Structure-activity relationships and mechanism of action of tetragomycin derivatives as inhibitors of Staphylococcus aureus staphyloxanthin biosynthesis.

Despite the main strategy to overcome bacterial resistance has focused on the development of more potent antimicrobial agents, the evolutionary pressure caused by such drugs makes this strategy limited. Molecules that interfere with virulence factors appear as a promising alternative though, as they cause reduced selective pressure. As a matter of fact, staphyloxanthin biosynthesis inhibition (STXBI) has been pursued as promising strategy to reduce S. aureus virulence. Herein, we report the inhibitory profile of 27 tetrangomycin derivatives over staphyloxanthin production. The experimental result showed that naphthoquinone dehydro-α-lapachone (25 - EC50 = 57.29 ± 1.15 µM) and 2-Isopropylnaphtho[2,3-b]furan-4,9-dione (26 EC50 = 82.10 ± 1.09 µM) are the most potent compounds and suggest that hydrogen acceptor groups and lipophilic moieties decorating the naphthoquinone ring are crucial for STXBI. In addition, we present an in situ analysis, through RAMAN spectroscopy, that is inexpensive and might be employed to probe the mechanism of action of staphyloxanthin biosynthesis inhibitors. Therefore, our molecular simplification strategies afforded promising lead compounds for the development of drugs that modulate S. aureus staphyloxanthin biosynthesis.

The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection.

Exported proteins of bacterial pathogens function both in essential physiological processes and in virulence. Past efforts to identify exported proteins were limited by the use of bacteria growing under laboratory (in vitro) conditions. Thus, exported proteins that are exported only or preferentially in the context of infection may be overlooked. To solve this problem, we developed a genome-wide method, named EXIT (exported in vivotechnology), to identify proteins that are exported by bacteria during infection and applied it to Mycobacterium tuberculosis during murine infection. Our studies validate the power of EXIT to identify proteins exported during infection on an unprecedented scale (593 proteins) and to reveal in vivo induced exported proteins (i.e., proteins exported significantly more during in vivo infection than in vitro). Our EXIT data also provide an unmatched resource for mapping the topology of M. tuberculosis membrane proteins. As a new approach for identifying exported proteins, EXIT has potential applicability to other pathogens and experimental conditions.IMPORTANCE There is long-standing interest in identifying exported proteins of bacteria as they play critical roles in physiology and virulence and are commonly immunogenic antigens and targets of antibiotics. While significant effort has been made to identify the bacterial proteins that are exported beyond the cytoplasm to the membrane, cell wall, or host environment, current methods to identify exported proteins are limited by their use of bacteria growing under laboratory (in vitro) conditions. Because in vitro conditions do not mimic the complexity of the host environment, critical exported proteins that are preferentially exported in the context of infection may be overlooked. We developed a novel method to identify proteins that are exported by bacteria during host infection and applied it to identify Mycobacterium tuberculosis proteins exported in a mouse model of tuberculosis.

Age-associated Epstein-Barr virus-specific T cell responses in seropositive healthy adults.

Epstein-Barr virus (EBV) is present in 95% of the world's adult population. The immune response participates in immune vigilance and persistent infection control, and this condition is maintained by both a good quality (functionality) and quantity of specific T cells throughout life. In the present study, we evaluated EBV-specific CD4(+) and CD8(+) T lymphocyte responses in seropositive healthy individuals younger and older than 50 years of age. The assessment comprised the frequency, phenotype, functionality and clonotypic distribution of T lymphocytes. We found that in both age groups a similar EBV-specific T cell response was found, with overlapping numbers of tumour necrosis factor (TNF)-α(+) T lymphocytes (CD4(+) and CD8(+)) within the memory and effector cell compartments, in addition to monofunctional and multi-functional T cells producing interleukin (IL)-2 and/or interferon (IFN)-γ. However, individuals aged more than 50 years showed significantly higher frequencies of IL-2-producing CD4(+) T lymphocytes in association with greater production of soluble IFN-γ, TNF-α and IL-6 than subjects younger than 50 years. A polyclonal T cell receptor (TCR)-variable beta region (Vß) repertoire exists in both age groups under basal conditions and in response to EBV; the major TCR families found in TNF-α(+) /CD4(+) T lymphocytes were Vß1, Vß2, Vß17 and Vß22 in both age groups, and the major TCR family in TNF-α(+) /CD8(+) T cells was Vß13·1 for individuals younger than 50 years and Vß9 for individuals aged more than 50 years. Our findings suggest that the EBV-specific T cell response (using a polyclonal stimulation model) is distributed throughout several T cell differentiation compartments in an age-independent manner and includes both monofunctional and multi-functional T lymphocytes.

