Specific short hairpin RNA-mediated inhibition of viral DNA packaging of human cytomegalovirus

AbstractTo clearly demonstrate the role of the human cytomegalovirus (HCMV) portal protein pUL104 RNA interference was applied. Expressing cell lines were constructed by transduction of shRNAs via the infection with retroviral vectors. After infection of these cells with HCMV AD169 the expression of pUL104 was reduced up to 80% for shRNA S1 and 54% for shRNA S2 at late times of infection compared to controls. In addition, the inhibitory effect was corresponding with a decrease in viral mRNA and plaque formations. Electron microscopic analysis demonstrated that infection of cells expressing pUL104-specific shRNAs resulted in the inhibition of formation of replicative particles

Identifizierung und Charakterisierung des HCMV Portal Proteins pUL104

Die HCMV DNA-Verpackung beruht auf der Spaltung konkatemerer DNA durch die virale Terminase, deren Interaktion mit einem im Kapsid vorliegenden Portalprotein und der anschließenden Translokation der DNA in das Kapsid. In der vorliegenden Arbeit konnte pUL104 als das HCMV Portalprotein identifiziert werden. Über indirekte Immunfluoreszenz konnte gezeigt werden, dass pUL104 über ein nicht lineares, endogenes NLS verfügt und bei solitärer Expression im Zellkern vorliegt. In infizierten Zellen wird pUL104 in die Replikationszentren rekrutiert und erscheint spät nach Infektion als Bestandteil der Virionen im Zytoplasma. Nach Aufreinigung eines monospezifischen polyklonalen Antikörpers konnte pUL104 in infizierten Zellen durch Immunpräzipitationen als Protein von 75 kDa identifiziert werden, das auch als Dimer von 150 kDa vorliegt. Unter nativen Bedingungen liegen beide Formen als Komplex vor. Bei einer Zellfraktionierung zeigte sich ebenfalls eine sowohl nukleäre als auch zytoplasmatische Verteilung. Weiterhin konnte pUL104 als Strukturprotein in extrazellulären Virionen nachgewiesen werden. Dies konnte durch Immunogoldfärbung von Ultradünnschnitten infizierter Zellen weiter eingegrenzt werden, wo sich pUL104 einzeln an Kapsiden beobachten ließ. Untersuchungen zur Struktur von pUL104 zeigten, dass rekombinant exprimiertes rpUL104 sowohl in der Sedimentation über einen Sucrosegradienten als auch in der Gelpermeationschromatographie als hochmolekularer Komplex von mindestens 670 kDa vorliegt. Auch bei Cross-Linking- und Einzelpartikel-Analysen liegt rpUL104 als Oligomer vor, wobei bei letzterem eine Ringstruktur mit einer tonnenförmigen Seitenansicht auszumachen war. Eine Interaktion mit pUL56, der großen Terminase-Untereinheit, ließ sich sowohl durch in vitro Bindungsexperimente als auch durch Co-Immunpräzipitationen in vivo nachweisen. Diese konnte durch den HCMV Inhibitor Cl4RB, ein Benzimidazol-D-Ribonukleosid, unterbunden werden. Weiterhin konnte eine Bindung an dsDNA gezeigt werden. Sowohl die strukturellen als auch die funktionalen Anforderungen an ein Portalprotein werden damit durch pUL104 erfüllt. Durch den Einsatz von UL104 spezifischer RNAi konnte eine Verminderung der pUL104 Expression und auch der HCMV Replikation erreicht werden. Die EM-Analyse von Ultradünnschnitten infizierter Zellen zeigte hierbei eine Akkumulation leerer Kapside im Zellkern sowie einen Mangel an infektiösen Virionen im Zytoplasma. Dies zeigt, dass pUL104 für die DNA-Verpackung notwendig ist. Zusammenfassend handelt es sich bei pUL104 um ein Kapsid-assoziertes Oligomer, das mit der HCMV Terminase interagiert und für die DNA-Verpackung essentiell ist

Oxazole-Bridged Combretastatin A-4 Derivatives with Tethered Hydroxamic Acids: Structure-Activity Relations of New Inhibitors of HDAC and/or Tubulin Function

