Dynamics of KSHV gene expression during de novo infection and the role of LANA in immune modulation

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic human herpes virus that has been linked to the development of multiple malignancies including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Like all members of the herpesvirus family, KSHV establishes a lifelong persistence in the infected host. However, immediately upon infection, the virus has to overcome many challenges in the hostile cellular environment before it can establish a long-term infection. The remarkable success of the virus in establishing lifelong persistence in the infected host indicates that the virus is well equipped to manipulate the host environment very efficiently, even before it has a chance to fully express its genome. In order to understand how KSHV manipulates the host environment immediately upon infection, we must first understand which molecules are packaged in the virions and which viral genes are expressed during the initial time points following entry of the virus into the host cells. In chapter I, we probe these questions by performing a comprehensive analysis of the viral transcriptome in the purified KSHV virions. We also examine the dynamics of viral transcriptome at very early time-points following de novo infection of multiple cell lines permissive to long-term infection of KSHV. For this comprehensive study of viral transcriptome analysis, we used a high throughput approach of next-generation RNA sequencing. The results of this study identified many viral transcripts that are packaged into the virions and the transcripts that are actively transcribed during the initial infection period. Overall, the results of this study lay a foundation for future research targeting the viral genes whose expression may be critical for establishing a successful viral infection. One critical aspect of KSHV infection is its ability to persist throughout the life of the host. This can be attributed to its ability to efficiently hide from the host's immune surveillance. In chapter II, we identify a novel mechanism that helps the virus hide from the radar of host immunity. Our data sheds light on how one of the viral proteins, Latency- associated nuclear antigen (LANA), reduces the expression of major histocompatibility class II (MHC II) molecules, which are the molecules that are critical for reporting viral antigens to the immune cells of the host. We demonstrated that LANA binds with the proteins of regulatory factor X (RFX) complex, which are essential components of MHC II transcription machinery. The association of LANA with RFX complex reduces binding of the transcription factor called class II transactivator (CIITA) to the highly conserved promoters of MHC II genes. Binding of the CIITA to MHC II promoters is absolutely necessary for the expression of MHC II genes; by reducing the association of CIITA with the promoters of MHC II genes, LANA inhibits the expression of MHC II genes. KSHV infection is known to induce multiple pro-angiogenic cytokines that aid in extensive angiogenesis associated with Kaposi's sarcoma tumors. In chapter III, we demonstrate that LANA induces expression of a secreted angiogenic cytokine epidermal growth factor-like multiple 7 (EGFL7). Our data shows that LANA induces expression of EGFL7 in B cells, suggesting that it may act in a paracrine fashion during KSHV infection. The results of this research provide insight into an additional mechanism used by KSHV to promote angiogenesis. Targeting EGFL7 in combination with other important angiogenic cytokines induced during KSHV infection may increase the effectiveness of currently available anti-angiogenic therapy for Kaposi's sarcoma

Semantic-driven matchmaking of web services using case-based reasoning

With the rapid proliferation of Web services as the medium of choice to securely publish application services beyond the firewall, the importance of accurate, yet flexible matchmaking of similar services gains importance both for the human user and for dynamic composition engines. In this paper, we present a novel approach that utilizes the case based reasoning methodology for modelling dynamic Web service discovery and matchmaking. Our framework considers Web services execution experiences in the decision making process and is highly adaptable to the service requester constraints. The framework also utilises OWL semantic descriptions extensively for implementing both the components of the CBR engine and the matchmaking profile of the Web services

Genetic background influences tumour development in heterozygous Men1 knockout mice

Multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disorder caused by MEN1 germline mutations, is characterised by parathyroid, pancreatic and pituitary tumours. MEN1 mutations also cause familial isolated primary hyperparathyroidism (FIHP), a milder condition causing hyperparathyroidism only. Identical mutations can cause either MEN1 or FIHP in different families, thereby implicating a role for genetic modifiers in altering phenotypic expression of tumours. We therefore investigated the effects of genetic background and potential for genetic modifiers on tumour development in adult Men1+/- mice, which develop tumours of the parathyroids, pancreatic islets, anterior pituitary, adrenal cortex and gonads, that had been backcrossed to generate C57BL/6 and 129S6/SvEv congenic strains. A total of 275 Men1+/- mice, aged 5–26 months were macroscopically studied, and this revealed that genetic background significantly influenced the development of pituitary, adrenal and ovarian tumours, which occurred in mice over 12 months of age and more frequently in C57BL/6 females, 129S6/SvEv males and 129S6/SvEv females, respectively. Moreover, pituitary and adrenal tumours developed earlier, in C57BL/6 males and 129S6/SvEv females, respectively, and pancreatic and testicular tumours developed earlier in 129S6/SvEv males. Furthermore, glucagon-positive staining pancreatic tumours occurred more frequently in 129S6/SvEv Men1+/- mice. Whole genome sequence analysis of 129S6/SvEv and C57BL/6 Men1+/- mice revealed >54,000 different variants in >300 genes. These included, Coq7, Dmpk, Ccne2, Kras, Wnt2b, Il3ra and Tnfrsf10a, and qRT-PCR analysis revealed that Kras was significantly higher in pituitaries of male 129S6/SvEv mice. Thus, our results demonstrate that Kras and other genes could represent possible genetic modifiers of Men1

