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Currently, there is an increasing tendency to migrate the management of communications and information systems onto the Web. This is making many traditional service support models obsolete. In addition, current security mechanisms are not sufficiently robust to protect each management system and/or subsystem from web-based intrusions, malware, and hacking attacks. This paper presents research challenges in autonomic management to provide self-protection mechanisms and tools by using trust and reputation concepts based on policy-based management to decentralize management decisions. This work also uses user-based reputation mechanisms to help enforce trust management in pervasive and communications services. The scope of this research is founded in social models, where the application of trust and reputation applied in communication systems helps detect potential users as well as hackers attempting to corrupt management operations and services. These so-called “cheating services” act as “attacks”, altering the performance and the security in communication systems by consumption of computing or network resources unnecessarily.

We investigated the regulation of the sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) axis and its role during pregnancy in the rat myometrium. SphK1 and SphK2 were coexpressed in myometrium during gestation. The levels and activity of SphK1/2 were modest at midgestation (d 12), increased at d 19 and progressively declined to low at postpartum. Similar patterns were observed for the phosphorylation of ERK and protein kinase C (PKC). Inhibition of PKC and ERK reduced SphK1/2 activity. In late pregnancy, levels of cyclooxygenase 2 (COX2) increased in parallel to SphK levels. Using a pharmacological approach, we demonstrated that in primary cultures of myometrial cells from d-19 pregnant rats, induction of COX2 was mediated by 4beta-phorbol 12,13-dibutyrate and IL-1beta through sequential activation of PKC, ERK1/2, and SphK1. S1P produced by SphK1 was released in the medium. Addition of S1P, IL-1beta or 4beta-phorbol 12,13-dibutyrate enhanced COX2 levels via Gi protein. Interestingly, S1P was also released by myometrial tissues at late gestation. This event was dependent on PKC/ERK/SphK1. By contrast, in d-12 myometrial tissues, the release of S1P was markedly reduced in association with low levels of SphK1 and COX2. However, prolonged incubation of myometrium from midgestation led to the induction of COX2. This effect was blocked by SphK inhibitors, providing evidence of the close relationship between SphK activity and COX2 induction in rat myometrium. Overall, our findings provided insight into the physiological relevance of the SphK activation and S1P release in uterine smooth muscle during gestation.

The ESCRT machinery functions in topologically equivalent membrane fission events, namely multivesicular body formation, the terminal stages of cytokinesis and HIV-1 release. Here, we show that the ESCRT-III-binding protein Alix is recruited to the midbody of dividing cells through binding Cep55 via an evolutionarily conserved peptide. Disruption of Cep55/Alix/ESCRT-III interactions causes formation of aberrant midbodies and cytokinetic failure, demonstrating an essential role for these proteins in midbody morphology and cell division. We also show that the C terminus of Alix encodes a multimerization activity that is essential for its function in Alix-dependent HIV-1 release and for interaction with Tsg101. Last, we demonstrate that overexpression of Chmp4b and Chmp4c differentially inhibits HIV-1 release and cytokinesis, suggesting possible reasons for gene expansion within the mammalian Class E VPS pathway.

Chemogenomics comprises a systematic relationship between targets and ligands that are used as target modulators in living systems such as cells or organisms. In recent years, data on small molecule-bioactivity relationships have become increasingly available, and consequently so have the number of approaches used to translate bioactivity data into knowledge. This review will focus on two aspects of chemogenomics. Firstly, in cases such as cell-based screens, the question of which target(s) a compound is modulating in order to cause the observed phenotype is crucial. In silico target prediction tools can suggest likely biological targets of small molecules via data mining in target-annotated chemical databases. This review presents some of the current tools available for this task and shows some sample applications relevant to a pharmaceutical industry setting. These applications are the prediction of false-positives in cell-based reporter gene assays, the prediction of targets by linking bioassay data with protein domain annotations, and the direct prediction of adverse reactions. Secondly, in recent years a shift from structure-derived chemical descriptors to biological descriptors has occurred. Here, the effect of a compound on a number of biological endpoints is used to make predictions about other properties, such as putative targets, associated adverse reactions, and pathways modulated by the compound. This review further summarizes these "performance" descriptors and their applications, focusing on gene expression profiles and high-content screening data. The advent of such biological fingerprints suggests that the field of drug discovery is currently at a crossroads, where single target bioassay results are supplanted by multidimensional biological fingerprints that reflect a new awareness of biological networks and polypharmacology.

