric vGAAP retains both its anti-apoptotic function and its effect on intracellular Ca(2+) stores. In conclusion, GAAP can oligomerize in a pH-regulated manner, and monomeric GAAP is functional.
The goal of this study is to contribute to the physics underlying the material properties of suspensions that exhibit shear thickening through the ultrasonic characterization of suspensions of cornstarch in a density-matched solution. Ultrasonic measurements at frequencies in the range of 4 to 8?MHz of the speed of sound and the frequency-dependent attenuation properties are reported for concentrations of cornstarch in a density-matched aqueous (cesium chloride brine) suspension, ranging up to 40% cornstarch. The speed of sound is found to range from 1483?±?10?m/s in pure brine to 1765?±?9?m/s in the 40% cornstarch suspension. The bulk modulus of a granule of cornstarch is inferred to be 1.2(±?0.1) × 10(10)?Pa. The attenuation coefficient at 5?MHz increases from essentially zero in brine to 12.0?±?1.2?dB/cm at 40% cornstarch.
Selective oxidation of ?-tertiary amine self-assembled thiol monolayers to tertiary amine N-oxides is shown to transform the adhesion of model proteins lysozyme and fibrinogen upon them. Efficient preparation of both secondary and tertiary linker amides as judged by X-ray photoelectron spectroscopy (XPS) and water droplet contact angle was achieved with an improved amide bond formation on gold quartz crystal microbalance (QCM) sensors using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl hexafluorophosphate methanaminium uronium (HATU). Oxidation with hydrogen peroxide was similarly assessed, and adhesion of lysozyme and fibrinogen from phosphate buffered saline was then assayed by QCM and imaged by AFM. Tertiary amine-functionalized sensors adsorbed multilayers of aggregated lysozyme, whereas tertiary amine N-oxides and triethylene glycol-terminated monolayers are consistent with small protein aggregates. The surface containing a dimethylamine N-oxide headgroup and ethyl secondary amide linker showed the largest difference in adsorption of both proteins. Oxidation of tertiary amine decorated surfaces therefore holds the potential for selective deposition of proteins and cells through masking and other patterning techniques.
Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma, a high incidence tumor in Chinese populations, in which tumor cells express the two EBV antigens EB nuclear antigen 1 (EBNA1) and latent membrane protein 2 (LMP2). Here, we report the phase I trial of a recombinant vaccinia virus, MVA-EL, which encodes an EBNA1/LMP2 fusion protein designed to boost T-cell immunity to these antigens. The vaccine was delivered to Hong Kong patients with nasopharyngeal carcinoma to determine a safe and immunogenic dose. The patients, all in remission more than 12 weeks after primary therapy, received three intradermal MVA-EL vaccinations at three weekly intervals, using five escalating dose levels between 5 × 10(7) and 5 × 10(8) plaque-forming unit (pfu). Blood samples were taken during prescreening, immediately before vaccination, one week afterward and at intervals up to one year later. Immunogenicity was tested by IFN-? ELIspot assays using complete EBNA1 and LMP2 15-mer peptide mixes and known epitope peptides relevant to patient MHC type. Eighteen patients were treated, three per dose level one to four and six at the highest dose, without dose-limiting toxicity. T-cell responses to one or both vaccine antigens were increased in 15 of 18 patients and, in many cases, were mapped to known CD4 and CD8 epitopes in EBNA1 and/or LMP2. The range of these responses suggested a direct relationship with vaccine dose, with all six patients at the highest dose level giving strong EBNA1/LMP2 responses. We concluded that MVA-EL is both safe and immunogenic, allowing the highest dose to be forwarded to phase II studies examining clinical benefit.