New inhibitors of tubulin polymerization and/or histone deacetylase (HDAC) activity were synthesized by attaching alkyl tethered hydroxamic acid appendages of varying length to oxazole-bridged combretastatin A-4 analogous caps. While their antiproliferative and microtubule disrupting effect was most pronounced for derivatives with short spacers, HDAC inhibition was strongest for those with longer spacers. These findings were further supported by computational methods such as structure-based docking experiments exploring the target interactions of the derivatives with varying linkers. For instance, compounds featuring short four-atom spacers between cap and hydroxamic acid inhibited the growth of various cancer cell lines and human endothelial hybrid cells with IC50 values in the low nanomolar range. In line with their ability to inhibit the microtubule assembly, four- and five-atom spacered hydroxamic acids caused an accumulation of 518A2 melanoma cells in G2/M phase, whereas a compound featuring a six-atom spacer and performing best in HDAC inhibition, induced a G1 arrest in these cells. All these beneficial anticancer activities together with their selectivity for cancer cells over non-malignant cells, point out the great potential of these novel pleiotropic HDAC and tubulin inhibitors as drug candidates for cancer therapy

A “Coiled-Coil” Motif Is Important for Oligomerization and DNA Binding Properties of Human Cytomegalovirus Protein UL77

Human cytomegalovirus (HCMV) UL77 gene encodes the essential protein UL77, its function is characterized in the present study. Immunoprecipitation identified monomeric and oligomeric pUL77 in HCMV infected cells. Immunostaining of purified virions and subviral fractions showed that pUL77 is a structural protein associated with capsids. In silico analysis revealed the presence of a coiled-coil motif (CCM) at the N-terminus of pUL77. Chemical cross-linking of either wild-type pUL77 or CCM deletion mutant (pUL77ΔCCM) implicated that CCM is critical for oligomerization of pUL77. Furthermore, co-immunoprecipitations of infected and transfected cells demonstrated that pUL77 interacts with the capsid-associated DNA packaging motor components, pUL56 and pUL104, as well as the major capsid protein. The ability of pUL77 to bind dsDNA was shown by an in vitro assay. Binding to certain DNA was further confirmed by an assay using biotinylated 36-, 250-, 500-, 1000-meric dsDNA and 966-meric HCMV-specific dsDNA designed for this study. The binding efficiency (BE) was determined by image processing program defining values above 1.0 as positive. While the BE of the pUL56 binding to the 36-mer bio-pac1 containing a packaging signal was 10.0±0.63, the one for pUL77 was only 0.2±0.03. In contrast to this observation the BE of pUL77 binding to bio-500 bp or bio-1000 bp was 2.2±0.41 and 4.9±0.71, respectively. By using pUL77ΔCCM it was demonstrated that this protein could not bind to dsDNA. These data indicated that pUL77 (i) could form homodimers, (ii) CCM of pUL77 is crucial for oligomerization and (iii) could bind to dsDNA in a sequence independent manner

Optimasi Portofolio Resiko Menggunakan Model Markowitz MVO Dikaitkan dengan Keterbatasan Manusia dalam Memprediksi Masa Depan dalam Perspektif Al-Qur`an

Risk portfolio on modern finance has become increasingly technical, requiring the use of sophisticated mathematical tools in both research and practice. Since companies cannot insure themselves completely against risk, as human incompetence in predicting the future precisely that written in Al-Quran surah Luqman verse 34, they have to manage it to yield an optimal portfolio. The objective here is to minimize the variance among all portfolios, or alternatively, to maximize expected return among all portfolios that has at least a certain expected return. Furthermore, this study focuses on optimizing risk portfolio so called Markowitz MVO (Mean-Variance Optimization). Some theoretical frameworks for analysis are arithmetic mean, geometric mean, variance, covariance, linear programming, and quadratic programming. Moreover, finding a minimum variance portfolio produces a convex quadratic programming, that is minimizing the objective function ðð¥with constraintsð ð 𥠥 ðandð´ð¥ = ð. The outcome of this research is the solution of optimal risk portofolio in some investments that could be finished smoothly using MATLAB R2007b software together with its graphic analysis

