PDB References: insulin, 3i3z; 3i40.Attempts to crystallize the noncovalent complex of recombinant human insulin with polysialic acid were carried out under normal and microgravity conditions. Both crystal types belonged to the same space group, I2 1 3, with unit-cell parameters a = b = c = 77.365 Å , = = = 90.00. The reported space group and unit-cell parameters are almost identical to those of cubic insulin reported in the PDB. The results of X-ray studies confirmed that the crystals obtained were cubic insulin crystals and that they contained no polysialic acid or its fragments. Electron-density maps were calculated using X-ray diffraction sets from earthgrown and microgravity-grown crystals and the three-dimensional structure of the insulin molecule was determined and refined. The conformation and secondary-structural elements of the insulin molecule in different crystal forms were compared.

Radiation therapy is one of the main methods of treating patients with non-small cell lung cancer (NSCLC). However, the resistance of tumor cells to exposure remains the main factor that limits successful therapeutic outcome. To study the molecular/cellular mechanisms of increased resistance of NSCLC to ionizing radiation (IR) exposure, we compared A549 (p53 wild-type) and H1299 (p53-deficient) cells, the two NSCLC cell lines. Using fractionated X-ray irradiation of these cells at a total dose of 60 Gy, we obtained the survived populations and named them A549IR and H1299IR, respectively. Further characterization of these cells showed multiple alterations compared to parental NSCLC cells. The additional 2 Gy exposure led to significant changes in the kinetics of γH2AX and phosphorylated ataxia telangiectasia mutated (pATM) foci numbers in A549IR and H1299IR compared to parental NSCLC cells. Whereas A549, A549IR, and H1299 cells demonstrated clear two-component kinetics of DNA double-strand break (DSB) repair, H1299IR showed slower kinetics of γH2AX foci disappearance with the presence of around 50% of the foci 8 h post-IR. The character of H2AX phosphorylation in these cells was pATM-independent. A decrease of residual γH2AX/53BP1 foci number was observed in both A549IR and H1299IR compared to parental cells post-IR at extra doses of 2, 4, and 6 Gy. This process was accompanied with the changes in the proliferation, cell cycle, apoptosis, and the expression of ATP-binding cassette sub-family G member 2 (ABCG2, also designated as CDw338 and the breast cancer resistance protein (BCRP)) protein. Our study provides strong evidence that different DNA repair mechanisms are activated by multifraction radiotherapy (MFR), as well as single-dose IR, and that the enhanced cellular survival after MFR is reliant on both p53 and 53BP1 signaling along with non-homologous end-joining (NHEJ). Our results are of clinical significance as they can guide the choice of the most effective IR regimen by analyzing the expression status of the p53–53BP1 pathway in tumors and thereby maximize therapeutic benefits for the patients while minimizing collateral damage to normal tissue.

Cancer stem cells (CSCs) play a critical role in the initiation, progression and therapy relapse of many cancers including non-small cell lung cancer (NSCLC). Here, we aimed to address the question of whether the FACS-sorted CSC-like (CD44+ &CD133+) vs. non-CSC (CD44−/CD133− isogenic subpopulations of p53wt A549 and p53null H1299 cells differ in terms of DNA-damage signaling and the appearance of “dormant” features, including polyploidy, which are early markers (predictors) of their sensitivity to genotoxic stress. X-ray irradiation (IR) at 5 Gy provoked significantly higher levels of the ATR-Chk1/Chk2-pathway activity in CD44−/CD133− and CD133+ subpopulations of H1299 cells compared to the respective subpopulations of A549 cells, which only excited ATR-Chk2 activation as demonstrated by the Multiplex DNA-Damage/Genotoxicity profiling. The CD44+ subpopulations did not demonstrate IR-induced activation of ATR, while significantly augmenting only Chk2 and Chk1/2 in the A549- and H1299-derived cells, respectively. Compared to the A549 cells, all the subpopulations of H1299 cells established an increased IR-induced expression of the γH2AX DNA-repair protein. The CD44−/CD133− and CD133+ subpopulations of the A549 cells revealed IR-induced activation of ATR-p53-p21 cell dormancy signaling-mediated pathway, while none of the CD44+ subpopulations of either cell line possessed any signs of such activity. Our data indicated, for the first time, the transcription factor MITF–FAM3C axis operative in p53-deficient H1299 cells, specifically their CD44+ and CD133+ populations, in response to IR, which warrants further investigation. The p21-mediated quiescence is likely the predominant surviving pathway in CD44−/CD133− and CD133+ populations of A549 cells as indicated by single-cell high-content imaging and analysis of Ki67- and EdU-coupled fluorescence after IR stress. SA-beta-galhistology revealed that cellular-stress-induced premature senescence (SIPS) likely has a significant influence on the temporary dormant state of H1299 cells. For the first time, we demonstrated polyploid giant and/or multinucleated cancer-cell (PGCC/MGCC) fractions mainly featuring the progressively augmenting Ki67low phenotype in CD44+ and CD133+ A549 cells at 24–48 h after IR. In contrast, the Ki67high phenotype enrichment in the same fractions of all the sorted H1299 cells suggested an increase in their cycling/heterochromatin reorganization activity after IR stress. Our results proposed that entering the “quiescence” state rather than p21-mediated SIPS may play a significant role in the survival of p53wt CSC-like NSCLC cells after IR. The results obtained are important for the selection of therapeutic schemes for the treatment of patients with NSCLC, depending on the functioning of the p53 system in tumor cells.