This study examines the interviewers use of immediacy during a dynamic interview to enhance the patients ability to process affective material and deepen personal exploration. Using a microprocess design, immediacy events were identified and rated using the Consensual Qualitative Rating method. Moment-to-moment in-session activity was rated by trained observers with a focus on measuring patient process using the Therapist-Patient Interaction Rating Scale and interviewer process using the Therapeutic Environment Scale. Five immediacy events were identified and were found to range in depth from mundane exchanges to more active exchanges with affective depth. Mundane events were characterized by little attention to the affective component of the here-and-now relationship, dismissive and unsupportive comments, and had either no effect, or a negative effect on patient process. In contrast, immediacy events characterized by even limited affect and acknowledging engagement between patient and interviewer were followed by greater patient disclosure and increased capacity to process emotional information. Thus, attention to the quality of the immediacy intervention in future research appears warranted.
Older adults exhibit a disproportionate deficit in their ability to recover contextual elements or source information about prior encounters with stimuli. A recent theoretical account, DRYAD, attributes this selective deficit to a global decrease in memory fidelity with age, moderated by weak representation of contextual information. The predictions of DRYAD are tested here in three experiments. We show that an age-related deficit obtains for whichever aspect of the stimulus subjects attention is directed away from during encoding (Experiment 1), suggesting a central role for attention in producing the age-related deficit in context. We also show that an analogous deficit can be elicited within young subjects with a manipulation of study time (Experiment 2), suggesting that any means of reducing memory fidelity yields an interaction of the same form as the age-related effect. Experiment 3 evaluates the critical prediction of DRYAD that endorsement probability in an exclusion task should vary nonmonotonically with memory strength. This prediction was confirmed by assessing the shape of the forgetting function in a continuous exclusion task. The results are consistent with the DRYAD account of aging and memory judgments and do not support the widely held view that aging entails the selective disruption of processes involved in encoding, storing, or retrieving contextual information.
In response to potential bioterrorism with smallpox, members of the Japanese Self-Defense Forces were vaccinated with vaccinia virus (VACV) strain LC16m8, an attenuated smallpox vaccine derived from VACV strain Lister. The serological response induced by LC16m8 to four virion-surface proteins and the intracellular mature virus (IMV) and extracellular enveloped virus (EEV) was investigated. LC16m8 induced antibody response against the IMV protein A27 and the EEV protein A56. LC16m8 also induced IMV-neutralizing antibodies, but unlike the VACV strain Lister, did not induce either EEV-neutralizing antibody or antibody to EEV protein B5, except after revaccination. Given that B5 is the only target for EEV-neutralizing antibody and that neutralization of both IMV and EEV give optimal protection against orthopoxvirus challenge, these data suggest that immunity induced by LC16m8 might be less potent than that deriving from strain Lister. This potential disadvantage should be balanced against the advantage of the greater safety of LC16m8.
A? (amyloid-? peptide) assembles to form amyloid fibres that accumulate in senile plaques associated with AD (Alzheimers disease). The major constituent, a 42-residue A?, has the propensity to assemble and form soluble and potentially cytotoxic oligomers, as well as ordered stable amyloid fibres. It is widely believed that the cytotoxicity is a result of the formation of transient soluble oligomers. This observed toxicity may be associated with the ability of oligomers to associate with and cause permeation of lipid membranes. In the present study, we have investigated the ability of oligomeric and fibrillar A?42 to simultaneously associate with and affect the integrity of biomimetic membranes in vitro. Surface plasmon field-enhanced fluorescence spectroscopy reveals that the binding of the freshly dissolved oligomeric 42-residue peptide binds with a two-step association with the lipid bilayer, and causes disruption of the membrane resulting in leakage from vesicles. In contrast, fibrils bind with a 2-fold reduced avidity, and their addition results in approximately 2-fold less fluorophore leakage compared with oligomeric A?. Binding of the oligomers may be, in part, mediated by the GM1 ganglioside receptors as there is a 1.8-fold increase in oligomeric A? binding and a 2-fold increase in permeation compared with when GM1 is not present. Atomic force microscopy reveals the formation of defects and holes in response to oligomeric A?, but not preformed fibrillar A?. The results of the present study indicate that significant membrane disruption arises from association of low-molecular-mass A? and this may be mediated by mechanical damage to the membranes by A? aggregation. This membrane disruption may play a key role in the mechanism of A?-related cell toxicity in AD.