ENDOCRINOLOGY IN THE TIME OF COVID-19 Clinical management of neuroendocrine neoplasms (NENs)

In viral pandemics, most specifically Covid-19, many patients with neuroendocrine neoplasms (NENs), including phaeochromocytomas, paragangliomas and medullary thyroid carcinoma, may develop Covid-19 in a mild or severe form, or be concerned about the influence of viral infection relative to their anti-tumoral therapy. In general, newly presenting patients should be assessed, and patients recently receiving chemotherapy, targeted therapy or radionuclide therapy, or showing tumour growth, should be closely followed. For previously diagnosed patients, who have indolent disease, some delay in routine follow-up or treatment may not be problematic. However, patients developing acute secretory syndromes due to functional neuroendocrine neoplasms (such as of the pancreas, intestine or lung), phaeochromocytomas and paragangliomas, will require prompt treatment. Patients with life-threatening Covid-19-related symptoms should be urgently treated and long-term anti-tumoral treatments may be temporarily delayed. In patients with especially aggressive NENs, a careful judgement should be made regarding the severity of any Covid-19 illness, tumour grade, and the immunosuppressant effects of any planned chemotherapy, immunotherapy (e.g. interferon-alpha), targeted therapy or related treatment. In other cases, especially patients with completely resected NENs, or who are under surveillance for a genetic disorder, a telephone or delayed consultation may be in order, balancing the risk of a delay against that of the possible development of Covid-19.Peer reviewe

Modeling study of human renal chloride channel (hCLC-5) mutations suggests a structural-functional relationship

Modeling study of human renal chloride channel (hCLC-5) mutations suggests a structural-functional relationship.BackgroundDent's disease, a renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, and nephrolithiasis, is due to inactivating mutations in the X-linked renal-specific chloride channel, hCLC-5. The x-ray crystal structures of two bacterial chloride channels (CLCs) have recently been established, thereby allowing us to construct a model for hCLC-5 and further examine the role of its mutations.MethodsThe data regarding 49 hCLC-5 mutations were reviewed. Thirty-four mutations that predicted absent or truncated channels were excluded. The remaining 15 mutations (one in-frame insertion and 14 missense mutations), 12 of which have been studied electrophysiologically, were assessed. The hCLC-5 sequence was aligned with the Salmonella typhimurium and Escherichia coli sequences and used to map the hCLC-5 mutations onto a three-dimensional model.ResultshCLC-5 is a homodimeric protein, with each subunit consisting of 18 helices. None of the missense mutations involved the chloride (Cl−) selectivity filter, but 12 of the 15 mutations were found to be clustered at the interface of the two subunits. Six of these mutations occurred in two of the helices that either form part of the interface or lie in close proximity to the interface, and three other mutations that did not lead to complete loss of Cl− conductance were at the edge of the interface.ConclusionThese results demonstrate a crucial role for the interaction between the two subunits at the interface of the homodimeric hCLC-5

A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin-biased signaling

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that signals through Gq/11and Gi/oto stimulate cytosolic calcium (Ca2+i) and mitogen-activated protein kinase (MAPK) signaling to control extracellular calcium homeostasis. Studies of loss- and gain-of-functionCASRmutations, which cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively, have revealed that the CaSR signals in a biased manner. Thus, some mutations associated with FHH1 lead to signaling predominantly through the MAPK pathway, whereas mutations associated with ADH1 preferentially enhance Ca2+iresponses. We report a previously unidentified ADH1-associated R680G CaSR mutation, which led to the identification of a CaSR structural motif that mediates biased signaling. Expressing CaSRR680Gin HEK 293 cells showed that this mutation increased MAPK signaling without altering Ca2+iresponses. Moreover, this gain of function in MAPK activity occurred independently of Gq/11and Gi/oand was mediated instead by a noncanonical pathway involving β-arrestin proteins. Homology modeling and mutagenesis studies showed that the R680G CaSR mutation selectively enhanced β-arrestin signaling by disrupting a salt bridge formed between Arg680and Glu767, which are located in CaSR transmembrane domain 3 and extracellular loop 2, respectively. Thus, our results demonstrate CaSR signaling through β-arrestin and the importance of the Arg680-Glu767salt bridge in mediating signaling bias