Proteins can aggregate in response to stresses, including hyperosmotic shock. Formation and disassembly of aggregates is a relatively slow process. We describe a novel instant response of the cell to hyperosmosis, during which chaperones and other proteins form numerous foci with properties uncharacteristic of classical aggregates. These foci appeared/disappeared seconds after shock onset/removal, in close correlation with cell volume changes. Genome-wide and targeted testing revealed chaperones, metabolic enzymes, P-body components and amyloidogenic proteins in the foci. Most of these proteins can form large assemblies and for some, the assembled state was pre-requisite for participation in foci. A genome-wide screen failed to identify genes whose absence prevented foci participation by Hsp70. Shapes of and interconnections between foci, revealed by super-resolution microscopy, indicated that the foci were compressed between other entities. Based on our findings, we suggest a new model of cytosol architecture as a collection of numerous gel-like regions suspended in a liquid network. This network is reduced in volume in response to hyperosmosis and forms small pockets between the gel-like regions.

We developed a novel asymmetric depth filtration (DF) approach to isolate extracellular vesicles (EVs) from biological fluids that outperforms ultracentrifugation and size‐exclusion chromatography in purity and yield of isolated EVs. By these metrics, a single‐step DF matches or exceeds the performance of multistep protocols with dedicated purification procedures in the isolation of plasma EVs. We demonstrate the selective transit and capture of biological nanoparticles in asymmetric pores by size and elasticity, low surface binding to the filtration medium, and the ability to cleanse EVs held by the filter before their recovery with the reversed flow all contribute to the achieved purity and yield of preparations. We further demonstrate the method's versatility by applying it to isolate EVs from different biofluids (plasma, urine, and cell culture growth medium). The DF workflow is simple, fast, and inexpensive. Only standard laboratory equipment is required for its implementation, making DF suitable for low‐resource and point‐of‐use locations. The method may be used for EV isolation from small biological samples in diagnostic and treatment guidance applications. It can also be scaled up to harvest therapeutic EVs from large volumes of cell culture medium.

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Radiotherapy is a primary treatment modality for patients with unresectable non-small cell lung cancer (NSCLC). Tumor heterogeneity still poses the central question of cancer radioresistance, whether the presence of a particular cell population inside a tumor undergoing a selective outgrowth during radio- and chemotherapy give rise to metastasis and tumor recurrence. In this study, we examined the impact of two different multifraction X-ray radiation exposure (MFR) regimens, fraction dose escalation (FDE) in the split course and the conventional hypofractionation (HF), on the phenotypic and molecular signatures of four MFR-surviving NSCLC cell sublines derived from parental A549 (p53 wild-type) and H1299 (p53-null) cells, namely A549FR/A549HR, H1299FR/H1299HR cells. We demonstrate that sublines surviving different MFR regimens in a total dose of 60 Gy significantly diverge in their molecular traits related to irradiation regimen and p53 status. The observed changes regarding radiosensitivity, transformation, proliferation, metabolic activity, partial epithelial-to-mesenchymal transition (EMT) program activation and 1D confined migratory behavior (wound healing). For the first time, we demonstrated that MFR exposure led to the significant decrease in the expression of p63 and p73, the p53-family members, in p53null cells, which correlated with the increase in cell polyploidy. We could not find significant differences in FRA1 expression between parental cells and their sublines that survived after any MFR regimen regardless of p53 status. In our study, the FDE regimen probably causes partial EMT program activation in MFR-survived NSCLC cells through either Vimentin upregulation in p53null or an aberrant N-cadherin upregulation in p53wt cells. The HF regimen likely less influences the EMT activation irrespectively of the p53 status of MFR-survived NSCLC cells. Our data highlight that both MFR regimens caused overall higher cell transformation of p53null H1299FR and H1299HR cells than their parental H1299 cells. Moreover, our results indicate that the FDE regimen raised the radioresistance and transformation of MFR-surviving NSCLC cells irrespectively of their p53 status, though the HF regimen demonstrated a similar effect on p53null NSCLC cells only. Our data once again emphasize that NSCLC therapy approaches should become more personalized according to radiation therapy (RT) regimen, tumor histology, and molecular status of critical proteins.

BackgroundSmall synthetic molecules provide valuable tools to agricultural biotechnology to circumvent the need for genetic engineering and provide unique benefits to modulate plant growth and development.ResultsWe developed a method to explore molecular mechanisms of plant growth by high-throughput phenotypic screening of haploid populations of pollen cells. These cells rapidly germinate to develop pollen tubes. Compounds acting as growth inhibitors or stimulators of pollen tube growth are identified in a screen lasting not longer than 8 h high-lighting the potential broad applicability of this assay to prioritize chemicals for future mechanism focused investigations in plants. We identified 65 chemical compounds that influenced pollen development. We demonstrated the usefulness of the identified compounds as promotors or inhibitors of tobacco and Arabidopsis thaliana seed growth. When 7 days old seedlings were grown in the presence of these chemicals twenty two of these compounds caused a reduction in Arabidopsis root length in the range from 4.76 to 49.20 % when compared to controls grown in the absence of the chemicals. Two of the chemicals sharing structural homology with thiazolidines stimulated root growth and increased root length by 129.23 and 119.09 %, respectively. The pollen tube growth stimulating compound (S-02) belongs to benzazepin-type chemicals and increased Arabidopsis root length by 126.24 %.ConclusionsIn this study we demonstrate the usefulness of plant pollen tube based assay for screening small chemical compound libraries for new biologically active compounds. The pollen tubes represent an ultra-rapid screening tool with which even large compound libraries can be analyzed in very short time intervals. The broadly applicable high-throughput protocol is suitable for automated phenotypic screening of germinating pollen resulting in combination with seed germination assays in identification of plant growth inhibitors and stimulators.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0875-4) contains supplementary material, which is available to authorized users.