Biomolecular imaging and electronic damage using X-ray free-electron lasers

Proposals to determine biomolecular structures from diffraction experiments using femtosecond X-ray free-electron laser (XFEL) pulses involve a conflict between the incident brightness required to achieve diffraction-limited atomic resolution and the electronic and structural damage induced by the illumination. Here we show that previous estimates of the conditions under which biomolecular structures may be obtained in this manner are unduly restrictive, because they are based on a coherent diffraction model that is not appropriate to the proposed interaction conditions. A more detailed imaging model derived from optical coherence theory and quantum electrodynamics is shown to be far more tolerant of electronic damage. The nuclear density is employed as the principal descriptor of molecular structure. The foundations of the approach may also be used to characterize electrodynamical processes by performing scattering experiments on complex molecules of known structure.Comment: 16 pages, 2 figure

Effects of radiation damage and inelastic scattering on single-particle imaging of hydrated proteins with an X-ray Free-Electron Laser

We present a computational case study of X-ray single-particle imaging of hydrated proteins on an example of 2-Nitrogenase–Iron protein covered with water layers of various thickness, using a start-to-end simulation platform and experimental parameters of the SPB/SFX instrument at the European X-ray Free-Electron Laser facility. The simulations identify an optimal thickness of the water layer at which the effective resolution for imaging the hydrated sample becomes significantly higher than for the non-hydrated sample. This effect is lost when the water layer becomes too thick. Even though the detailed results presented pertain to the specific sample studied, the trends which we identify should also hold in a general case. We expect these findings will guide future single-particle imaging experiments using hydrated proteins

Cover to Volume 3

The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-κB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth

Epidermal neural crest stem cell transplantation as a promising therapeutic strategy for ischemic stroke

Introduction: Cell-based therapy is considered as promising strategy to cure stroke. However, employing appropriate type of stem cell to fulfill many therapeutic needs of cerebral ischemia is still challenging. In this regard, the current study was designed to elucidate therapeutic potential of epidermal neural crest stem cells (EPI-NCSCs) compared to bone marrow mesenchymal stem cells (BM-MSCs) in rat model of ischemic stroke. Methods: Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) for 45 minutes. Immediately after reperfusion, EPI-NCSCs or BM-MSCs were transplanted via intra-arterial or intravenous route. A test for neurological function was performed before ischemia and 1, 3, and 7 days after MCAO. Also, infarct volume ratio and relative expression of 15 selected target genes were evaluated 7 days after transplantation. Results: EPI-NCSCs transplantation (both intra-arterial and intravenous) and BM-MSCs transplantation (only intra-arterial) tended to result in a better functional outcome, compared to the MCAO group; however, this difference was not statistically significant. The infarct volume ratio significantly decreased in NCSC-intra-arterial, NCSC-intravenous and MSC-intra-arterial groups compared to the control. EPI-NCSCs interventions led to higher expression levels of Bdnf, nestin, Sox10, doublecortin, β-III tubulin, Gfap, and interleukin-6, whereas neurotrophin-3 and interleukin-10 were decreased. On the other hand, BM-MSCs therapy resulted in upregulation of Gdnf, β-III tubulin, and Gfap and down-regulation of neurotrophin-3, interleukin-1, and interleukin-10. Conclusion: These findings highlight the therapeutic effects of EPI-NCSCs transplantation, probably through simultaneous induction of neuronal and glial formation, as well as Bdnf over-expression in a rat model of ischemic strok

Sex differences in in-hospital mortality following a first acute myocardial infarction: Symptomatology, delayed presentation, and hospital setting