Ontology-based discovery of time-series data sources for landslide early warning system

YesModern early warning system (EWS) requires sophisticated knowledge of the natural hazards, the urban context and underlying risk factors to enable dynamic and timely decision making (e.g., hazard detection, hazard preparedness). Landslides are a common form of natural hazard with a global impact and closely linked to a variety of other hazards. EWS for landslides prediction and detection relies on scientific methods and models which requires input from the time series data, such as the earth observation (EO) and urban environment data. Such data sets are produced by a variety of remote sensing satellites and Internet of things sensors which are deployed in the landslide prone areas. To this end, the automatic discovery of potential time series data sources has become a challenge due to the complexity and high variety of data sources. To solve this hard research problem, in this paper, we propose a novel ontology, namely Landslip Ontology, to provide the knowledge base that establishes relationship between landslide hazard and EO and urban data sources. The purpose of Landslip Ontology is to facilitate time series data source discovery for the verification and prediction of landslide hazards. The ontology is evaluated based on scenarios and competency questions to verify the coverage and consistency. Moreover, the ontology can also be used to realize the implementation of data sources discovery system which is an essential component in EWS that needs to manage (store, search, process) rich information from heterogeneous data sources

The role of the central stellar cluster in active galactic nuclei. I. Semi-analytical model

The subject of the paper is the role of the massive stellar cluster in the activity phenomenon and in the structure of active galactic nuclei. We introduce a simple model of stellar dynamics in the internal part of the cluster, which allows us to include both the star-disk and the star-star interactions. It is shown that the properties of the distribution of stars in the vicinity of the black hole are determined both by the interaction of the stars with the accretion disk and by the pair gravitational and contact interaction between the stars. We calculate the distribution of stars in the central parts of the cluster and we discuss possible effects of stellar mass-loss due to the star-disk interaction. Finally, we study the implications of the central cluster for active galactic nuclei activity. We model the broad line region assuming that the gaseous wakes, following stars after each disk crossing, play the role of the broad line region clouds, and we calculate the corresponding line profiles. We also analyze the contribution of star-star and star-disk collisions to active galactic nuclei variability.Comment: Accepted for publication in Astronomy and Astrophysic

Sequence analysis and transcript expression of the MEN1 gene in sporadic pituitary tumours

The majority of pituitary tumours are monoclonal in origin and arise sporadically or occasionally as part of multiple endocrine neoplasia type 1 (MEN1). Whilst a multi-step aetiology involving both oncogenes and tumour suppressor genes has been proposed for their development, the target(s) of these changes are less clearly defined. Both familial and sporadic pituitary tumours have been shown to harbour allelic deletion on 11q13, which is the location of the recently cloned MEN1 gene. We investigated 23 sporadic pituitary tumours previously shown to harbour allelic deletion on 11q13 with the marker PYGM centromeric and within 50 kb of the MEN1 locus. In addition, the use of intragenic polymorphisms in exon 9 and at D11S4946, and of telomeric loci at D11S4940 and D11S4936, revealed that five of 20 tumours had loss of heterozygosity (LOH) telomeric to the menin gene. However, the overall pattern of loss in informative cases was indicative of non-contiguous deletion that brackets the menin gene. Sequence analysis of all MEN1 coding exons and flanking intronic sequence, in tumours and matched patient leucocyte DNA, did not reveal mutation(s) in any of the 23 tumours studied. A benign polymorphism in exon 9 was encountered at the expected frequency, and in seven patients heterozygous for the polymorphism the tumour showed retention of both copies of the menin gene. Reverse transcription polymerase chain reaction analysis of ten evaluable tumours and four normal pituitaries revealed the presence of the menin transcript. Whilst these findings suggest that gene silencing is unlikely to be mechanistic in sporadic pituitary tumorigenesis, they do not exclude changes in the level or stability of the transcript or translation to mature protein. Our study would support and extend very recent reports of a limited role for mutations in the MEN1 gene in sporadic pituitary tumours. Alternatively, these findings may point to an, as yet, unidentified tumour suppressor gene in this region