Improving prison health care requires a robust measurement dashboard that addresses multiple domains of care. We sought to identify tested indicators of clinical quality and access that prison health managers could use to ascertain gaps in performance and guide quality improvement. We used the RAND/UCLA modified Delphi method to select the best indicators for correctional health. An expert panel rated 111 indicators on validity and feasibility. They voted to retain 79 indicators in areas such as access, cardiac conditions, geriatrics, infectious diseases, medication monitoring, metabolic diseases, obstetrics/gynecology, screening/prevention, psychiatric disorders/substance abuse, pulmonary conditions, and urgent conditions. Prison health institutions, like all other large health institutions, need robust measurement systems. The indicators presented here provide a basic library for prison health managers developing such systems.
Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol.2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery 2006, 5, 993). However, the structure-function relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structure-function determination of these species (Poo et al. Nature 1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a "nested trap" and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins.
Frontal lobe memory disorders are distinguished from hippocampal memory disorders by poor organization of encoding and retrieval, among other things. Because the verbal Selective Reminding Test (SRT) has a metamemory ("remembering-to-remember") component, it may be useful in distinguishing frontal from temporal lobe memory disorders in patients with intractable epilepsy. Thirty-four patients with frontal lobe epilepsy (FLE) and 34 with temporal lobe epilepsy (TLE) underwent a comprehensive neuropsychological evaluation that included multiple memory and executive function tests. Patients with FLE performed significantly worse than those with TLE on SRT measures and Wechsler Memory Scale, Third Edition, Logical Memory (LM II), but not on other verbal and nonverbal memory tests. Furthermore, SRT and LM-II were significantly correlated with executive function measures. These findings have both theoretical and practical implications: (1) the memory impairment observed in frontal lobe disorders may be due, in part, to deficits in organizational strategy, monitoring, and remembering-to-remember, and (2) SRT and LM-II may be useful tests to differentiate frontal from temporal lobe memory disorders.
The 2010 RAD-AID Conference on International Radiology for Developing Countries was a multidisciplinary meeting to discuss data, experiences, and models pertaining to radiology in the developing world, where widespread shortages of imaging services reduce health care quality. The theme of this years conference was sustainability, with a focus on establishing and maintaining imaging services in resource-limited regions. Conference presenters and participants identified 4 important components of sustainability: (1) sustainable financing models for radiology development, (2) integration of radiology and public health, (3) sustainable clinical models and technology solutions for resource-limited regions, and (4) education and training of both developing and developed world health care personnel.
Clinical imaging of the coronary arteries in the cardiac catheterization laboratory using intravascular ultrasound (IVUS) is known to display a three-layered appearance, corresponding to the intima/plaque, media and adventitia. It is not known whether ultrasonic anisotropy arising from these tissues may alter this pattern in future IVUS systems that insonify in the forward direction or obliquely. In anticipation of such devices, the current study was carried out by imaging fresh human coronary arteries in two orthogonal directions in vitro. Twenty-six sites from 12 arteries were imaged with a side-looking IVUS system, and with an acoustic microscope both radially and axially. Side-looking IVUS and radial acoustic microscopy scans demonstrated the typical "bright-dark-bright" pattern of the backscatter, with the media being significantly darker than the other two layers. Images obtained in the axial orientation exhibited a markedly different pattern, with the relative brightness of the media significantly larger than that of the intima/plaque.
