In Silico Design of Fusion Toxin DT389GCSF and a Comparative Study

Background: Chemotherapy and radiotherapy have negative effects on normal tissues and they are very expensive and lengthy treatments. These disadvantages have recently attracted researchers to the new methods that specifically affect cancerous tissues and have lower damage to normal tissues. One of these methods is the use of intelligent recombinant fusion toxin. The fusion toxin DTGCSF, which consists of linked Diphtheria Toxin (DT) and Granulocyte Colony Stimulate Factor (GCSF), was first studied by Chadwick et al. in 1993 where HATPL linker provided the linking sequence between GCSF and the 486 amino acid sequences of DT. Methods: In this study, the fusion toxin DT389GCSF is evaluated for functional structure in silico. With the idea of the commercial fusion toxin of Ontak, the DT in this fusion protein is designed incomplete for 389 amino acids and is linked to the beginning of the GCSF cytokine via the SG4SM linker (DT389GCSF). The affinity of the DT389GCSF as a ligand with GCSF-R as receptor was compared with DT486GCSF as a ligand with GCSF-R as receptor. Both DT486GCSF and its receptor GCSF-R have been modeled by Easy Modeler2 software. Our fusion protein (DT389GCSF) and GCSF-R are modeled through Modeller software; all of the structures were confirmed by server MDWEB and VMD software. Then, the interaction studies between two proteins are done using protein-protein docking (HADDOCK 2.2 web server) for both the fusion protein in this study and DT486GCSF. Results: The HADDOCK results demonstrate that the interaction of DT389GCSF with GCSF-R is very different and has a more powerful interaction than DT486GCSF with GCSF-R. Conclusion: HADDOCK web server is operative tools for evaluation of protein–protein interactions, therefore, in silico study of DT389GCSF will help with studying the function and the structure of these molecules. Moreover, DT389GCSF may have important new therapeutic applications.

Regulation of Coagulation by Polyphosphate

Polyphosphate (polyP) is a biomolecule comprised of repeating units of inorganic phosphate residues linked by energy-rich phosphoanhydride bonds. PolyP is highly conserved throughout evolution and plays a vital role in the survival of prokaryotes and single-cell eukaryotes1 by functioning in processes such as motility, virulence and stress. Its role in mammalian systems was less well defined, however, identification of polyP in the dense granules of human platelets2 prompted analysis of the function of polyP in hemostasis. Stimulation of platelets by conventional agonists such as ADP, thrombin and collagen induces release of polyP, and promotes coagulation by acting at several points in the cascade3. These include 1) providing a surface for activation of the contact pathway protein factor XII, 2) augmenting conversion of factor V to factor Va which participates in the prothrombinase complex and 3) accelerating thrombin-mediated activation of factor XI4. The net outcome is a reduction in the lag time to thrombin formation thereby driving clot formation. The effect of polyP on thrombin generation has down-stream repercussions in terms of clot breakdown, as it augments the early activation of thrombin activatable fibrinolysis inhibitor (TAFI). There has been a degree of uncertainty and debate over whether polyP is capable of reaching adequate concentrations within the plasma milieu to elicit these profound effects on haemostasis. However, the observations that polyP can bind directly to fibrin5, allowing it to be incorporated into the forming clot, localise the biomolecule to its site of action. In addition, binding alters the structure of the fibrin clot altering its susceptibility to degradation by fibrinolytic enzymes6. These observations led to the hypothesis that polyP operates as a timed switch, being released from platelets upon activation thereby allowing rapid repair of an injury while simultaneously preventing clot breakdown until sufficient wound healing has occurred3. The ability of polyP to promote FXII-dependent coagulation has been reported in several studies but has been contested, as shorter soluble polymers, such as those found in platelets, are relatively inefficient in this process. Recent work from our group7 and others8 documents the retention of polyP on the surface of degranulated platelets. This pool of polyP is of longer chain length than the soluble secreted form and is complexed to divalent metal ions8 thereby augmenting its ability to stimulate factor XII activation. These studies highlight the potential that polyP may exert its biological cofactor activity when anchored to a surface such as platelets, fibrin and potentially other blood cells. Many questions remain as to how this polymer regulates its plethora of functions and whether the binding surface impacts on the target reaction and the half-life of the polymer. However, taken together these in vitro and in vivo studies have delineated a role for polyP in hemostasis and highlight the requirement for more efficient tools to quantify this interesting biomolecule in subsets of patients. References Kornberg A. Inorganic polyphosphate: a molecule of many functions. Prog Mol Subcell Biol. 1999;23:1-18. Ruiz FA, Lea CR, Oldfield E, Docampo R. Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. J Biol Chem. 2004;279(43):44250-44257. Smith SA, Mutch NJ, Baskar D, Rohloff P, Docampo R, Morrissey JH. Polyphosphate modulates blood coagulation and fibrinolysis. Proc Natl Acad Sci U S A. 2006;103(4):903-908. Choi SH, Smith SA, Morrissey JH. Polyphosphate is a cofactor for the activation of factor XI by thrombin. Blood. 2011;118(26):6963-6970. Whyte CS, Chernysh IN, Domingues MM, Connell S, Weisel JW, Ariens RA, Mutch NJ. Polyphosphate delays fibrin polymerisation and alters the mechanical properties of the fibrin network. Thromb Haemost. 2016;116(5):897-903. Mutch NJ, Engel R, Uitte de Willige S, Philippou H, Ariens RA. Polyphosphate modifies the fibrin network and down-regulates fibrinolysis by attenuating binding of tPA and plasminogen to fibrin. Blood. 2010;115(19):3980-3988. Mitchell JL, Lionikiene AS, Georgiev G, Klemmer A, Brain C, Kim PY, Mutch NJ. Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity. Blood. 2016;128(24):2834-2845. Verhoef JJ, Barendrecht AD, Nickel KF, Dijkxhoorn K, Kenne E, Labberton L, McCarty OJ, Schiffelers R, Heijnen HF, Hendrickx AP, Schellekens H, Fens MH, de Maat S, Renne T, Maas C. Polyphosphate nanoparticles on the platelet surface trigger contact system activation. Blood. 2017;129(12):1707-1717. Disclosures No relevant conflicts of interest to declare.