[Analysis of the positive predictive value of the subcategories of BI-RADS(®) 4 lesions: preliminary results in 880 lesions]. / Análisis del valor predictivo positivo de las subcategorías BI-RADS(®)4: resultados preliminares en 880 lesiones.

OBJECTIVES: The positive predictive values (PPV) of the subcategories of BI-RADS(®) 4 lesions (A/B/C) vary widely, and their correlation with specific descriptors has yet to be defined. We aimed to analyze the PPV of the subcategories and of the mammographic and ultrasonographic descriptors assigned to each. MATERIAL AND METHODS: We analyzed 880 histologically confirmed lesions prospectively classified as BI-RADS(®) 4 A/B/C between 2003 and 2010. The statistical analysis included significance tests, contingency tables, and relative risk (RR) ratios, calculated for 545 mammographic lesions and 627 ultrasonographic lesions. RESULTS: The PPV was 8.8% for subcategory 4A, 18.9% for subcategory 4B, and 58.3% for subcategory 4C. The correlation between PPV and lesions was what we expected, with three exceptions: a) the PPV of 4A was greater than that of 4B in nodules that were irregular or had uncircumscribed margins on ultrasonography and in microcalcifications with segmental distribution on mammography, b) BI-RADS(®) 3 lesions classified as BI-RADS(®) 4, and c) identical lesions classified in distinct subcategories. In the contingency table analysis, the mammographic lesions were 4B/C and the ultrasonographic lesions were 4B. On mammography, the RR was significant for nodules with irregular shape (RR=3.205) and for those with spiculated margins (RR=2.469), as well as for microcalcifications that were pleomorphic (RR=2.531) or amorphous (RR=0.334), and for those with segmental (RR=1.895). On ultrasonography, the RR were significant for all the descriptors, with values greater than 1 for irregular shape (RR=1.977) and uncircumscribed margins (RR=2.277). CONCLUSIONS: Our results corroborate previous reports. The exceptions can be explained by aspects related to variability and nonradiological factors that might influence the classification and PPV. Mathematical models should be developed to enable the objective classification and these should include factors not related to imaging.

Percutaneous microductectomy with a directional vacuum-assisted system guided by ultrasonography for the treatment of breast discharge: experience in 63 cases.

BACKGROUND: Vacuum-assisted devices are becoming a useful tool in the diagnosis and treatment of breast pathology. Recent publications show good results in percutaneous removal of benign lesions. PURPOSE: To discuss our experience in percutaneous excision of ductal lesions with a vacuum-assisted, ultrasound-guided directional system. MATERIAL AND METHODS: From January 2003 to July 2006, 63 patients with pathological nipple discharge and intraductal lesion identifiable on imaging were studied at two reference centers. Percutaneous excision with a vacuum-assisted device was offered as an alternative to surgery. RESULTS: A total of 71 lesions were diagnosed in 63 patients with a mean age of 52 years. All the patients presented nipple discharge. Mammography was normal in 65 cases (92%). Galactography showed an intraductal lesion in 67 cases (94%). Mean lesion size on ultrasonography was 7.4 mm (2-26 mm). Percutaneous excision was performed in 45 lesions (63%), while surgical excision was indicated in 26 lesions. The histopathological results in the 45 lesions biopsied demonstrated intraductal papilloma in 30 cases, dilated duct with papillomatous projections in 11, ductal ectasia with no papillary lesion in three, and a nonspecific benign result in one. Excision was considered complete in 41 lesions (91%). Clinical signs of discharge were resolved in 39 patients (95% of cases treated percutaneously). Mild complications occurred in four cases. CONCLUSION: Percutaneous excision of ductal lesions with an ultrasound-guided, vacuum-assisted device is a safe procedure with high diagnostic and therapeutic value for the management of breast discharge.

Probabilistic prediction of unknown metabolic and signal-transduction networks.

Regulatory networks provide control over complex cell behavior in all kingdoms of life. Here we describe a statistical model, based on representing proteins as collections of domains or motifs, which predicts unknown molecular interactions within these biological networks. Using known protein-protein interactions of Saccharomyces cerevisiae as training data, we were able to predict the links within this network with only 7% false-negative and 10% false-positive error rates. We also use Markov chain Monte Carlo simulation for the prediction of networks with maximum probability under our model. This model can be applied across species, where interaction data from one (or several) species can be used to infer interactions in another. In addition, the model is extensible and can be analogously applied to other molecular data (e.g., DNA sequences).

