Dimer-dimer stacking interactions are important for nucleic acid binding by the archaeal chromatin protein Alba

Archaea use a variety of small basic proteins to package their DNA. One of the most widespread and highly conserved is the Alba (Sso10b) protein. Alba interacts with both DNA and RNA in vitro, and we show in the present study that it binds more tightly to dsDNA (double-stranded DNA) than to either ssDNA (single-stranded DNA) or RNA. The Alba protein is dimeric in solution, and forms distinct ordered complexes with DNA that have been visualized by electron microscopy studies; these studies suggest that, on binding dsDNA, the protein forms extended helical protein fibres. An end-to-end association of consecutive Alba dimers is suggested by the presence of a dimer-dimer interface in crystal structures of Alba from several species, and by the strong conservation of the interface residues, centred on Are and Phe(60). In the present study we map perturbation of the polypeptide backbone of Alba upon binding to DNA and RNA by NMR, and demonstrate the central role of Phe(60) in forming the dimer dimer interface. Site-directed spin labelling and pulsed ESR are used to confirm that an end-to-end, dimer dimer interaction forms in the presence of dsDNA.Publisher PDFPeer reviewe

Novel retargeting strategies in the design of oncolytic herpes simplex viruses

La terapia virale oncolitica sfrutta l’abilità dei virus di infettare e uccidere le cellule e si propone come cura per i tumori che non rispondono agli attuali approcci terapeutici. Un herpes simplex 1 (HSV-1) attenuato, denominato T-Vec, è l’unico virus oncolitico approvato per l’impiego in clinica. In questo virus, la sicurezza è stata ottenuta a spese della virulenza. Per superare i limiti dell’attenuazione, una strategia alternativa è quella di alterare lo spettro d’ospite del virus. Il legame della glicoproteina D (gD) di HSV-1 ad uno dei suoi recettori attiva le glicoproteine gH/gL e gB, che eseguono la fusione. Finora gD è stata l’unica glicoproteina che ha permesso di reindirizzare con successo il tropismo di HSV-1. Il lavoro di questa tesi prevede lo sviluppo di HSV-1 oncolitici, reindirizzati a recettori tumore-specifici, per condurli alla fase traslazionale. Il tropismo di HSV-1 è stato modificato ingegnerizzando un ligando eterologo in gH (nel virus R-809) o in gB (in R-909). Il ligando eterologo è un frammento anticorpale variabile a catena singola (scFv) diretto contro HER2, recettore sovra-espresso in diversi tumori. Il reindirizzamento ottenuto tramite gB o gH conferisce ad HSV-1 proprietà molto simili al reindirizzamento conseguito tramite gD, in termini di replicazione virale e attività oncolitica in vitro. Le modificazioni in gB o gH sono state combinate con quelle in gD, ottenendo, per la prima volta, due HSV-1 reindirizzati contemporaneamente verso due recettori distinti. R-805, diretto contemporaneamente verso HER2 ed EGFR, è stato caratterizzato in vitro e si è messo a punto un modello in vivo col quale studiare la sua efficacia oncolitica. R-313 è risultato in grado di utilizzare alternativamente due recettori, per infettare le cellule tumorali e le cellule produttrici non tumorali, in quanto la produzione di un vettore oncolitico in una linea tumorale non è compatibile con la sperimentazione clinica.Oncolytic virotherapy exploits the ability of viruses to infect and kill cells and is envisioned as treatment for tumors that respond poorly to the current therapeutic approaches. The attenuated herpes simplex virus 1 (HSV-1), named T-Vec, is the only oncolytic virus approved so far for clinical practice. Safety was obtained at the expense of virulence. To overcome the attenuation limits, an alternative strategy consists in altering the host range of a virus. The binding of membrane-bound glycoprotein D (gD) of HSV-1 to one of its receptors activates the downstream glycoproteins gH/gL and gB. The latter executes the fusion virion-cell. So far, gD was the only glycoprotein that successfully enabled the retargeting of HSV-1. The work of this thesis focuses on the the development of oncolytic herpes simplex viruses (o-HSVs), retargeted to tumor-specific receptors, in order to bring retargeted o-HSVs to the translational phase. The tropism of HSV-1 has been modified by engineering a heterologous ligand in gH (in virus R-809) or in gB (in R-909). The selected heterologous ligand was a single chain variable fragment antibody (scFv) directed against HER2, a receptor overexpressed in several cancers. The retargeting achieved via gB or gH confers to HSV-1 very similar properties to the retargeting achieved via gD, in terms of virus growth and oncolytic activity in vitro. The changes in gB or gH were combined with those in gD, leading, for the first time, to two HSV-1 simultaneously redirected to two distinct receptors. R-805, directed simultaneously to HER2 and EGFR, has been characterized in vitro. An in vivo model to study its oncolytic efficacy was developed. R-313 was designed to enable the production of an oncolytic vector into a non-tumor cell line. R-313 is capable to use alternately two receptors, and to infect the tumor cells and the non-tumor-producing cells

