Risks associated with air emboli introduced during cardiac surgery have been highlighted by reports of postoperative neuropsychological dysfunction, myocardial dysfunction, and mortality. Presently, there are no standard effective methods for quantifying potential emboli in the bloodstream during cardiac surgery. Our objective was to develop software that can automatically detect and quantify air bubbles within the ascending aorta and/or cardiac chambers during cardiac surgery in real time.
We created a software algorithm (“Detection of Emboli using Transesophageal Echocardiography for Counting, Total volume, and Size estimation”, or DETECTS™) to identify and measure potential emboli present during cardiac surgery using two-dimensional ultrasound. An in vitro experiment was used to validate the accuracy of DETECTS™ at identifying and measuring air emboli. An experimental rig was built to correlate the ultrasound images to high definition camera images of air bubbles created in water by an automatic bubbler system. There was a correlation between true bubble size and the size reported by DETECTS™ in our in vitro experiment (r = 0.76). We also tested DETECTS™ using TEE images obtained during cardiac surgery, and provide visualization of the software interface.
While monitoring the heart during cardiac surgery using existing ultrasound technology and DETECTS™, the operative team can obtain real-time data on the number and volume of potential air emboli. This system will potentially allow de-airing techniques to be evaluated and improved upon. This could lead to reduced air in the cardiac chambers after cardiopulmonary bypass, possibly reducing the risk of neurological dysfunction following cardiac surgery.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-7120-13-3) contains supplementary material, which is available to authorized users.
Cardiac surgery; Cardiopulmonary bypass; Air emboli; Cerebral emboli; Neurological dysfunction; Myocardial dysfunction; Ultrasound; Transcranial doppler; Transesophageal echocardiography
The bifunctional enzyme thymidylate synthase–dihydrofolate reductase (TS–DHFR) from the protozoal parasite Cryptosporidium hominis is a potential molecular target for the design of antiparasitic therapies for AIDS-related opportunistic infections. The enzyme exists as a homodimer with each monomer containing a unique swap domain known as a “crossover helix” that binds in a cleft on the adjacent DHFR active site. This crossover helix is absent in species containing monofunctional forms of DHFR such as human. An in-depth understanding of protein–protein interactions between the crossover helix and adjacent DHFR active site that might modulate enzyme integrity or function would allow for insights into rational design of species-specific allosteric inhibitors. Mutational analysis coupled with structural studies and biophysical and kinetic characterization of crossover helix mutants identifies this domain as essential for full enzyme stability and catalytic activity, and pinpoints these effects to distinct faces of the crossover helix important in protein–protein interactions. Moreover, targeting this helical protein interaction with α-helix mimetics of the crossover helix leads to selective inhibition and destabilization of the C. hominis TS–DHFR enzyme, thus validating this region as a new avenue to explore for species-specific inhibitor design.
The adaptor protein Grb2 links cell-surface receptors, such as Her2, to the multisite docking proteins Gab1 and 2, leading to cell growth and proliferation in breast and other cancers. Gab2 interacts with the C-terminal SH3 domain (SH3C) of Grb2 through atypical RxxK motifs within polyproline II or 310 helices. A virtual screen was conducted for putative binders of the Grb2 SH3C domain. Of the top hits, 34 were validated experimentally by surface plasmon resonance spectroscopy and isothermal titration calorimetry. A subset of these molecules was found to inhibit the Grb2–Gab2 interaction in a competition assay, with moderate to low affinities (5: IC50 320 µM). The most promising binders were based on a dihydro-s-triazine scaffold, and are the first small molecules reported to target the Grb2 SH3C protein-interaction surface.
SH3 Domain; Grb2; Gab2; Virtual screening; Protein-protein interaction; Inhibitors
Improved methods are required to screen drug candidates for their influences on protein interactions. There is also a compelling need for miniaturization of screening assays, with attendant reductions in reagent consumption and assay costs.