Energy-efficient house: economic, ecological and social justification in Ukrainian conditions

The main goal of the article is the efficiency justification of energy-efficient house (EEH) from the different points of view: economic, ecological and social. In this case, the EEH under the green economy context was considered by the authors. In addition, according to the Ukrainian ongoing condition, the key preconditions of EEH implementation among the Ukrainian households were allocated. Besides, the main approaches to define EEH are analyzed and systematized by the authors. On this basis, the main bullet points and features of EEH were indicated. The authors determined the EEH opportunities for spreading among the Ukrainian households. It should be noted, that the lack of awareness among the civil society provokes the slow temp of the EEH enlarging in Ukraine. At the same time, the European experience showed that the huge part of their households can be characterized as energy-effective. With the purpose of understand the efficiency of EEH, the authors had estimated the economic benefits of installed solar collector in the household as one of the parts of EEH. According to the results, the authors allocate the restraining factors of the EEH spreading in Ukraine. Thus, the great payback period is one of them. In addition, the high level of the currency rate has negative impact on the payback period. From the other side the continuously increasing of the utility bills have been indicated as a negative stimulate factor. In order to increase the awareness of the EEH benefits under the Ukrainian civil society, the main economic, ecological and social benefits of EEH were systematized by the authors.

Multiplication of Ribosomal P-Stalk Proteins Contributes to the Fidelity of Translation

ABSTRACT The P-stalk represents a vital element within the ribosomal GTPase-associated center, which represents a landing platform for translational GTPases. The eukaryotic P-stalk exists as a uL10-(P1-P2)2 pentameric complex, which contains five identical C-terminal domains, one within each protein, and the presence of only one such element is sufficient to stimulate factor-dependent GTP hydrolysis in vitro and to sustain cell viability. The functional contribution of the P-stalk to the performance of the translational machinery in vivo, especially the role of P-protein multiplication, has never been explored. Here, we show that ribosomes depleted of P1/P2 proteins exhibit reduced translation fidelity at elongation and termination steps. The elevated rate of the decoding error is inversely correlated with the number of the P-proteins present on the ribosome. Unexpectedly, the lack of P1/P2 has little effect in vivo on the efficiency of other translational GTPase (trGTPase)-dependent steps of protein synthesis, including translocation. We have shown that loss of accuracy of decoding caused by P1/P2 depletion is the major cause of translation slowdown, which in turn affects the metabolic fitness of the yeast cell. We postulate that the multiplication of P-proteins is functionally coupled with the qualitative aspect of ribosome action, i.e., the recoding phenomenon shaping the cellular proteome.