ESCRT and ESCRT-associated proteins are required during the assembly and release of many RNA viruses, including HIV. Two new papers provide structures for the ESCRT-associated protein ALIX/AIP1 and demonstrate how this protein interacts with HIV Gag. One of these studies provides the clearest evidence to date that ESCRT-III mediates key events in virus release and indicates that there are cellular proteins involved in this process still to be discovered.

ChemBank (http://chembank.broad.harvard.edu/) is a public, web-based informatics environment developed through a collaboration between the Chemical Biology Program and Platform at the Broad Institute of Harvard and MIT. This knowledge environment includes freely available data derived from small molecules and small-molecule screens and resources for studying these data. ChemBank is unique among small-molecule databases in its dedication to the storage of raw screening data, its rigorous definition of screening experiments in terms of statistical hypothesis testing, and its metadata-based organization of screening experiments into projects involving collections of related assays. ChemBank stores an increasingly varied set of measurements derived from cells and other biological assay systems treated with small molecules. Analysis tools are available and are continuously being developed that allow the relationships between small molecules, cell measurements, and cell states to be studied. Currently, ChemBank stores information on hundreds of thousands of small molecules and hundreds of biomedically relevant assays that have been performed at the Broad Institute by collaborators from the worldwide research community. The goal of ChemBank is to provide life scientists unfettered access to biomedically relevant data and tools heretofore available primarily in the private sector.

During cytokinesis, as dividing animal cells pull apart into two daughter cells, the final stage, termed abscission, requires breakage of the midbody, a thin membranous stalk connecting the daughter cells. This membrane fission event topologically resembles the budding of viruses, such as HIV-1, from infected cells. We found that two proteins involved in HIV-1 budding—tumor susceptibility gene 101 (Tsg101), a subunit of the endosomal sorting complex required for transport I (ESCRT-I), and Alix, an ESCRT-associated protein—were recruited to the midbody during cytokinesis by interaction with centrosome protein 55 (Cep55), a centrosome and midbody protein essential for abscission. Tsg101, Alix, and possibly other components of ESCRT-I were required for the completion of cytokinesis. Thus, HIV-1 budding and cytokinesis use a similar subset of cellular components to carry out topologically similar membrane fission events.

Defective human immunodeficiency virus type 1 (HIV-1) assembly in murine cells is accompanied by poor plasma membrane binding and proteolytic processing of the HIV-1 Gag precursor. Here, we show that such defects are induced by the propensity of the HIV-1 MA globular head to inhibit membrane binding and particle assembly, particularly at the low expression levels observed in murine cells. Simple additions to or deletion of the MA globular head can improve the yield of infectious virions from murine cells by >50-fold. Expression level and autoinhibition can be important confounding variables in studies of HIV-1 assembly and contribute to defects encountered in murine cells.

Many enveloped viruses exploit the class E vacuolar protein-sorting (VPS) pathway to bud from cells, and use peptide motifs to recruit specific class E VPS factors. Homologous to E6AP COOH terminus (HECT) ubiquitin ligases have been implicated as cofactors for PPXY motif–dependent budding, but precisely which members of this family are responsible, and how they access the VPS pathway is unclear. Here, we show that PPXY-dependent viral budding is unusually sensitive to inhibitory fragments derived from specific HECT ubiquitin ligases, namely WWP1 and WWP2. We also show that WWP1, WWP2, or Itch ubiquitin ligase recruitment promotes PPXY-dependent virion release, and that this function requires that the HECT ubiquitin ligase domain be catalytically active. Finally, we show that several mammalian HECT ubiquitin ligases, including WWP1, WWP2, and Itch are recruited to class E compartments induced by dominant negative forms of the class E VPS ATPase, VPS4. These data indicate that specific HECT ubiquitin ligases can link PPXY motifs to the VPS pathway to induce viral budding.