We used sensitive, miniaturized proximity ligation assays (PLAs) to monitor binding of vascular endothelial growth factor A (VEGF-A) to 2 of its receptors, VEGFR-1 and VEGFR-2. We measured the effects of proteins and low molecular weight compounds capable of disrupting these interactions and compared the results with those obtained by immunoblot analysis. We analyzed 6 different inhibitors: a DNA aptamer, a mixed DNA/ RNA aptamer, a monoclonal VEGF-A neutralizing antibody, a monoclonal antibody directed against VEGFR-2, a recombinant competitive protein, and a low molecular weight synthetic molecule.
The PLAs were successful for monitoring the formation and inhibition of VEGF-A–receptor complexes, and the results correlated well with those obtained by measuring receptor phosphorylation. The total PLA time is just 3 hours, with minimal manual work and reagent additions. The method allows evaluation of the apparent affinity [half-maximal inhibitory concentration (IC50)] from a dose–response curve.
The PLA may offer significant advantages over conventional methods for screening the interactions of ligands with their receptors. The assay may prove useful for parallel analyses of large numbers of samples in the screening of inhibitor libraries for promising agents. The technique pro vides dose–response curves, allowing IC50 values to be calculated.
Akt has emerged as a critical target for the development of anti-cancer therapies. It has been found to be amplified, overexpressed, or constitutively activated in numerous human malignancies with oncogenesis derived from the simultaneous promotion of cell survival and suppression of apoptosis. A valuable alternative to the more common ATP-mimetic based chemotherapies is a substrate-mimetic approach, which has the potential advantage of inherent specificity of the substrate-binding pocket. In this paper we present the development of high affinity non-peptidic, substrate-mimetic inhibitors based on the minimum GSK3β substrate sequence. Optimization of initial peptidic leads resulted in the development of several classes of small molecule inhibitors, which have comparable potency to the initial peptidomimetics, while eliminating the remaining amino acid residues. We have identified the first non-peptidic substrate-mimetic lead inhibitors of Akt 29a–b, which have affinities of 17 and 12 μM, respectively. This strategy has potential to provide a useful set of molecular probes to assist in the validation of Akt as a potential target for anti-cancer drug design.
Akt; Protein kinase B; Substrate-mimetic; Cancer; Inhibitor
Protein farnesylation and geranylgeranylation, together referred to as prenylation, are lipid post-translational modifications that are required for the transforming activity of many oncogenic proteins, including some RAS family members. This observation prompted the development of inhibitors of farnesyltransferase (FT) and geranylgeranyltransferase 1 (GGT1) as potential anticancer drugs. In this Review, we discuss the mechanisms by which FT and GGT1 inhibitors (FTIs and GGTIs, respectively) affect signal transduction pathways, cell cycle progression, proliferation and cell survival. In contrast to their preclinical efficacy, only a small subset of patients responds to FTIs. Identifying tumours that depend on farnesylation for survival remains a challenge, and strategies to overcome this are discussed. One GGTI has recently entered the clinic, and the safety and efficacy of GGTIs await results from clinical trials.
The Chicago Area Patient-Centered Outcomes Research Network (CAPriCORN) represents an unprecedented collaboration across diverse healthcare institutions including private, county, and state hospitals and health systems, a consortium of Federally Qualified Health Centers, and two Department of Veterans Affairs hospitals. CAPriCORN builds on the strengths of our institutions to develop a cross-cutting infrastructure for sustainable and patient-centered comparative effectiveness research in Chicago. Unique aspects include collaboration with the University HealthSystem Consortium to aggregate data across sites, a centralized communication center to integrate patient recruitment with the data infrastructure, and a centralized institutional review board to ensure a strong and efficient human subject protection program. With coordination by the Chicago Community Trust and the Illinois Medical District Commission, CAPriCORN will model how healthcare institutions can overcome barriers of data integration, marketplace competition, and care fragmentation to develop, test, and implement strategies to improve care for diverse populations and reduce health disparities.
