Identifying allosteric fluctuation transitions between different protein conformational states as applied to Cyclin Dependent Kinase 2

BACKGROUND: The mechanisms underlying protein function and associated conformational change are dominated by a series of local entropy fluctuations affecting the global structure yet are mediated by only a few key residues. Transitional Dynamic Analysis (TDA) is a new method to detect these changes in local protein flexibility between different conformations arising from, for example, ligand binding. Additionally, Positional Impact Vertex for Entropy Transfer (PIVET) uses TDA to identify important residue contact changes that have a large impact on global fluctuation. We demonstrate the utility of these methods for Cyclin-dependent kinase 2 (CDK2), a system with crystal structures of this protein in multiple functionally relevant conformations and experimental data revealing the importance of local fluctuation changes for protein function. RESULTS: TDA and PIVET successfully identified select residues that are responsible for conformation specific regional fluctuation in the activation cycle of Cyclin Dependent Kinase 2 (CDK2). The detected local changes in protein flexibility have been experimentally confirmed to be essential for the regulation and function of the kinase. The methodologies also highlighted possible errors in previous molecular dynamic simulations that need to be resolved in order to understand this key player in cell cycle regulation. Finally, the use of entropy compensation as a possible allosteric mechanism for protein function is reported for CDK2. CONCLUSION: The methodologies embodied in TDA and PIVET provide a quick approach to identify local fluctuation change important for protein function and residue contacts that contributes to these changes. Further, these approaches can be used to check for possible errors in protein dynamic simulations and have the potential to facilitate a better understanding of the contribution of entropy to protein allostery and function

The triple combination of tenofovir, emtricitabine and efavirenz shows synergistic anti-HIV-1 activity in vitro: a mechanism of action study

<p>Abstract</p> <p>Background</p> <p>Tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and efavirenz (EFV) are the three components of the once-daily, single tablet regimen (Atripla) for treatment of HIV-1 infection. Previous cell culture studies have demonstrated that the double combination of tenofovir (TFV), the parent drug of TDF, and FTC were additive to synergistic in their anti-HIV activity, which correlated with increased levels of intracellular phosphorylation of both compounds.</p> <p>Results</p> <p>In this study, we demonstrated the combinations of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV synergistically inhibit HIV replication in cell culture and synergistically inhibit HIV-1 reverse transcriptase (RT) catalyzed DNA synthesis in biochemical assays. Several different methods were applied to define synergy including median-effect analysis, MacSynergy^®II and quantitative isobologram analysis. We demonstrated that the enhanced formation of dead-end complexes (DEC) by HIV-1 RT and TFV-terminated DNA in the presence of FTC-triphosphate (TP) could contribute to the synergy observed for the combination of TFV+FTC, possibly through reduced terminal NRTI excision. Furthermore, we showed that EFV facilitated efficient formation of stable, DEC-like complexes by TFV- or FTC-monophosphate (MP)-terminated DNA and this can contribute to the synergistic inhibition of HIV-1 RT by TFV-diphosphate (DP)+EFV and FTC-TP+EFV combinations.</p> <p>Conclusion</p> <p>This study demonstrated a clear correlation between the synergistic antiviral activities of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV combinations and synergistic HIV-1 RT inhibition at the enzymatic level. We propose the molecular mechanisms for the TFV+FTC+EFV synergy to be a combination of increased levels of the active metabolites TFV-DP and FTC-TP and enhanced DEC formation by a chain-terminated DNA and HIV-1 RT in the presence of the second and the third drug in the combination. This study furthers the understanding of the longstanding observations of synergistic anti-HIV-1 effects of many NRTI+NNRTI and certain NRTI+NRTI combinations in cell culture, and provides biochemical evidence that combinations of anti-HIV agents can increase the intracellular drug efficacy, without increasing the extracellular drug concentrations.</p

Am Anfang war die Kohle 125 Jahre Harpener Aktiengesellschaft

Bibliothek Weltwirtschaft Kiel A149,400 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Factors Influencing the Movement Biology of Migrant Songbirds Confronted With an Ecological Barrier