Endosomal sorting complex required for transport-I (ESCRT-I) is one of three defined protein complexes in the class E vacuolar protein sorting (VPS) pathway required for the sorting of ubiquitinated transmembrane proteins into internal vesicles of multivesicular bodies. In yeast, ESCRT-I is composed of three proteins, VSP23, VPS28, and VPS37, whereas in mammals only Tsg101(VPS23) and VPS28 were originally identified as ESCRT-I components. Using yeast two-hybrid screens, we identified one of a family of human proteins (VPS37C) as a Tsg101-binding protein. VPS37C can form a ternary complex with Tsg101 and VPS28 by binding to a domain situated toward the carboxyl terminus of Tsg101 and binds to another class E VPS factor, namely Hrs. In addition, VPS37C is recruited to aberrant endosomes induced by overexpression of Tsg101, Hrs, or dominant negative form of the class E VPS ATPase, VPS4. Enveloped viruses that encode PTAP motifs to facilitate budding exploit ESCRT-I as an interface with the class E VPS pathway, and accordingly, VPS37C is recruited to the plasma membrane along with Tsg101 by human immunodeficiency virus, type 1 (HIV-1) Gag. Moreover, direct fusion of VPS37C to HIV-1 Gag obviates the requirement for a PTAP motif to induce virion release. Depletion of VPS37C from cells does not inhibit murine leukemia virus budding, which is not mediated by ESCRT-I, however, if murine leukemia virus budding is engineered to be ESCRT-I-dependent, then it is inhibited by VPS37C depletion, and this inhibition is accentuated if VPS37B is simultaneously depleted. Thus, this study identifies VPS37C as a functional component of mammalian ESCRT-I.

Many enveloped viruses encode late assembly domains, or L domains, that facilitate virion egress. PTAP-type L domains act by recruiting the ESCRT-I (endosomal sorting complex required for transport I) component Tsg101, and YPXL/LXXLF-type L domains recruit AIP-1/ALIX, both of which are class E vacuolar protein sorting (VPS) factors, normally required for the generation of vesicles within endosomes. The binding cofactors for PPXY-type L domains have not been unambiguously resolved but may include Nedd4-like ubiquitin ligases. Largely because they act as autonomous binding sites for host factors, L domains are generally transferable and active in a context-independent manner. Ebola virus matrix protein (EbVP40) contains two overlapping L-domain motifs within the sequence IL PTAPPEY MEA. Here, we show that both motifs are required for efficient EbVP40 budding. However, upon transplantation into two different retroviral contexts, the relative contributions of the PTAP and PPEY motifs differ markedly. In a murine leukemia virus carrying the EbVP40 sequence, both motifs contributed to overall L domain activity, and budding proceeded in a partly Tsg101-independent manner. Conversely, when transplanted into the context of human immunodeficiency virus type 1 (HIV-1), EbVP40 L-domain activity was entirely due to a PTAP-Tsg101 interaction. In fact, a number of PPXY-type L domains were inactive in the context of HIV-1. Surprisingly, PTAP and YPXL-type L domains that simulated HIV-1 budding reduced the amount of ubiquitin conjugated to Gag, while inactive PPXY-type L domains increased Gag ubiquitination. These observations suggest that active L domains recruit deubiquitinating enzymes as a consequence of class E VPS factor recruitment. Moreover, context-dependent L-domain function may reflect distinct requirements for host functions during the morphogenesis of different viral particles or the underlying presence of additional, as yet undiscovered L domains.

Human immunodeficiency virus type 1 (HIV-1) encodes a PTAP motif within the p6 domain of Gag that recruits Tsg101 and associated factors to facilitate virion budding. In this study, we use trans -complementation assays to demonstrate that the PTAP motif acts synergistically with additional p6 sequences to mediate the formation of infectious extracellular HIV-1 virions. These studies suggest that Tsg101 recruitment is necessary but not sufficient to account for late-budding activity exhibited by HIV-1 p6.