PURPOSE: Thiosulfate has been shown to reduce the risk of cyanide toxicity during nitroprusside administration. Admixtures containing both agents may provide a safe and effective alternative to more expensive agents used to reduce blood pressure in the critically ill patient. This study determined the physical and chemical stability of a 1:10 nitroprusside:thiosulfate admixture, stored up to 48 hours. The economic consequences of a shift toward using thiosulfate and nitroprusside, and away from higher cost alternatives, are considered. METHODS: Seven samples of 50 mg nitroprusside and 500 mg thiosulfate were prepared and stored away from light, at room temperature, and in a refrigerator prepared in D5W and NS. Each sample was analyzed via a novel high-performance liquid chromatographic (HPLC) method at time 0, 8, 24, and 48 hours. The method was tested and passed specifications for linearity, reproducibility, and accuracy. A visual inspection by 9 licensed pharmacists was used to demonstrate physical stability. A cost evaluation comparing nitroprusside and thiosulfate to alternative agents was completed. RESULTS: The concentration of both nitroprusside and thiosulfate remain greater than 95% of the initial concentration through 48 hours. Physical compatibility was confirmed in all samples tested through 72 hours. CONCLUSION: The combination of nitroprusside and thiosulfate is chemically and physically stable as a single compounded dose for up to 48 hours when stored at room temperature and protected from light. The admixture represents an inexpensive option to other higher cost alternatives such as nicardipine or clevidipine.
Ischemic mitral regurgitation (MR) results from displacement of the papillary muscles caused by ischemic ventricular distortion. Progressive left ventricular (LV) remodeling has challenged therapy. Our hypothesis is that repositioning of the papillary muscles can be achieved by injection of polyvinyl-alcohol (PVA) hydrogel polymer into the myocardium in chronic MR despite advanced LV remodeling.
School shootings are traumatic events that cause a community to question itself, its values, and its educational systems. In this article Bryan Warnick, Benjamin Johnson, and Samuel Rocha explore the meanings of school shootings by examining three recent books on school violence. Topics that grow out of these books include (1) how school shootings might be seen as ceremonial rituals, (2) how schools come to be seen as appropriate places for shootings, and (3) how advice to educators relating to school shootings might change the practice of teaching. The authors present various ways of understanding school shootings that may eventually prove helpful, but they also highlight the problems, tensions, and contradictions associated with each position. In the end, the authors argue, the circumstances surrounding school shootings demonstrate the need for the "tragic sense" in education. This need for the tragic sense, while manifest in many different areas of schooling, is exemplified most clearly in targeted school shootings.
Tethered bilayer lipid membranes (tBLM) are formed on 1) pure tether lipid triethyleneoxythiol cholesterol (EO(3)C) or on 2) mixed self-assembled monolayers (SAMs) of EO(3)C and 6-mercaptohexanol (6MH). While EO(3)C is required to form a tBLM with high resistivity, 6MH dilutes the cholesterol content in the lower leaflet of the bilayer forming ionic reservoirs required for submembrane hydration. Here we show that these ionic reservoirs are required for ion transport through gramicidin or valinomycin, most likely due to the thermodynamic requirements of ions to be solvated once transported through the membrane. Unexpectedly, electrochemical impedance spectroscopy (EIS) shows an increase of capacitance upon addition of gramicidin, while addition of valinomycin decreases the membrane resistance in the presence of K(+) ions. We hypothesise that this is due to previously reported phase separation of EO(3)C and 6MH on the surface. This results in ionic reservoirs on the nanometre scale, which are not fully accounted for by the equivalent circuits used to describe the system.
The cholesterol-dependent cytolysins (CDCs) are a family of beta-barrel pore-forming toxins secreted by Gram-positive bacteria. These toxins are produced as water-soluble monomeric proteins that after binding to the target cell oligomerize on the membrane surface forming a ring-like pre-pore complex, and finally insert a large beta-barrel into the membrane (about 250 A in diameter). Formation of such a large transmembrane structure requires multiple and coordinated conformational changes. The presence of cholesterol in the target membrane is absolutely required for pore-formation, and therefore it was long thought that cholesterol was the cellular receptor for these toxins. However, not all the CDCs require cholesterol for binding. Intermedilysin, secreted by Streptoccocus intermedius only binds to membranes containing a protein receptor, but forms pores only if the membrane contains sufficient cholesterol. In contrast, perfringolysin O, secreted by Clostridium perfringens, only binds to membranes containing substantial amounts of cholesterol. The mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not understood at the molecular level. The C-terminus of perfringolysin O is involved in cholesterol recognition, and changes in the conformation of the loops located at the distal tip of this domain affect the toxin-membrane interactions. At the same time, the distribution of cholesterol in the membrane can modulate toxin binding. Recent studies support the concept that there is a dynamic interplay between the cholesterol-binding domain of the CDCs and the excess of cholesterol molecules in the target membrane.