Whether or not a migratory songbird embarks on a long-distance flight across an ecological barrier is likely a response to a number of endogenous and exogenous factors. During autumn 2008 and 2009, we used automated radio tracking to investigate how energetic condition, age, and weather influenced the departure timing and direction of Swainson’s thrushes (Catharus ustulatus) during migratory stopover along the northern coast of the Gulf of Mexico. Most birds left within 1 h after sunset on the evening following capture. Those birds that departed later on the first night or remained longer than 1 day were lean. Birds that carried fat loads sufficient to cross the Gulf of Mexico generally departed in a seasonally appropriate southerly direction, whereas lean birds nearly always flew inland in a northerly direction. We did not detect an effect of age or weather on departures. The decision by lean birds to reorient movement inland may reflect the suitability of the coastal stopover site for deposition of fuel stores and the motivation to seek food among more extensive forested habitat away from the barrier

Image1_Single cell phototransfection of mRNAs encoding SARS-CoV2 spike and nucleocapsid into human astrocytes results in RNA dependent translation interference.TIF

Multi-RNA co-transfection is starting to be employed to stimulate immune responses to SARS-CoV-2 viral infection. While there are good reasons to utilize such an approach, there is little background on whether there are synergistic RNA-dependent cellular effects. To address this issue, we use transcriptome-induced phenotype remodeling (TIPeR) via phototransfection to assess whether mRNAs encoding the Spike and Nucleocapsid proteins of SARS-CoV-2 virus into single human astrocytes (an endogenous human cell host for the virus) and mouse 3T3 cells (often used in high-throughput therapeutic screens) synergistically impact host cell biologies. An RNA concentration-dependent expression was observed where an increase of RNA by less than 2-fold results in reduced expression of each individual RNAs. Further, a dominant inhibitory effect of Nucleocapsid RNA upon Spike RNA translation was detected that is distinct from codon-mediated epistasis. Knowledge of the cellular consequences of multi-RNA transfection will aid in selecting RNA concentrations that will maximize antigen presentation on host cell surface with the goal of eliciting a robust immune response. Further, application of this single cell stoichiometrically tunable RNA functional genomics approach to the study of SARS-CoV-2 biology promises to provide details of the cellular sequalae that arise upon infection in anticipation of providing novel targets for inhibition of viral replication and propagation for therapeutic intervention.</p

Changes in the interfacial tension of chlorinated solvents following flow through UK soils and shallow aquifer material

The interfacial tension (IFT) that arises at the interface between water and an immiscible organic liquid is a key parameter affecting the transport and subsequent fate of the organic liquid in water-saturated porous media. In this paper, data are presented that show how contact between a range of soil types and chlorinated hydrocarbon solvent (CHS) dense nonaqueous phase liquids (DNAPLs) can affect DNAPL/water IFT values. The soils examined are indicative of U.K. soil types and shallow aquifer materials. The solvents investigated were tetrachloroethylene (PCE) and trichloroethylene (TCE). Lab grade, recovered field DNAPL and industrial waste chlorinated solvent mixtures were used. The data from batch and column experiments invariably revealed that water/DNAPL IFT values change following contact with unsaturated soils. In the majority of cases, the IFT values increase following soil exposure. However, after contact with an organic-rich soil, the I FT of the lab grade solvents decreased. The experimental evidence suggests that these reductions are linked to the removal of organic material from the soil and its subsequent incorporation into the solvent. IFT increases in the case of lab solvents are shown to be linked to the removal of stabilizers (added by the manufacturers to obviate degradation) that are removed by adsorption to soil mineral surfaces. Similarly, it is conjectured that adsorption of surface-active compounds from the industrial waste samples to soil surfaces is responsible for increases in the IFT in these samples. Finally, it was observed that invading CHSs are capable of dissolving and subsequently mobilizing in-situ soil contaminants. GC/MS analysis revealed these mobilized soil contaminants to be polyaromatic hydrocarbons and phthalate esters