Birth of scale-free molecular networks and the number of distinct DNA and protein domains per genome.

MOTIVATION: Current growth in the field of genomics has provided a number of exciting approaches to the modeling of evolutionary mechanisms within the genome. Separately, dynamical and statistical analyses of networks such as the World Wide Web and the social interactions existing between humans have shown that these networks can exhibit common fractal properties-including the property of being scale-free. This work attempts to bridge these two fields and demonstrate that the fractal properties of molecular networks are linked to the fractal properties of their underlying genomes. RESULTS: We suggest a stochastic model capable of describing the evolutionary growth of metabolic or signal-transduction networks. This model generates networks that share important statistical properties (so-called scale-free behavior) with real molecular networks. In particular, the frequency of vertices connected to exactly k other vertices follows a power-law distribution. The shape of this distribution remains invariant to changes in network scale: a small subgraph has the same distribution as the complete graph from which it is derived. Furthermore, the model correctly predicts that the frequencies of distinct DNA and protein domains also follow a power-law distribution. Finally, the model leads to a simple equation linking the total number of different DNA and protein domains in a genome with both the total number of genes and the overall network topology. AVAILABILITY: MatLab (MathWorks, Inc.) programs described in this manuscript are available on request from the authors. CONTACT: ar345@columbia.edu.

A knowledge model for analysis and simulation of regulatory networks.

MOTIVATION: In order to aid in hypothesis-driven experimental gene discovery, we are designing a computer application for the automatic retrieval of signal transduction data from electronic versions of scientific publications using natural language processing (NLP) techniques, as well as for visualizing and editing representations of regulatory systems. These systems describe both signal transduction and biochemical pathways within complex multicellular organisms, yeast, and bacteria. This computer application in turn requires the development of a domain-specific ontology, or knowledge model. RESULTS: We introduce an ontological model for the representation of biological knowledge related to regulatory networks in vertebrates. We outline a taxonomy of the concepts, define their 'whole-to-part' relationships, describe the properties of major concepts, and outline a set of the most important axioms. The ontology is partially realized in a computer system designed to aid researchers in biology and medicine in visualizing and editing a representation of a signal transduction system.

Eosinophilic dermatitis with edema in nine dogs, compared with eosinophilic cellulitis in humans.

A unique eosinophilic dermatitis with edema in dogs is characterized by extremely erythematous coalescing macules and plaques with associated edema, and is similar to eosinophilic cellulitis (Wells' syndrome) in humans. Histopathologic features include a profound eosinophilic dermal infiltrate, focal areas of collagen fiber degeneration surrounded by eosinophils (flame figures), dilated vessels, and dermal edema. Etiopathogenesis is unknown, but a hypersensitivity reaction to medications, arthropod bites, or other foreign antigens is suspected.

Capture of rare cells in suspension with antibody-coated polystyrene beads.

A method for the separation of one cell type present in small number from a predominant mixture of cell types using macroscopic polystyrene beads is demonstrated. An antibody specific to murine leukocytes (CD45) was adsorbed to the surface of the beads. Beads and murine hybridoma B cells were placed in test tubes and periodically inverted at fixed time intervals, causing the beads to settle through the suspension under creeping flow conditions. Capture was dependent upon interception: the captured cells must have traveled along streamlines that brought them to within a cell radius of the bead surface. B cells attached to 99-micrometer beads (maximum shear rate 8.1 s-1) were captured with greater efficiency but in lesser quantity than those attached to 170-micrometer beads (maximum shear rate 13.9 s-1). Cell capture unexpectedly reached a plateau in less than 2 h, a phenomenon that appears to involve changes in both the cells and the beads. Capture of cells was effective out to dilutions of 1:10 000 with purity in the captured population of better than 74%. This method allows for the study of physical parameters important for cell attachment and capture as well as for practical separation of rare cells.

Cavernous sinus involvement in head and neck cancer.

Spread of tumor to intracranial structures is an infrequent and late manifestation of head and neck cancers. We recently encountered six patients with a distinct clinical syndrome due to involvement of the cavernous sinus, which forms the basis of this report. This syndrome was a source of significant morbidity and mortality, with a mean survival of only 4 months. The diagnosis is often elusive, but is now made more commonly than previously. Whether this reflects increased incidence (due to alterations in the natural history of disease by therapy) or improved diagnosis (due to modern imaging modalities) is unknown. Cavernous sinus involvement may be the first evidence of distant disease in head and neck cancer. Although survival is poor, palliation is worthwhile. Awareness of this syndrome can lead to earlier diagnosis and alteration of treatment.