Comparative Analysis of Limit Bearing Capacity of a Continuous Beam Depending on the Character of the Load

Determination of the bearing capacity of a structure, as well as the assessment related tothe structure failure is very valuable, not only as a simple control of beam bearing capacity,but also as a significant basis and factor in designing of structures. When the structure isexposed to the action of a proportionally increasing load, by applying the limit analysis it ispossible to determine the limit failure load which is one of the bearing capacity indicators. Inthe case when beam systems are exposed to repeated load, the limit theorems do not yield the adequate solutions, thus the adaptation theorems which made safe limit load determination possible were developed simultaneously. Applying the limit and shakedown analysis, the analysis of bearing capacity of a continuous beam with two spans was conducted in the paper.Also displayed is the difference between the values of failure forces depending on the character of load and the beam span value in order to assess justification for application of the shakedown method in the analysis of the limit bearing capacity of the beams

Molecular docking study on the interaction of Rhodopsin-like receptor with tetra-coordinated gold(III) complex

The pharmacologic properties of gold compounds have been known since the end of the 19th century. In the last decade, gold complexes have received increased attention due to the variety of their applications. Rhodopsin-like receptors are a family of proteins that belong to the largest group of G protein-coupled receptors (GPCRs). In this paper, the molecular interactions between active binding sites of the Rhodopsin-like receptor (RLR) and synthesized gold(III) complex ([Au(DPP)Cl2]+ where DPP=4,7-diphenyl-1,10-phenanthroline) were investigated by molecular docking simulations. The crystal structure of investigated receptor RLR (PDB ID: 4A4M) was extracted from RCSB Protein Data Bank in PDB format. The native bound ligand (11-cis-retinal) was extracted from receptor and binding pocket analysis was performed. Re-docking was performed with the gold(III) complex to generate the same docking pose as found in co-crystallized form of receptor. The binding energy of gold(III) complex to RLR was found to be -35.35 kJ/mol, as opposed to 11-cis-retinal which of about - 40.5 kJ/mol. The obtained results of revealed that gold(III) complex binds at the same binding pockets to RLR, as well as native bound ligand, by weak non-covalent interactions. The most prominent interactions are hydrogen bonds, alkyl-π, and π-π interactions. The preliminary results suggest that gold(III) complex showed good binding affinity against RLR, as well as native bound ligand, 11-cisretinal, as evident from the free binding energy (ΔGbind in kJ/mol).The authors are grateful to the Ministry of Education, Science and Technological Development of the Republic of Serbia (Agreement No. 451-03-9/2021-14/200378 and Agreement No. 451-03-68/2021-14/200122) for financial supportPublishe

Binding of Platinum(II) to Some Biologicaly Important Thiols

The reactions between [Pt(terpy)Cl+ and thiols, such as glutathione, L-cysteine, D-penicillamine and thioglycolic acid have been Studied by conventional UV-VIS spectrophotometry and H NMR spectroscopy. The second-ordero rate constants, K2, are similar for these four thiols, varying between 1.06 x 10-2 and 6.10 x 10+3 M-1 s-1 at 25°C. The activation entropies have large negative values between -100 and -200 J mol-1 which are compatible with an associative A mechanism. However, L-methionine, as thioether ligand, is unreactive under the same experimental conditions. The obtained results have been analyzed in relation to the antitumor activity and toxicity of platinum(II) complexes

Tumor necrosis factor-alpha as differential diagnostic marker for patients with fever of unknown origin

© 2019, University of Kragujevac, Faculty of Science. All rights reserved. Febrile conditions of unidentified origin are still unknown in modern medicine despite the development of diagnostic procedures. There are various agents of long-term temperature encompassing numerous infectious or non-infectious diseases. The aim of this study was to determine if there was a statistically significant difference in the values of proinfl ammatory cytokines (IL-1, TNFa, IL-6) in patients who meet the criteria for febrile conditions of unidentified origin, between the group of infectious, malignant, rheumatic, “other” diseases and undiagnosed patients. The study was conducted in the Immunology laboratory of the Center for Molecular Medicine and Stem Cells Research of the Faculty of Medical Sciences in Kragujevac. Blood samples were taken from patients tested at the Clinic for Infectious Diseases, of the Clinical Center of Kragujevac, in the period from 2014 to 2016. The study included 70 patients. The measured values of the level of TNFa showed significantly higher values in a group of malignant diseases than in the group of infectious diseases, while the values of IL-1 and IL-6 did not show statistical significance. TNFa can improve diagnosing in case of patients with an unknown febrile condition, which can shorten the length of the hospital stay and reduce the volume of performance of diagnostic procedures

The simultaneous insertion of two ligands in gD for the cultivation of oncolytic HSVs in non-cancer cells and the retargeting to cancer receptors