Tricyclic pyrazole tetrazoles which are potent partial agonists of the high affinity niacin receptor, GPR109a, have been discovered and optimized. One of these compounds has proven to be effective at lowering free fatty acids in vitro and in vivo.
The RAD-AID Conference on International Radiology for Developing Countries was an assembly of individuals and organizations interested in improving access to medical imaging services in developing countries where the availability of radiology has been inadequate for both patient care and public health programs. The purpose of the meeting was to discuss data, experiences, and models pertaining to radiology in the developing world and to evaluate potential opportunities for future collaboration. Conference participants included radiologists, technologists, faculty members of academic medical institutions, and leadership of nongovernmental organizations involved in international health care and social entrepreneurship. Four main themes from the conference are presented in this white paper as important factors for the implementation and optimization of radiology in the developing world: (1) ensuring the economic sustainability of radiologic services through financial and administrative training support of health care personnel; (2) designing, testing, and deploying clinical strategies adapted for regions with limited resources; (3) structuring and improving the role of American radiology residents interested in global health service projects; and (4) implementing information technology models to support digital imaging in the developing world.
Cerebrospinal fluid (CSF) sodium levels have been reported to rise during episodic migraine. Since migraine frequently starts in early morning or late afternoon, we hypothesized that natural sodium chronobiology may predispose susceptible persons when extracellular CSF sodium increases. Since no mammalian brain sodium rhythms are known, we designed a study of healthy humans to test if cation rhythms exist in CSF.
Valacyclovir, the L-valyl ester prodrug of acyclovir (ACV), is widely prescribed to treat infections caused by varicella-zoster virus or herpes simplex virus. Rarely, treatment is complicated by reversible neuropsychiatric symptoms. By mechanisms not fully understood, this occurs more frequently in the setting of renal impairment. We characterized the steady-state pharmacokinetics of ACV and its metabolites 9-[(carboxymethoxy)methyl]guanine (CMMG) and 8-hydroxy-acyclovir (8-OH-ACV) in cerebrospinal fluid (CSF) and the systemic circulation. We administered multiple doses of high-dose valacyclovir to 6 subjects with normal renal function and 3 subjects with chronic renal impairment (creatinine clearance [CrCl], approximately 15 to 30 ml/min). Dosages were 2,000 mg every 6 h and 1,500 mg every 12 h, respectively. Indwelling intrathecal catheters allowed serial CSF sampling throughout the dosing interval. The average steady-state concentrations of acyclovir, CMMG, and 8-OH-ACV were greater in both the systemic circulation and the CSF among subjects with impaired renal function than among subjects with normal renal function. However, the CSF penetration of each analyte, reflected by the CSF-to-plasma area under the concentration-time curve over the 6- or 12-h dosing interval (AUC(tau)) ratio, did not differ based on renal function. Renal impairment does not alter the propensity for ACV or its metabolites to distribute to the CSF, but the higher concentrations in the systemic circulation, as a result of reduced elimination, are associated with proportionally higher concentrations in CSF.
Few studies have examined access to a regular source of medical care and preventive medical care among adults being treated for substance abuse. This report describes the prevalence of a usual source of care, use of preventive care services, and self-reported chronic conditions among adults in a publicly funded substance abuse treatment program.