Background: Women generally wait longer than men prior to seeking treatment for acute myocardial infarction (AMI). They are more likely to present with atypical symptoms, and are less likely to be admitted to coronary or intensive care units (CCU or ICU) compared to similarly-aged males. Women are more likely to die during hospital admission. Sex differences in the associations of delayed arrival, admitting ward, and mortality have not been thoroughly investigated. Methods: Focusing on presenting symptoms and time of presentation since symptom onset, we evaluated sex differences in in-hospital mortality following a first AMI in 4859 men and women presenting to three emergency departments (ED) from December 2008 to February 2014. Sex-specific risk of mortality associated with admission to either CCU/ICU or medical wards was calculated after adjusting for age, socioeconomic status, triage-assigned urgency of presentation, blood pressure, heart rate, presenting symptoms, timing of presentation since symptom onset, and treatment in the ED. Sex-specific age-adjusted attributable risks were calculated.Results: Compared to males, females waited longer before seeking treatment, presented more often with atypical symptoms, and were less likely to be admitted to CCU or ICU. Age-adjusted mortality in CCU/ICU or medical wards was higher among females (3.1 and 4.9 % respectively in CCU/ICU and medical wards in females compared to 2.6 and 3.2 % in males). However, after adjusting for variation in presenting symptoms, delayed arrival and other risk factors, risk of death was similar between males and females if they were admitted to CCU or ICU. This was in contrast to those admitted to medical wards. Females admitted to medical wards were 89 % more likely to die than their male counterparts. Arriving in the ED within 60 min of onset of symptoms was not associated with in-hospital mortality. Among males, 2.2 % of in-hospital mortality was attributed to being admitted to medical wards rather than CCU or ICU, while for females this age-adjusted attributable risk was 4.1 %. Conclusions: Our study stresses the need to reappraise decision making in patient selection for admission to specialised care units, whilst raising awareness of possible sex-related bias in management of patients diagnosed with an AMI

X-ray multiphoton-induced Coulomb explosion images complex single molecules

Following structural dynamics in real time is a fundamental goal towards a better understanding of chemical reactions. Recording snapshots of individual molecules with ultrashort exposure times is a key ingredient towards this goal, as atoms move on femtosecond (10-15 s) timescales. For condensed-phase samples, ultrafast, atomically resolved structure determination has been demonstrated using X-ray and electron diffraction. Pioneering experiments have also started addressing gaseous samples. However, they face the problem of low target densities, low scattering cross sections and random spatial orientation of the molecules. Therefore, obtaining images of entire, isolated molecules capturing all constituents, including hydrogen atoms, remains challenging. Here we demonstrate that intense femtosecond pulses from an X-ray free-electron laser trigger rapid and complete Coulomb explosions of 2-iodopyridine and 2-iodopyrazine molecules. We obtain intriguingly clear momentum images depicting ten or eleven atoms, including all the hydrogens, and thus overcome a so-far impregnable barrier for complete Coulomb explosion imaging—its limitation on molecules consisting of three to five atoms. In combination with state-of-the-art multi-coincidence techniques and elaborate theoretical modelling, this allows tracing ultrafast hydrogen emission and obtaining information on the result of intramolecular electron rearrangement. Our work represents an important step towards imaging femtosecond chemistry via Coulomb explosion

Characterization of megahertz X ray laser beams by multishot desorption imprints in PMMA

Proper diagnostics of intense free electron laser FEL X ray pulses is indisputably important for experimental data analysis as well as for the protection of beamline optical elements. New challenges for beam diagnostic methods are introduced by modern FEL facilities capable of delivering powerful pulses at megahertz MHz repetition rates. In this paper, we report the first characterization of a defocused MHz 13.5 nm beam generated by the free electron laser in Hamburg FLASH using the method of multi pulse desorption imprints in poly methyl methacrylate PMMA . The beam fluence profile is reconstructed in a novel and highly accurate way that takes into account the nonlinear response of material removal to total dose delivered by multiple pulses. The algorithm is applied to experimental data of single shot ablation imprints and multi shot desorption imprints at both low 10 Hz and high 1 MHz repetition rates. Reconstructed response functions show a great agreement with the theoretical desorption response function mode

Ensemble and fuzzy techniques applied to imbalanced traffic congestion datasets a comparative study