Patient Centered; Clinical Data Network; Outcomes Research
Leafhoppers (Hemiptera: Cicadellidae) are pests of many temperate crops, including grapevines (Vitis species). Uncontrolled populations can induce direct and indirect damage to crops due to feeding that results in significant yield losses and increased mortality in infected vineyards due to virus, bacteria, or phytoplasmas vectored by leafhoppers. The main objective of this work was to determine the diversity of leafhoppers found in vineyards of the three main Canadian production provinces, i.e., in British Columbia, Ontario, and Quebec. Approximately 18,000 specimens were collected in 80 commercial vineyards from 2006 to 2008. We identified 54 genera and at least 110 different species associated with vineyards, among which 22 were predominant and represented more than 91% of all the leafhoppers. Species richness and diversity were estimated by both Shannon's and Pielou's indices. For each province, results indicated a temporal variation in species composition. Color photographs provide a tool to quickly identify 72 leafhoppers commonly associated with vineyards.
Cicadellidae; Eythroneura spp.; Macrosteles spp.; Empoasca spp.; Vitis spp
The ICAM-1 mediated brain endothelial cell signalling pathway induced by adherent lymphocytes is a central element in facilitating lymphocyte migration through the tight endothelial barrier of the brain. Rho proteins, which must undergo post-translational prenylation to be functionally active, have been shown to be an essential component of this signalling cascade. In this study we have evaluated the effect of inhibiting protein prenylation in brain endothelial cells on their ability to support T lymphocyte migration. Endothelial cells treated in vitro with protein prenylation inhibitors (PTI) resulted in a significant reduction in transendothelial T lymphocyte migration. To determine the therapeutic potential of this approach an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis, was induced in Biozzi ABH mice. Animals treated prior to disease onset with PTI exhibited a dramatic and significant reduction in both leucocyte infiltration into the central nervous system (CNS) and clinical presentation of disease compared to untreated animals. These studies demonstrate, for the first time, the potential for pharmacologically targeting CNS endothelial cell signalling responses, and particularly endothelial Rho proteins, as a means of attenuating leucocyte recruitment to the CNS.
Leucocyte recruitment; Rho proteins; EAE
Growth of new dendritic spines contributes to experience-dependent circuit plasticity in the cerebral cortex. Yet the signaling mechanisms leading to new spine outgrowth remain poorly defined. Increasing evidence supports that the proteasome is an important mediator of activity-dependent neuronal signaling. We therefore tested the role of the proteasome in activity-dependent spinogenesis. Using pharmacological manipulations, glutamate uncaging, and two-photon imaging of GFP-transfected hippocampal pyramidal neurons, we demonstrate that acute inhibition of the proteasome blocks activity-induced spine outgrowth. Remarkably, mutation of serine 120 to alanine of the Rpt6 protea-somal subunit in individual neurons was sufficient to block activity-induced spine outgrowth. Signaling through NMDA receptors and CaMKII, but not PKA, is required to facilitate spine outgrowth. Moreover, abrogating CaMKII binding to the NMDA receptor abolished activity-induced spinogenesis. Our data support a model in which neural activity facilitates spine outgrowth via an NMDA receptor- and CaM-KII-dependent increase in local proteasomal degradation.
A DNA quadruplex system that exhibits ultrafast intramolecular energy transfer is discussed.
The ubiquitin-proteasome system (UPS) is most widely known for its role in intracellular protein degradation; however, in the decades since its discovery, ubiquitination has been associated with the regulation of a wide variety of cellular processes. The addition of ubiquitin tags, either as single moieties or as polyubiquitin chains, has been shown not only to mediate degradation by the proteasome and the lysosome, but also to modulate protein function, localization, and endocytosis. The UPS plays a particularly important role in neurons, where local synthesis and degradation work to balance synaptic protein levels at synapses distant from the cell body. In recent years, the UPS has come under increasing scrutiny in neurons, as elements of the UPS have been found to regulate such diverse neuronal functions as synaptic strength, homeostatic plasticity, axon guidance, and neurite outgrowth. Here we focus on recent advances detailing the roles of the UPS in regulating the morphogenesis of axons, dendrites, and dendritic spines, with an emphasis on E3 ubiquitin ligases and their identified regulatory targets.
To present a partnership-based and community-oriented approach designed to ease provider anxiety and facilitate the implementation of electronic health records (EHR) in resource-limited primary care settings.