Many individuals involved in the sport of powerlifting believe that the squat and deadlift have such similar lifting characteristics that the lifts yield comparable training results. The aim of this study was to compare and contrast biomechanical parameters between the conventional style deadlift and the back squat performed by 25 lifters competing in regional powerlifting championship. The 3-dimensional analysis incorporated 4 60 Hz synchronized video cameras for collecting data from 25 participants. Parameters were quantified at the sticking point specific to each lift. Kinematic variables were calculated at the hip, knee, and ankle. Paired (samples) t-tests were used to detect significant differences in the kinematic mean scores for the different lift types. The statistical analysis revealed significant differences exist between the squat (0.09 m/s) and the deadlift (0.20 m/s) vertical bar velocities. Differences were found for angular position of the hip, knee, and ankle between lifts. The sticking point thigh angles were quantified as 32.54 +/- 3.02 and 57.42 +/- 4.57 for the squat and deadlift, respectively. Trunk angles were 40.58 +/- 6.29 (squat) and 58.30 +/- 7.15 (deadlift). The results indicate the back squat represents a synergistic or simultaneous movement, whereas the deadlift demonstrates a sequential or segmented movement. The kinematic analysis of the squat and the conventional deadlift indicate that the individual lifts are markedly different (p < 0.01), implying that no direct or specific cross-over effect exists between the individual lifts.
Vascular endothelial growth factor (VEGF), known as a primary mediator of tumor-induced angiogenesis, is now understood to have a role in tumor-associated immunosuppression. Initially, VEGF was identified to alter the growth and maturation of the immature granulocyte-macrophage progenitors, and more recently it has been noted that it prevents dendritic cell (DC) precursors from developing into mature, antigen-presenting DC. VEGF is associated with recruitment of macrophages to the tumor stroma and VEGF inhibition of myeloid progenitor maturation is associated with the development tumor associated macrophages (TAM) which possess immunosuppressive capacity as well. Therapies intended to inhibit VEGF or VEGF receptors have demonstrated improved anti-tumor immunity and enhanced responses to cancer vaccines.
In patients with left ventricular infarction or dilatation, leaflet tethering by displaced papillary muscles frequently induces mitral regurgitation, which doubles mortality. Little is known about the biological potential of the mitral valve (MV) to compensate for ventricular remodeling. We tested the hypothesis that MV leaflet surface area increases over time with mechanical stretch created by papillary muscle displacement through cell activation, not passive stretching.
Peptidoglycan precursors containing D-Cys at position 4 were polymerised to form a synthetic peptidoglycan layer, which could be fluorescently labelled, providing a new method to monitor peptidoglycan transglycosylation and transpeptidation.
In the northern hemisphere winter of 2003-04 antigenic variant strains (A/Fujian/411/02 -like) of influenza A H3N2 emerged. Circulation of these strains in the UK was accompanied by an unusually high number of laboratory confirmed influenza associated fatalities in children. This study was carried out to better understand risk factors associated with fatal cases of influenza in children.
Ultra-small gold nanoclusters (AuNCs) have unique size-dependent optical, electrical and chemical properties. They have emerged as a new nanomaterial with broad applications in optoelectronics, catalysis, biosensing, and bioimaging. Several strategies have been exploited to prepare AuNCs of different "magic number" sizes, using different templates e.g. dendrimers, polyethyleneimines, peptides, and more recently, proteins. Notwithstanding, almost all bio-template-protected AuNCs reported so far exhibit fairly low fluorescence quantum yields (QYs), typically <5%, which is especially true for AuNCs prepared using the protein templates. In this paper, we report a facile, one-pot aqueous synthesis of highly fluorescent AuNCs using bovine pancreatic ribonuclease A (RNase-A) as the bio-template. The as-prepared AuNCs not only fluoresce strongly at the near-infrared (NIR) region (?(em) = 682 nm), but also exhibit an elevated QY of ?12%. Additionally, the RNase-A-encapsulated AuNC (RNase-A-AuNC) displays an exceptionally large Stokes shift of ?210 nm as well as a single dominant fluorescence lifetime of ?1.5 ?s, about three orders of magnitude longer than biological autofluorescence. Furthermore, by coupling vitamin B(12) (VB(12)) to the RNase-A-AuNC, we develop a multifunctional nanoplatform that is suitable for simultaneous targeting and imaging of cancer at the cellular level using Caco-2 cell lines as an in vitro model. Since VB(12) has effective uptake pathways in the digestive system, this nanoplatform may have potential for targeted oral drug delivery in vivo.