Class imbalance is among the most persistent complications which may confront the traditional supervised learning task in real-world applications. Among the different kind of classification problems that have been studied in the literature, the imbalanced ones, particularly those that represents real-world problems, have attracted the interest of many researchers in recent years. In order to face this problems, different approaches have been used or proposed in the literature, between then, soft computing and ensemble techniques. In this work, ensembles and fuzzy techniques have been applied to real-world traffic datasets in order to study their performance in imbalanced real-world scenarios. KEEL platform is used to carried out this study. The results show that different ensemble techniques obtain the best results in the proposed datasets. Document type: Part of book or chapter of boo

Joint Effects of Febrile Acute Infection and an Interferon-γ Polymorphism on Breast Cancer Risk

BACKGROUND: There is an inverse relationship between febrile infection and the risk of malignancies. Interferon gamma (IFN-γ) plays an important role in fever induction and its expression increases with incubation at fever-range temperatures. Therefore, the genetic polymorphism of IFN-γ may modify the association of febrile infection with breast cancer risk. METHODOLOGY AND PRINCIPAL FINDINGS: Information on potential breast cancer risk factors, history of fever during the last 10 years, and blood specimens were collected from 839 incident breast cancer cases and 863 age-matched controls between October 2008 and June 2010 in Guangzhou, China. IFN-γ (rs2069705) was genotyped using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry platform. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated using multivariate logistic regression. We found that women who had experienced ≥1 fever per year had a decreased risk of breast cancer [ORs and 95% CI: 0.77 (0.61-0.99)] compared to those with less than one fever a year. This association only occurred in women with CT/TT genotypes [0.54 (0.37-0.77)] but not in those with the CC genotype [1.09 (0.77-1.55)]. The association of IFN-γ rs2069705 with the risk of breast cancer was not significant among all participants, while the CT/TT genotypes were significantly related to an elevated risk of breast cancer [1.32 (1.03-1.70)] among the women with <1 fever per year and to a reduced risk of breast cancer [0.63 (0.40-0.99)] among women with ≥1 fever per year compared to the CC genotype. A marked interaction between fever frequencies and the IFN-γ genotypes was observed (P for multiplicative and additive interactions were 0.005 and 0.058, respectively). CONCLUSIONS: Our findings indicate a possible link between febrile acute infection and a decreased risk of breast cancer, and this association was modified by IFN-γ rs2069705

Chronic oxytocin-driven alternative splicing of Crfr2α induces anxiety

The neuropeptide oxytocin (OXT) has generated considerable interest as potential treatment for psychiatric disorders, including anxiety and autism spectrum disorders. However, the behavioral and molecular consequences associated with chronic OXT treatment and chronic receptor (OXTR) activation have scarcely been studied, despite the potential therapeutic long-term use of intranasal OXT. Here, we reveal that chronic OXT treatment over two weeks increased anxiety-like behavior in rats, with higher sensitivity in females, contrasting the well-known anxiolytic effect of acute OXT. The increase in anxiety was transient and waned 5 days after the infusion has ended. The behavioral effects of chronic OXT were paralleled by activation of an intracellular signaling pathway, which ultimately led to alternative splicing of hypothalamic corticotropin-releasing factor receptor 2α (Crfr2α), an important modulator of anxiety. In detail, chronic OXT shifted the splicing ratio from the anxiolytic membrane-bound (mCRFR2α) form of CRFR2α towards the soluble CRFR2α (sCRFR2α) form. Experimental induction of alternative splicing mimicked the anxiogenic effects of chronic OXT, while sCRFR2α-knock down reduced anxiety-related behavior of male rats. Furthermore, chronic OXT treatment triggered the release of sCRFR2α into the cerebrospinal fluid with sCRFR2α levels positively correlating with anxiety-like behavior. In summary, we revealed that the shifted splicing ratio towards expression of the anxiogenic sCRFR2α underlies the adverse effects of chronic OXT treatment on anxiety