Insertion of a single chain antibody (scFv) to HER2 (human epidermal growth factor receptor 2) in gD, gH, or gB gives rise to herpes simplex viruses (HSVs) specifically retargeted to HER2-positive cancer cells, hence in highly specific non-attenuated oncolytic agents. Clinical grade virus production can not rely on cancer cells. Recently, we developed a double retargeting strategy whereby gH carries the GCN4 peptide for retargeting to the non-cancer producer Vero-GCN4R cell line, and gD carries the scFv to HER2 for cancer retargeting. Here, we engineered double retargeted recombinants, which carry both the GCN4 peptide and the scFv to HER2 in gD. Novel, more advantageous detargeting strategies were devised, so as to optimize the cultivation of the double-retargeted recombinants. Nectin1 detargeting was achieved by deletion of aa 35-39, 214-223, or 219-223, and replacement of the deleted sequences with one of the two ligands. The latter two deletions were not attempted before. All recombinants exhibited the double retargeting to HER2 and to the Vero-GCN4R cells, as well as detargeting from the natural receptors HVEM and nectin1. Of note, some recombinants grew to higher yields than others. The best performing recombinants carried a gD deletion as small as 5 amino acids, and grew to titers similar to those exhibited by the singly retargeted R-LM113, and by the non-retargeted R-LM5. This study shows that double retargeting through insertion of two ligands in gD is feasible and, when combined with appropriate detargeting modifications, can result in recombinants highly effectivein vitroandin vivo.IMPORTANCEThere is increasing interest in oncolytic viruses, following FDA and EMA approval of the oncolytic HSV OncovexGM-CSF, and, mainly, because they greatly boost the immune response to the tumor and can be combined with immunotherapeutic agents, particularly immune checkpoint inhibitors. A strategy to gain high cancer specificity and avoid virus attenuation is to retarget the virus tropism to cancer-specific receptors of choice. However, cultivation of retargeted oncolytics in cells expressing the cancer receptor may not be approvable by regulatory agencies. We devised a strategy for their cultivation in non-cancer cells. Here, we describe a double retargeting strategy, based on the simultaneous insertion of two ligands in gD, one for retargeting to a producer, universal Vero cell derivative, one for retargeting to the HER2 cancer receptor. These insertions were combined with novel, minimally-disadvantageous detargeting modifications. The current and accompanying studies teach how to best achieve the clinical-grade cultivation of retargeted oncolytics

Leptin immunoexpression and innervation in rat interscapular brown adipose tissue of cold-acclimated rats: the effects of L-arginine and L-NAME.

The aim of the present study was to explore the effect of nitric oxide on leptin immunoexpression and innervation in interscapular brown adipose tissue (IBAT) of room- and cold- acclimated rats. Animals acclimated both to room-temperature (22 +/- 1 degrees C) and cold (4 +/- 1 degrees C) were treated with L-arginine, a substrate for nitric oxide synthases (NOSs), or N?-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOSs, for 45 days. Leptin expression and localization in brown adipocytes was studied by immunohistochemistry, and innervation stained by the Bodian method. Strong leptin immunopositivity was observed in brown adipocytes cytoplasm of all room-acclimated groups, but nuclear leptin positivity was found only in L-NAME treated rats. In cold-acclimated control and L-NAME treated rats leptin immunopositivity was absent, while L-arginine treatment reversed the cold-induced suppression of leptin expression. Comparing to control, L-arginine, and even more L-NAME, at 22 +/- 1 degrees C induced greater innervation. In conclusion, L-arginine treatment changes leptin expression pattern on cold in rat IBAT

Mechanism of DNA loading by the DNA repair helicase XPD

Funding: Welcome Trust Programme Grant [WT091825MA to M.F.W., J.H.N.]; Wellcome Trust [099149/Z/12/Z]; Royal Society Wolfson Merit Award (to M.F.W., J.H.N.). Funding for open access charge: Wellcome Trust [WT091825MA].The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5′ to 3′ helicase with an essential iron–sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase from Thermoplasma acidophilum has been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD from Sulfolobus acidocaldiarius that lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD.Publisher PDFPeer reviewe

Ekonomski parametri u proizvodnji mleka na poljoprivrednom gazdinstvu

This article presents the economic analysis of two farms A and B for the period 2013-2015. Based on the data for the three years, the income and value of realized production, variable costs and gross margin were calculated, and values were measured per dairy cow for easier comparison. Efficiency in milk production largely depends on the cost of feeding dairy cows, as well on the selling price of milk. These parameters changed in the research period on both farms and significantly impacted the values of realized gross margin.U radu je data ekonomska analiza dva poljoprivredna gazdinstva A i B za period od 2013-2015. godine. Na osnovu praćenih podataka za tri godine izračunati su prihodi i vrednosti ostvarene proizvodnje, varijabilni troškovi i bruto marža, a radi lakše komparacije iskazani su rezultati po muznom grlu. Efikasnost u proizvodnji mleka u velikoj meri zavisi od troškova ishrane muznih krava, kao i prodajne cene mleka, ovi parametri su bili promenljivi u posmatranom periodu na oba gazdinstva i značajno su uticali na ostvarene vrednosti bruto marže