Materials and Methods
The approach, referred to as partnership model, was developed and iteratively refined through the research team's previous work on implementing health information technology (HIT) in over 30 safety net practices. This paper uses two case studies to illustrate how the model was applied to help two nurse-managed health centers (NMHC), a particularly vulnerable primary care setting, implement EHR and get prepared to meet the meaningful use criteria.
The strong focus of the model on continuous quality improvement led to eventual implementation success at both sites, despite difficulties encountered during the initial stages of the project.
There has been a lack of research, particularly in resource-limited primary care settings, on strategies for abating provider anxiety and preparing them to manage complex changes associated with EHR uptake. The partnership model described in this paper may provide useful insights into the work shepherded by HIT regional extension centers dedicated to supporting resource-limited communities disproportionally affected by EHR adoption barriers.
NMHC, similar to other primary care settings, are often poorly resourced, understaffed, and lack the necessary expertise to deploy EHR and integrate its use into their day-to-day practice. This study demonstrates that implementation of EHR, a prerequisite to meaningful use, can be successfully achieved in this setting, and partnership efforts extending far beyond the initial software deployment stage may be the key.
Collaborative technologies; community health care; developing/using clinical decision support (other than diagnostic) and guideline systems; electronic health records (E05.318.308.940.968.625.500); health information technology for economic and clinical health act (N03.706.615.049); human–computer interaction and human-centered computing; improving healthcare workflow and process efficiency; nurse-managed health centers; personal health records and self-care systems; qualitative/ethnographic field study; regional extension centers; social/organizational study; system implementation and management issues; systems supporting patient–provider interaction
Nomenclatural benchmarking is the periodic realignment of species names with species theories and is necessary for the accurate and uniform use of Linnaean binominals in the face of changing species limits. Gaining access to types, often for little more than a cursory examination by an expert, is a major bottleneck in the advance and availability of biodiversity informatics. For the nearly two million described species it has been estimated that five to six million name-bearing type specimens exist, including those for synonymized binominals. Recognizing that examination of types in person will remain necessary in special cases, we propose a four-part strategy for opening access to types that relies heavily on digitization and that would eliminate much of the bottleneck: (1) modify codes of nomenclature to create registries of nomenclatural acts, such as the proposed ZooBank, that include a requirement for digital representations (e-types) for all newly described species to avoid adding to backlog; (2) an “r” strategy that would engineer and deploy a network of automated instruments capable of rapidly creating 3-D images of type specimens not requiring participation of taxon experts; (3) a “K” strategy using remotely operable microscopes to engage taxon experts in targeting and annotating informative characters of types to supplement and extend information content of rapidly acquired e-types, a process that can be done on an as-needed basis as in the normal course of revisionary taxonomy; and (4) creation of a global e-type archive associated with the commissions on nomenclature and species registries providing one-stop-shopping for e-types. We describe a first generation implementation of the “K” strategy that adapts current technology to create a network of Remotely Operable Benchmarkers Of Types (ROBOT) specifically engineered to handle the largest backlog of types, pinned insect specimens. The three initial instruments will be in the Smithsonian Institution(Washington, DC), Natural History Museum (London), and Museum National d’Histoire Naturelle (Paris), networking the three largest insect collections in the world with entomologists worldwide. These three instruments make possible remote examination, manipulation, and photography of types for more than 600,000 species. This is a cybertaxonomy demonstration project that we anticipate will lead to similar instruments for a wide range of museum specimens and objects as well as revolutionary changes in collaborative taxonomy and formal and public taxonomic education.
Types; typification; digital imaging; biodiversity informatics; taxonomy; nomenclature; natural history museums
Surveillance designed to detect changes in the type-specific distribution of HPV in cervical intraepithelial neoplasia grade 3 (CIN-3) is necessary to evaluate the effectiveness of the Australian vaccination programme on cancer causing HPV types. This paper develops a protocol that eliminates the need to calculate required sample size; sample size is difficult to calculate in advance because HPV’s true type-specific prevalence is imperfectly known.