The final step in the life cycle of enveloped viruses is a membrane fusion event that releases the nascent virion from the host cell. Studies on the morphogenesis of human immunodeficiency virus type 1 (HIV-1) were the first to suggest that a virus-encoded function within the Gag protein was necessary for this late budding step to proceed efficiently (15). Subsequent investigations have shown that all retroviruses examined encode a so-called late-budding (L) domain within Gag whose disruption results in a phenotype characterized by normal virion assembly, with the exception of particle release (27, 37-39, 41). Moreover, at least some otherwise unrelated rhabdo-, filo-, and possibly reoviruses also encode sequences with proven or putative (based on sequence homology) L-domain activity (11, 22, 33, 34). Viral L domains fall into one of three classes, based on characteristic sequence motifs. The majority contain either PT/SAP or PPXY tetrapeptide motifs (15, 18, 38, 39, 41). In some, both motifs are present within a relatively short linear sequence. Conversely, equine infectious anemia virus (EIAV) encodes an unusual L-domain sequence that does not conform to the PT/SAP or PPXY consensus sequences but instead contains a critical YPDL motif that is essential for function (27). In several cases, viral L domains are functionally interchangeable and can exert their activity when positioned at different locations within a retroviral Gag protein (22, 24, 40). In fact, an HIV-1 strain that is noninfectious due to inactivating mutations in the PTAP-type L domain can be complemented in trans by coexpression of a Gag protein fused to either a homologous (PTAP) or heterologous (YPDL) L domain (22). These observations strongly suggest that L domains act by recruiting host cell factors to facilitate egress, rather than by directly influencing virion morphogenesis. In fact, specific host cell factors have been implicated in mediating the activity of each class of viral L domain. For EIAV, the YPDL motif conforms to the YXXL consensus sequence that mediates endocytosis of certain transmembrane proteins via interaction with the AP2 clathrin adaptor (10). Accordingly, EIAV p9 was shown to bind to the AP50 subunit of the AP-2 complex in vitro and induce AP-2 relocalization to sites of viral assembly in infected cells (28). The PPXY L-domain motif found in some retroviruses, rhabdoviruses, and filoviruses is similar to the consensus motif that binds WW domains (13). In fact, the L-domain sequences of Rous sarcoma virus, Ebola virus, and vesicular stomatitis virus have each been reported to bind to a particular class of WW domain-containing proteins, namely, the Nedd4-like E2 ubiquitin ligases (16, 17, 20), and a physiological Nedd4 binding site has been reported to exhibit viral L-domain activity (33). The most compelling evidence that a specific cellular protein mediates the activity of viral L domains exists for Tsg101. A variety of physiological activities have been attributed to this protein, but it appears likely that its primary function is in endosomal protein sorting (3, 7, 8, 19). Tsg101 has been demonstrated to interact with the HIV-1 PTAP-type L domain by using various protein-protein interaction assays (14, 22, 35), and we and others have established that Tsg101 recruitment is indeed the mechanism by which the HIV-1 L domain mediates particle budding. Specifically, there is an almost perfect correlation between the abilities of HIV-1 p6 mutants to bind Tsg101 and to mediate virion production (14, 18, 22, 35). In addition, depletion of Tsg101 by using small interfering RNA results in an HIV-1 budding defect that recapitulates the phenotype of a PTAP-defective virus, and reintroduction of Tsg101 restores viral budding (14). Moreover, tethering Tsg101 to sites of HIV-1 particle assembly by fusion to Gag reverses the HIV-1 budding defect that results from mutational inactivation of the PTAP L domain (22). It is also evident that the PTAP-type L domain encoded by Ebola virus, a filovirus, recruits Tsg101 to particle assembly sites to facilitate budding (22). Tsg101 is a member of a family of mammalian proteins whose orthologs in yeast are required for the formation of multivesicular bodies. In yeast, the Tsg101 ortholog is termed Vps23, and genetic elimination of this factor, or any one of approximately 15 other factors, results in the so-called class E phenotype, in which aberrant enlarged endosomes lacking internal vesicles accumulate (29). Several interactions between members of this group of proteins have been documented, and class E factors participate in the formation of at least three protein complexes, termed ESCRT-I, -II and -III (1, 2, 19). These complexes select a subset of transmembrane proteins for delivery to the endosomal lumen and mediate intraluminal vesicle budding. Tsg101 is one subunit of mammalian ESCRT-I (3), which also includes VPS28 (8) and, possibly, an as-yet-unidentified mammalian ortholog of Vps37, an additional component of the yeast ESCRT-I complex (19). The precise mechanisms by which class E Vps factors mediate vesicle budding are not well understood, but ESCRT-I, -II, and -III may act sequentially. Ultimately, an ATPase, Vps4, binds to components of ESCRT-III and mediates dissociation of this and perhaps other class E factors from the endosome (4, 5). Catalytically inactive VPS4 mutants have a dominant negative (DN) phenotype and induce the formation of aberrant endosomes lacking internal vesicles on which class E factors accumulate in both yeast and mammalian cells (4, 5, 8). Despite the fact that Tsg101 depletion does not affect the activity of the PPXY-type L domain encoded by murine leukemia virus (MuLV), evidence that both PTAP- and PPXY-type L domains utilize class E vacuolar protein sorting (VPS) proteins derives from the observation that the budding of both HIV-1 and MuLV is arrested at late stage by DN VPS4 (14). Thus, the processes of vesicle budding at endosomes and retroviral budding at the plasma membrane are topologically equivalent and can both be blocked by DN VPS4, and at least one viral L-domain type recruits a known component of the ESCRT-I complex. Because of this, an attractive but as-yet-unproven hypothesis is that the same set of factors mediates both processes in a mechanistically analogous manner. We have previously shown that a C-terminal Tsg101 domain (residues 250 to 390) is largely sufficient to mediate HIV-1 budding, provided that it is artificially recruited to sites of particle assembly (22). Thus, it appears that the N-terminal 250 amino acids are dispensable and serve only to mediate Tsg101 recruitment to particle budding sites. In this study, we present evidence that HIV-1 particle budding is highly dependent on the integrity of the Tsg101 C-terminal domain. Sequences that mediate VPS28 binding and a putative leucine zipper are each required. The integrity of other C-terminal Tsg101 sequences that mediate a newly described homomultimerization activity is also important for HIV-1 budding. Together, these findings suggest that an intact ESCRT-I complex is necessary for PTAP-type L-domain function. Moreover, minimal Gag binding and multimerizing subdomains of Tsg101 act as DN inhibitors of the HIV-1 p6 (PTAP-type) late budding domain but do not affect budding mediated by EIAV (YPDL-type) or MuLV (PPXY-type) L domains. However, because the EIAV L domain is inhibited by DN VPS4, these data suggest that each of the three known classes of L domains accesses a common pathway involving class E VPS factors but that the PTAP type is unique in requiring an intact ESCRT-I complex to exploit this cellular function during viral budding.