Two distinct and scalable enantioselective approaches to the tricyclic indole (R)-2-(7-hydroxy-2,3-dihydro-1H-pyrrolo[1,2-a]indol-1-yl)acetate, an important synthon for a preclinical S1P(1) receptor agonist, are reported. Route 1 employs a modified version of Smiths modular 2-substituted indole synthesis as the key transformation. Route 2 involves a highly enantioselective CuH-catalyzed 1,4-hydrosilylation as the stereodefining step. Both routes can be performed without chromatography to provide multigram quantities of the tricycle in ?98% ee.
Emerging applications based on optical beams carrying orbital angular momentum (OAM) will probably require photonic integrated devices and circuits for miniaturization, improved performance, and enhanced functionality. We demonstrate silicon-integrated optical vortex emitters, using angular gratings to extract light confined in whispering gallery modes with high OAM into free-space beams with well-controlled amounts of OAM. The smallest device has a radius of 3.9 micrometers. Experimental characterization confirms the theoretical prediction that the emitted beams carry exactly defined and adjustable OAM. Fabrication of integrated arrays and demonstration of simultaneous emission of multiple identical optical vortices provide the potential for large-scale integration of optical vortex emitters on complementary metal-oxide-semiconductor compatible silicon chips for wide-ranging applications.
Micron sized, lipid stabilized bubbles of gas are of interest as contrast agents for ultra-sound (US) imaging and increasingly as delivery vehicles for targeted, triggered, therapeutic delivery. Microfluidics provides a reproducible means for microbubble production and surface functionalisation. In this study, microbubbles are generated on chip using flow-focussing microfluidic devices that combine streams of gas and liquid through a nozzle a few microns wide and then subjecting the two phases to a downstream pressure drop. While microfluidics has successfully demonstrated the generation of monodisperse bubble populations, these approaches inherently produce low bubble counts. We introduce a new micro-spray flow regime that generates consistently high bubble concentrations that are more clinically relevant compared to traditional monodisperse bubble populations. Final bubble concentrations produced by the micro-spray regime were up to 10(10) bubbles mL(-1). The technique is shown to be highly reproducible and by using multiplexed chip arrays, the time taken to produce one millilitre of sample containing 10(10) bubbles mL(-1) was ?10 min. Further, we also demonstrate that it is possible to attach liposomes, loaded with quantum dots (QDs) or fluorescein, in a single step during MBs formation.
Proximal femoral fracture is the most common reason for emergency orthopedic admission in the United Kingdom with an annual cost of £ 1.7 billion to the National Health Service. Surgical site infection (SSI) after proximal femoral fracture increases patient morbidity and mortality. Methicillin-resistant Staphylococcus aureus (MRSA) poses a particular risk in this patient cohort as a large proportion of these patients are residents of long-term care facilities and are therefore transient or chronic carriers of MRSA. We recorded the effect of three stages of care bundle development on the infection and specifically the MRSA rate after hemiarthroplasty over an 8-year period.