Transcranial Doppler in reversible migrainous vasospasm causing cerebellar infarction: report of a case.

A young woman with a history of classic migraine suffered a large cerebellar infarction. Comprehensive evaluation did not disclose any potential causes for the stroke and she had no other risk factors. Transcranial Doppler (TCD) was useful in identifying vasospasm of the vertebral arteries. Following treatment with propranolol, angiography and repeat TCD revealed resolution of the vasospastic disorder. TCD is a useful noninvasive tool in detecting vasospasm associated with ischemic lesions in certain patients with migraine.

Meso-diencephalic infarction: a not so rare form of stroke.

26 patients admitted with clinical pictures consistent with meso-diencephalic infarction were studied using computerized axial tomography, magnetic resonance imaging and angiography. All of the patients presented symptoms in two of three major categories which comprise the "top of the basilar" syndrome. Lesions in the territory of the basilar-communicating artery were demonstrated in 22 of them. The remaining patients died before adequate neuroimaging documentation was obtained. Overall prognosis appeared relatively good since 80% of the patients recovered completely or incompletely from the ictus. Meso-diencephalic infarction is a very important and not uncommon subtype of cerebrovascular disorder.

Transcranial Doppler ultrasonographic assessment of intermittent light stimulation at different frequencies.

Seven normal adult volunteers underwent intermittent photic stimulation at frequencies of 5-60 Hz while their posterior cerebral arteries were monitored using transcranial Doppler ultrasound. Baseline measurements were obtained under conditions of total darkness, and sampling was also done during continuous illumination. Overall variation in mean flow velocity between complete darkness and continuous illumination was 9.8%, but the maximal change (expressed as percentage deviation from baseline) occurred consistently when stimulation was undertaken at frequencies of 10 (21%) and 20 (19%) Hz (p = 0.05). Frequencies higher than 20 Hz resulted in mean flow velocity variations that were not significantly different from that found during continuous illumination. The optimal frequency of intermittent visual stimulation required to induce measurable changes in posterior cerebral artery Doppler characteristics appears to be in the range 10-20 Hz.

Transcranial Doppler determination of cerebral perfusion in patients undergoing CPR: methodology and preliminary findings.

STUDY OBJECTIVES: To correlate cerebral arterial blood velocity measurements as determined by transcranial Doppler ultrasound with various resuscitation maneuvers performed in an uncontrolled manner in a series of cardiac arrest patients undergoing standard resuscitation. TYPE OF PARTICIPANTS: Any patient undergoing resuscitation for a nontraumatic cardiac arrest was eligible for the study. INTERVENTIONS: Resuscitation was carried out while flow velocities were monitored in various intracranial arteries by transcranial Doppler ultrasound. Vessel identification was based on the angle and depth of insonation. MEASUREMENTS AND MAIN RESULTS: The internal carotid artery was located more consistently than either middle cerebral or ophthalmic arteries and provided superior waveforms for analysis. Alterations in flow velocity were noted and correlated to various therapeutic interventions. Flow velocities were 70% of normal during the early phase of resuscitation but deteriorated over time until there was no evidence of net forward flow. Even early in CPR, flow was demonstrated only during systole. CONCLUSION: Transcranial Doppler ultrasound appears to be a potentially promising means of determining cerebral flow in patients undergoing CPR.

Chimeric immunoglobulin-T cell receptor proteins form functional receptors: implications for T cell receptor complex formation and activation.

We constructed chimeric receptor chains in which an immunoglobulin heavy chain variable region (VH) from a phosphorylcholine-specific antibody is substituted for T cell receptor (Tcr) alpha and beta V regions. We demonstrate that the VH region joined to either the C alpha or the C beta region can form stable chimeric proteins in EL4 T cells. Both chimeric receptor chains associate with CD3 polypeptides in functional receptor complexes and respond to phosphorylcholine coupled to Sepharose beads. The VH-C alpha chimeric chain associates with the EL4 beta chain, while the VH-C beta chimeric protein appears to form either a homodimer or a heterodimer with the native EL4 beta chain. Thus, functional receptor complexes can be formed using two C beta regions, and the C alpha region may not be required for CD3 association and surface expression of Tcr complexes.