A fast dynamic mode of the EF‐G‐bound ribosome

A key intermediate in translocation is an ‘unlocked state’ of the pre‐translocation ribosome in which the P‐site tRNA adopts the P/E hybrid state, the L1 stalk domain closes and ribosomal subunits adopt a ratcheted configuration. Here, through two‐ and three‐colour smFRET imaging from multiple structural perspectives, EF‐G is shown to accelerate structural and kinetic pathways in the ribosome, leading to this transition. The EF‐G‐bound ribosome remains highly dynamic in nature, wherein, the unlocked state is transiently and reversibly formed. The P/E hybrid state is energetically favoured, but exchange with the classical P/P configuration persists; the L1 stalk adopts a fast dynamic mode characterized by rapid cycles of closure and opening. These data support a model in which P/E hybrid state formation, L1 stalk closure and subunit ratcheting are loosely coupled, independent processes that must converge to achieve the unlocked state. The highly dynamic nature of these motions, and their sensitivity to conformational and compositional changes in the ribosome, suggests that regulating the formation of this intermediate may present an effective avenue for translational control.     

Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells,with Limited Impact on Cancer Hallmark Functions

Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca²⁺ activated monovalent cation channel that contributes to the pathophysiology of several diseases.For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells.Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays.To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na⁺. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype.In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells.     

Biosensors based on peptide exposure show single molecule conformations in live cells

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Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Abstract Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. THe immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica . The monoclonal antibody belonged to IgG₁ isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant ( P < 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant ( P < 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant ( P < 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

Determination of field output correction factors of radiophotoluminescence glass dosimeter and CC01 ionization chamber and validation against IAEA-AAPM TRS-483 code of practice

PurposeTo determine the field output correction factors of the radiophotoluminescence glass dosimeter (RPLGD) in parallel and perpendicular orientations with reference to CC01, the ionization chamber.MethodsThe dose to a small water volume and the sensitive volume of the RPLGD and the IBA-CC01 were determined for 6-MV, 100-cm SAD, 10-cm depth using egs_chamber user-code. The RPLGD in perpendicular and parallel orientations to the beam axis were studied. The field output correction factors of each detector for 0.5 × 0.5 to 10 × 10 cm² field sizes were determined. These field output correction factors were validated by comparing field output factors against data determined from IAEA-AAPM TRS-483 code of practice.ResultsThe field output correction factors of all detectors were within 5% for field sizes down to 0.8 × 0.8 cm². For 0.5 × 0.5 cm², the field output correction factors of CC01, RPLGD in perpendicular and parallel orientations differed from unity by 14%, 19%, and 5%, respectively. The percentage difference between field output factors determined using RPLGD and CC01 data, corrected using the field output correction factors determined in this work and measurements with CC01 data corrected using TRS-483, was less than 3% for all field sizes, except for the smallest field size of RPLGD in perpendicular orientation and the CC01.ConclusionsThe field output correction factors of RPLGD and CC01 are reported. The validation proves that RPLGD in parallel orientation combined with the field output correction factors is the most suitable for determining the field output factors for the smallest field used in this study.     

Population dynamics of two small pelagic fish in the central-south area off Chile: delayed density-dependence and biological interaction

It is widely believed that environmental variability is the main cause for fluctuations in commercially exploited small pelagic fish populations around the world. Nevertheless, density-dependent factors also can drive population dynamics. In this paper, we analyzed thirteen years of a relative abundance index of two clupeoids fish populations coexisting in the central-south area off Chile, namely the common sardine, Strangomera bentincki, and anchovy, Engraulis ringens. We applied the classical diagnostic tools of time series analysis to the observed time-series. Also, the realized per capita population growth rate was studied with the aim of detecting the feedback structure that is characterizing the population dynamics of the two species. The analysis suggests that population fluctuations of the two species have an important density-dependent component, displaying first-order (direct density-dependent) and second-order (delayed density-dependent) simultaneously. The density-dependent component explained 70.5 and 55.6 % of the realized per capita population growth rate of common sardine and anchovy, respectively. The deterministic skeleton model showed an asymptotic convergence to equilibrium density. In presence of a stochastic environment, fluctuations were reproduced for the species showing a component of fluctuation with a period of 4 year. The intrinsic dynamics of each species is typical of interacting species resulting from trophic interactions. It is postulated that the second-order dynamics of S. bentincki and E. ringens in central-south Chile, may be the result from interactions with a specialist predator (the fishing fleet), interacting with exogenous environmental factors.     

Reaction of small heat-shock proteins to different kinds of cellular stress in cultured rat hippocampal neurons

Upregulation of small heat-shock proteins (sHsps) in response to cellular stress is one mechanism to increase cell viability. We previously described that cultured rat hippocampal neurons express five of the 11 family members but only upregulate two of them (HspB1 and HspB5) at the protein level after heat stress. Since neurons have to cope with many other pathological conditions, we investigated in this study the expression of all five expressed sHsps on mRNA and protein level after sublethal sodium arsenite and oxidative and hyperosmotic stress. Under all three conditions, HspB1, HspB5, HspB6, and HspB8 but not HspB11 were consistently upregulated but showed differences in the time course of upregulation. The increase of sHsps always occurred earlier on mRNA level compared with protein levels. We conclude from our data that these four upregulated sHsps (HspB1, HspB5, HspB6, HspB8) act together in different proportions in the protection of neurons from various stress conditions.     

Estimation of extremely small field radiation dose for brain stereotactic radiotherapy using the Vero4DRT system

The purpose of this study was a dosimetric validation of the Vero4DRT for brain stereotactic radiotherapy (SRT) with extremely small fields calculated by the treatment planning system (TPS) iPlan (Ver.4.5.1; algorithm XVMC). Measured and calculated data (e.g. percentage depth dose [PDD], dose profile, and point dose) were compared for small square fields of 30 × 30, 20 × 20, 10 × 10 and 5 × 5 mm² using ionization chambers of 0.01 or 0.04 cm³ and a diamond detector. Dose verifications were performed using an ionization chamber and radiochromic film (EBT3; the equivalent field sizes used were 8.2, 8.7, 8.9, 9.5, and 12.9 mm²) for five brain SRT cases irradiated with dynamic conformal arcs.The PDDs and dose profiles for the measured and calculated data were in good agreement for fields larger than or equal to 10 × 10 mm² when an appropriate detector was chosen. The dose differences for point doses in fields of 30 × 30, 20 × 20, 10 × 10 and 5 × 5 mm² were +0.48%, +0.56%, −0.52%, and +11.2% respectively. In the dose verifications for the brain SRT plans, the mean dose difference between the calculated and measured doses were −0.35% (range, −0.94% to +0.47%), with the average pass rates for the gamma index under the 3%/2 mm criterion being 96.71%, 93.37%, and 97.58% for coronal, sagittal, and axial planes respectively.The Vero4DRT system provides accurate delivery of radiation dose for small fields larger than or equal to 10 × 10 mm².