Changes in the cholesterol content of cell membranes affect many physiological and pathological events, including the formation of arterial plaques, the entry of virus into cells, and receptor organization. Measuring the trafficking and distribution of cholesterol is essential to understanding how cells regulate sterol levels in membranes. Perfringolysin O (PFO) is a cytolysin secreted by Clostridium perfringens that requires cholesterol in the target membrane for binding. The specificity of PFO for high levels of cholesterol makes the toxin an attractive tool for studying the distribution and trafficking of cholesterol in cells. However, the use of the native toxin is limited given that binding is triggered only above a determined cholesterol concentration. To this end, we have identified mutations in PFO that altered the threshold for how much cholesterol is required to trigger binding. The cholesterol threshold among different PFO derivatives varied up to 10 mol % sterol, and these variations were not dependent on the lipid composition of the membrane. We characterized the binding of these PFO derivatives on murine macrophage-like cells whose cholesterol content was reduced or augmented. Our findings revealed that engineered PFO derivatives differentially associated with these cells in response to changes in cholesterol levels in the plasma membrane.
G-protein coupled receptor (GPCR) GPR109a is a molecular target for nicotinic acid and is expressed in adipocytes, spleen, and immune cells. Nicotinic acid has long been used for the treatment of dyslipidemia due to its capacity to positively affect serum lipids to a greater extent than other currently marketed drugs. We report a series of tricyclic pyrazole carboxylic acids that are potent and selective agonists of GPR109a. Compound R,R-19a (MK-1903) was advanced through preclinical studies, was well tolerated, and presented no apparent safety concerns. Compound R,R-19a was advanced into a phase 1 clinical trial and produced a robust decrease in plasma free fatty acids. On the basis of these results, R,R-19a was evaluated in a phase 2 study in humans. Because R,R-19a produced only a weak effect on serum lipids as compared with niacin, we conclude that the beneficial effects of niacin are most likely the result of an undefined GPR109a independent pathway.
The Golgi anti-apoptotic protein (GAAP) is a hydrophobic Golgi protein that regulates intracellular calcium fluxes and apoptosis. GAAP is highly conserved throughout eukaryotes and some strains of vaccinia virus (VACV) and camelpox virus. Based on sequence, phylogeny, and hydrophobicity, GAAPs were classified within the transmembrane Bax inhibitor-containing motif (TMBIM) family. TMBIM members are anti-apoptotic and were predicted to have seven-transmembrane domains (TMDs). However, topology prediction programs are inconsistent and predicted that GAAP and other TMBIM members have six or seven TMDs. To address this discrepancy, we mapped the transmembrane topology of viral (vGAAP) and human (hGAAP), as well as Bax inhibitor (BI-1). Data presented show a six-, not seven-, transmembrane topology for vGAAP with a putative reentrant loop at the C terminus and both termini located in the cytosol. We find that this topology is also conserved in hGAAP and BI-1. This places the charged C terminus in the cytosol, and mutation of these charged residues in hGAAP ablated its anti-apoptotic function. Given the highly conserved hydrophobicity profile within the TMBIM family and recent phylogenetic data indicating that a GAAP-like protein may have been the ancestral progenitor of a subset of the TMBIM family, we propose that this vGAAP topology may be used as a model for the remainder of the TMBIM family of proteins. The topology described provides valuable information on the structure and function of an important but poorly understood family of proteins.
During the Northern Hemisphere winter of 2003-2004 the emergence of a novel influenza antigenic variant, A/Fujian/411/2002-like(H3N2), was associated with an unusually high number of fatalities in children. Seventeen fatal cases in the UK were laboratory confirmed for Fujian/411-like viruses. To look for phylogenetic patterns and genetic markers that might be associated with increased virulence, sequencing and phylogenetic analysis of the whole genomes of 63 viruses isolated from fatal cases and non fatal "control" cases was undertaken. The analysis revealed the circulation of two main genetic groups, I and II, both of which contained viruses from fatal cases. No associated amino acid substitutions could be linked with an exclusive or higher occurrence in fatal cases. The Fujian/411-like viruses in genetic groups I and II completely displaced other A(H3N2) viruses, but they disappeared after 2004. This study shows that two A(H3N2) virus genotypes circulated exclusively during the winter of 2003-2004 in the UK and caused an unusually high number of deaths in children. Host factors related to immune state and differences in genetic background between patients may also play important roles in determining the outcome of an influenza infection.
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