Tissue factor-dependent activation of tritium-labeled factor IX and factor X in human plasma

Recent investigations have suggested that the activation of factor IX by factor VII/tissue factor may be an important alternative route to the generation of factor Xa. Accordingly, we have compared the tissue factor-dependent activation of tritium-labeled factor IX and factor X in a human plasma system and have studied the role of proteases known to stimulate factor VII activity. Plasma was defibrinated by heating and depleted of its factors IX and X by passing it through antibody columns. Addition of human brain thromboplastin, Ca2+, and purified 3H- labeled factor X to the plasma resulted, after a short lag, in burst- like activation of the factor X, measured as the release of radiolabeled activation peptide. The progress of activation was slowed by both heparin and a specific inhibitor of factor Xa, suggesting a feedback role for this enzyme, but factor X activation could not be completely abolished by such inhibitors. In the case of 3H-factor IX activation, the rate also increased for approximately 3 min after addition of thromboplastin, but was not subsequently curtailed. A survey of proteases implicated as activators of factor VII in other settings showed that both factor Xa and (to a much smaller extent) factor IXa could accelerate the activation of factor IX. However, factor Xa was unique in obliterating activation when present at concentrations greater than approximately 1 nM. Heparin inhibited the tissue factor-dependent activation of factor IX almost completely, apparently through the effect of antithrombin on the feedback reactions of factors Xa and IXa on factor VII. These results suggest that a very tight, biphasic control of factor VII activity exists in human plasma, which is modulated mainly by factor Xa. Variation of the factor IX or factor X concentrations permitted kinetic parameters for each activation to be derived. At saturation of factor VIIa/tissue factor, factor IX activation was significantly more rapid than was previously found in bovine plasma under similar conditions. The activation of factor X at saturation was slightly more rapid than in bovine plasma, despite the presence of heparin.

Tissue factor-dependent activation of tritium-labeled factor IX and factor X in human plasma

Recent investigations have suggested that the activation of factor IX by factor VII/tissue factor may be an important alternative route to the generation of factor Xa. Accordingly, we have compared the tissue factor-dependent activation of tritium-labeled factor IX and factor X in a human plasma system and have studied the role of proteases known to stimulate factor VII activity. Plasma was defibrinated by heating and depleted of its factors IX and X by passing it through antibody columns. Addition of human brain thromboplastin, Ca2+, and purified 3H- labeled factor X to the plasma resulted, after a short lag, in burst- like activation of the factor X, measured as the release of radiolabeled activation peptide. The progress of activation was slowed by both heparin and a specific inhibitor of factor Xa, suggesting a feedback role for this enzyme, but factor X activation could not be completely abolished by such inhibitors. In the case of 3H-factor IX activation, the rate also increased for approximately 3 min after addition of thromboplastin, but was not subsequently curtailed. A survey of proteases implicated as activators of factor VII in other settings showed that both factor Xa and (to a much smaller extent) factor IXa could accelerate the activation of factor IX. However, factor Xa was unique in obliterating activation when present at concentrations greater than approximately 1 nM. Heparin inhibited the tissue factor-dependent activation of factor IX almost completely, apparently through the effect of antithrombin on the feedback reactions of factors Xa and IXa on factor VII. These results suggest that a very tight, biphasic control of factor VII activity exists in human plasma, which is modulated mainly by factor Xa. Variation of the factor IX or factor X concentrations permitted kinetic parameters for each activation to be derived. At saturation of factor VIIa/tissue factor, factor IX activation was significantly more rapid than was previously found in bovine plasma under similar conditions. The activation of factor X at saturation was slightly more rapid than in bovine plasma, despite the presence of heparin.