Impacts of the Tropical Pacific/Indian Oceans on the Seasonal Cycle of the West African Monsoon

The current consensus is that drought has developed in the Sahel during the second half of the twentieth century as a result of remote effects of oceanic anomalies amplified by local land–atmosphere interactions. This paper focuses on the impacts of oceanic anomalies upon West African climate and specifically aims to identify those from SST anomalies in the Pacific/Indian Oceans during spring and summer seasons, when they were significant. Idealized sensitivity experiments are performed with four atmospheric general circulation models (AGCMs). The prescribed SST patterns used in the AGCMs are based on the leading mode of covariability between SST anomalies over the Pacific/Indian Oceans and summer rainfall over West Africa. The results show that such oceanic anomalies in the Pacific/Indian Ocean lead to a northward shift of an anomalous dry belt from the Gulf of Guinea to the Sahel as the season advances. In the Sahel, the magnitude of rainfall anomalies is comparable to that obtained by other authors using SST anomalies confined to the proximity of the Atlantic Ocean. The mechanism connecting the Pacific/Indian SST anomalies with West African rainfall has a strong seasonal cycle. In spring (May and June), anomalous subsidence develops over both the Maritime Continent and the equatorial Atlantic in response to the enhanced equatorial heating. Precipitation increases over continental West Africa in association with stronger zonal convergence of moisture. In addition, precipitation decreases over the Gulf of Guinea. During the monsoon peak (July and August), the SST anomalies move westward over the equatorial Pacific and the two regions where subsidence occurred earlier in the seasons merge over West Africa. The monsoon weakens and rainfall decreases over the Sahel, especially in August.Peer reviewe

Search for heavy resonances decaying to two Higgs bosons in final states containing four b quarks

A search is presented for narrow heavy resonances X decaying into pairs of Higgs bosons (H) in proton-proton collisions collected by the CMS experiment at the LHC at root s = 8 TeV. The data correspond to an integrated luminosity of 19.7 fb(-1). The search considers HH resonances with masses between 1 and 3 TeV, having final states of two b quark pairs. Each Higgs boson is produced with large momentum, and the hadronization products of the pair of b quarks can usually be reconstructed as single large jets. The background from multijet and t (t) over bar events is significantly reduced by applying requirements related to the flavor of the jet, its mass, and its substructure. The signal would be identified as a peak on top of the dijet invariant mass spectrum of the remaining background events. No evidence is observed for such a signal. Upper limits obtained at 95 confidence level for the product of the production cross section and branching fraction sigma(gg -> X) B(X -> HH -> b (b) over barb (b) over bar) range from 10 to 1.5 fb for the mass of X from 1.15 to 2.0 TeV, significantly extending previous searches. For a warped extra dimension theory with amass scale Lambda(R) = 1 TeV, the data exclude radion scalar masses between 1.15 and 1.55 TeV

Search for supersymmetry in events with one lepton and multiple jets in proton-proton collisions at root s=13 TeV

Peer reviewe

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Combined searches for the production of supersymmetric top quark partners in proton-proton collisions at root s=13 TeV

A combination of searches for top squark pair production using proton-proton collision data at a center-of-mass energy of 13 TeV at the CERN LHC, corresponding to an integrated luminosity of 137 fb(-1) collected by the CMS experiment, is presented. Signatures with at least 2 jets and large missing transverse momentum are categorized into events with 0, 1, or 2 leptons. New results for regions of parameter space where the kinematical properties of top squark pair production and top quark pair production are very similar are presented. Depending on themodel, the combined result excludes a top squarkmass up to 1325 GeV for amassless neutralino, and a neutralinomass up to 700 GeV for a top squarkmass of 1150 GeV. Top squarks with masses from 145 to 295 GeV, for neutralino masses from 0 to 100 GeV, with a mass difference between the top squark and the neutralino in a window of 30 GeV around the mass of the top quark, are excluded for the first time with CMS data. The results of theses searches are also interpreted in an alternative signal model of dark matter production via a spin-0 mediator in association with a top quark pair. Upper limits are set on the cross section for mediator particle masses of up to 420 GeV