A truncated sequential sampling plan that collects a variable sample size was designed to detect changes in the type-specific distribution of HPV in CIN-3. Computer simulation to evaluate the accuracy of the plan at classifying the prevalence of an HPV type as low (< 5%), moderate (5-15%), or high (> 15%) and the average sample size collected was conducted and used to assess its appropriateness as a surveillance tool.
The plan classified the proportion of CIN-3 lesions positive for an HPV type very accurately, with >90% of simulations correctly classifying a simulated data-set with known prevalence. Misclassifying an HPV type of high prevalence as being of low prevalence, arguably the most serious kind of potential error, occurred < 0.05 times per 100 simulations. A much lower sample size (21–22 versus 40–48) was required to classify samples of high rather than low or moderate prevalence.
Truncated sequential sampling enables the proportion of CIN-3 due to an HPV type to be accurately classified using small sample sizes. Truncated sequential sampling should be used for type-specific HPV surveillance in the vaccination era.
Protein fiber formation is associated with diseases ranging from Alzheimer’s to type II diabetes. For many systems, including islet amyloid polypeptide (IAPP) from type II diabetes, fibrillogenesis can be catalyzed by lipid bilayers. Paradoxically, amyloid fibers are β-sheet rich while membrane stabilized states are α helical. Here, a small molecule α helix mimetic, IS5, is shown to inhibit bilayer catalysis of fibrillogenesis and to rescue IAPP induced toxicity in cell culture. Importantly, IAPP:IS5 interactions localize to the putative α helical region of IAPP revealing that α helical states are on-pathway to fiber formation. IAPP is not normally amyloidogenic as its cosecreted partner, insulin, prevents self-assembly. Here, we show that IS5 inhibition is synergistic with insulin. IS5 therefore represents a new approach to amyloid inhibition as the target is an assembly intermediate that may additionally restore functional IAPP expression.
The Akt activation inhibitor triciribine and the farnesyltransferase inhibitor tipifarnib have modest to little activity in clinical trials when used as single agents. In this manuscript pre-clinical data demonstrate that the combination is more effective than single agents both in cultured cells and in vivo. Combination index data analysis demonstrates that this combination is highly synergistic at inhibiting anchorage-dependent growth of breast cancer cells. This synergistic interaction is also observed with structurally unrelated inhibitors of Akt (MK-2206) and farnesyltransferase (FTI-2153). The triciribine/tipifarnib synergistic effects are seen with several cancer cell lines including those from breast, leukemia, multiple myeloma and lung tumors with different genetic alterations such as K-Ras, B-Raf, PI3K, p53 and pRb mutations, PTEN, pRB and Ink4a deletions and ErbB receptor overexpression. Furthermore, the combination is synergistic at inhibiting anchorage-independent growth and at inducing apoptosis in breast cancer cells. The combination is also more effective at inhibiting the Akt/mTOR/S6 kinase pathway. In an ErbB2-driven breast tumor transgenic mouse model the combination, but not single agent, treatment with triciribine and tipifarnib induces significant breast tumor regression. Our findings warrant further investigation of the combination of farnesyltransferase and Akt inhibitors.
Several cell wall constituents, including melanins or melanin-like compounds, have been implicated in the pathogenesis of a wide variety of microbial diseases caused by diverse species of pathogenic bacteria, fungi, and helminthes. Among these microorganisms, the dimorphic fungal pathogen Paracoccidioides brasiliensis produces melanin in its conidial and yeast forms. In the present study, melanin particles from P. brasiliensis were injected into BALB/c mice in order to produce monoclonal antibodies (MAbs). We identified five immunoglobulin G1 (IgG1) κ-chain and four IgM melanin-binding MAbs. The five IgG1 κ-chain isotypes are the first melanin-binding IgG MAbs ever reported. The nine MAbs labeled P. brasiliensis conidia and yeast cells both in vitro and in pulmonary tissues. The MAbs cross-reacted with melanin-like purified particles from other fungi and also with commercial melanins, such as synthetic and Sepia officinalis melanin. Melanization during paracoccidioidomycosis (PCM) was also further supported by the detection of IgG antibodies reactive to melanin from P. brasiliensis conidia and yeast in sera and bronchoalveolar lavage fluids from P. brasiliensis-infected mice, as well as in sera from human patients with PCM. Serum specimens from patients with other mycoses were also tested for melanin-binding antibodies by enzyme-linked immunosorbent assay, and cross-reactivities were detected for melanin particles from different fungal sources. These results suggest that melanin from P. brasiliensis is an immunologically active fungal structure that activates a strong IgG humoral response in humans and mice.