By following an analysis of the state of the art in the convergence of cloud computing and the Internet of Things (IoT), this paper presents design principles for the IoT in cloud environments. A framework for on-demand establishment of IoT services based on the automated formulation of societies of internet-connected objects is described and the interactions between architecture modules are explained in detail to validate this approach. The main building blocks of the functional framework and its operational components follow the utility-driven cloud-based computing model. The framework leverages well-known technologies (i.e. linked sensor data) and standards (notably the W3C semantic sensor networks ontology). Finally, an example for service formulation and delivery of services for a smart campus scenario is provided and discussed. This paper also introduces some experiment results using the utility-driven service formulation model for mobile applications.

Optical coherence tomography (OCT) is a high-resolution, cross-sectional imaging technique that allows detailed assessment of retinal thickness and morphologic evaluation of the retinal layers. This technology has developed quickly over the past two decades. OCT imaging has rapidly been integrated into routine ophthalmic clinical practice and trials. It has complemented fluorescein angiography in many instances, especially in the diagnosis and management of retinal disorders, including diabetic macular edema and age-related macular degeneration. With OCT, the exact localization of pathologic features can be visualized in segmentation maps of the retina, and this has allowed OCT to be used to evaluate specific features that may serve as predictive factors in the prognosis and follow up of these pathologies. Therefore, it has become an important clinical and research tool for the diagnosis, follow up, treatment, and assessment of new treatment modalities for all diseases that affect the posterior pole of the eye.

Optical coherence tomography (OCT) is now a standard of care in ophthalmology and is considered essential for the diagnosis and monitoring of many retinal diseases. One of the major advances obtained with OCT was the understanding of the pathophysiology of macular holes. Non-full-thickness macular holes have been revisited because high-resolution OCT images can detect a lamellar macular defect that is not always visible clinically, and surgery has been advocated by some authors. OCT can be valuable in determining the need for and/or timing of surgical intervention on epiretinal membranes or vitreomacular traction syndrome. In addition, we can use this technology as a predictive factor in the prognosis and follow-up of the most common posterior pole pathologies.