The endemic human pathogenic fungus Histoplasma capsulatum is a major fungal pathogen with a broad variety of clinical presentations, ranging from mild, focal pulmonary disease to life-threatening systemic infections. Although azoles, such as itraconazole and voriconazole, and amphotericin B have significant activity against H. capsulatum, about 1 in 10 patients hospitalized due to histoplasmosis die. Hence, new approaches for managing disease are being sought. Over the past 10 years, studies have demonstrated that monoclonal antibodies (mAbs) can modify the pathogenesis of histoplasmosis. Disease has been shown to be impacted by mAbs targeting either fungal cell surface proteins or host co-stimulatory molecules. This review will detail our current knowledge regarding the impact of antibody therapy on histoplasmosis.
Histoplasma capsulatum; histoplasmosis; antibody; histone 2B; heat shock protein 60; M antigen; co-stimulation
Guanine (G) rich G4T4G4 DNA and homologous PNA strands tend to form antiparallel dimeric quadruplexes, which can be hybridized to form a stable parallel DNA2PNA2 hybrid quadruplex. In contrast, the same DNA strands functionalised with large planar aromatic groups at the 5′-end lead to the formation of parallel DNA quadruplex, which cannot be perturbed by homologous PNA strands. Conformation and composition of the DNA quadruplexes can be programed by π-π-stacking interaction.
A potential anti-HIV and HCV drug candidate is highly desirable as coinfection has become a worldwide public health challenge. A potent compound based on a tetrabutoxy-calixarene scaffold that possesses dual inhibition for both HIV and is described. Structural activity relationship studies demonstrate the effects of lower-rim alkylation in maintaining cone conformation and upper-rim interating head groups on the calixarene play key roles for its potent dual antivial activities.
New chemotherapeutics are urgently needed to combat malaria. We previously reported on a novel series of antimalarial, ethylenediamine-based inhibitors of protein farnesyltransferase (PFT). In the current study, we designed and synthesized a series of second generation inhibitors, wherein the core ethylenediamine scaffold was varied in order to examine both the homology model of Plasmodium falciparum PFT (PfPFT) and our predicted inhibitor binding mode. We identified several PfPFT inhibitors (PfPFTIs) that are selective for PfPFT versus the mammalian isoform of the enzyme (up to 136-fold selectivity), that inhibit the malarial enzyme with IC50 values down to 1 nM, and that block the growth of P. falciparum in infected whole cells (erythrocytes) with ED50 values down to 55 nM. The structure–activity data for these second generation, ethylenediamine-inspired PFT inhibitors were rationalized by consideration of the X-ray crystal structure of mammalian PFT and the homology model of the malarial enzyme.
A potent class of anticancer, human farnesyltransferase (hFTase)
inhibitors has been identified by “piggy-backing” on potent,
antimalarial inhibitors of Plasmodium falciparum
farnesyltransferase (PfFTase). On the basis of a 4-fold
substituted ethylenediamine scaffold, the inhibitors are structurally simple and
readily derivatized, facilitating the extensive structure–activity
relationship (SAR) study reported herein. Our most potent inhibitor is compound
1f, which exhibited an in vitro hFTase IC50 value of
25 nM and a whole cell H-Ras processing IC50 value of 90 nM.
Moreover, it is noteworthy that several of our inhibitors proved highly
selective for hFTase (up to 333-fold) over the related prenyltransferase enzyme
geranylgeranyltransferase-I (GGTase-I). A crystal structure of inhibitor
1a co-crystallized with farnesyl pyrophosphate (FPP) in the
active site of rat FTase illustrates that the para-benzonitrile
moiety of 1a is stabilized by a
interaction with the Y361β residue, suggesting a
structural explanation for the observed importance of this component of our