Determination of interproton distances from NOESY spectra in the active site of paramagnetic metalloenzymes: cyanide-inhibited horseradish peroxidase

Two dimensional (2D) nuclear Overhauser effect (NOE) or NOESY experiments are performed on cyanide inhibited horseradish peroxidase in order to assess the prospects for obtaining quantitative interproton distances for the hyperfine shifted and paramagnetically relaxed active site signals in an intermediate sized (-44 kDa) paramagnetic metalloenzyme. This protein represents an ideal test case for such experiments because a series of structurally defined proton pairs on the heme and axial His 170 have been previously assigned. The relaxation properties of hyperfine shifted signals relevant to the experimental setup of 2D experiments and interpretation of both 1D and 2D NOE data are also investigated. NOESY spectra as a function of mixing time show that quantitative rise curves can be obtained that clearly differentiate between primary and secondary NOES even among the most strongly relaxed protons, but this requires very short mixing times in the range 0.5-3.0 ms. The sensitivity of the weak cross peaks at these short mixing times is improved by the relatively rapid pulse repetition rate and concommitant increase in the number of scans allowed by the rapid relaxation of active site protons. The paramagnetic relaxation influence, as well as the size of the protein, results in rise curves that are linear to only 1.5 ms for geminal protons and to only 3-5 ms for more weakly dipolar coupled proton pairs. However, the cross peak intensities in the linear region are shown to yield cross relaxation rates and internuclear distances for a series of assigned and orientationally invariant proton pairs that are in good agreement with their known distances. The patterns of NOESY rise curves are used both to determine the orientations of one propionate and both vinyl groups relative to the heme and to show that the axial His exhibits an orientation relative to its helix that is similar but not identical to that in crystallographically characterized cytochrome c peroxidase. Selective and nonselective 1D as well as 2D selective relaxation rate measurements for hyperfine shifted signals show that only selective or intrinsic relaxation rates can be used to optimize the setup of NOESY experiments and interpret 1D NOE data. The results of the study indicate that NOESY spectra can be expected to yield valuable quantitative structural information on the hyperfine shifted active site residues in a variety of cyanide-inhibited heme peroxidases

Importance of Spin-Orbit Interaction for the Electron Spin Relaxation in Organic Semiconductors

Despite the great interest organic spintronics has recently attracted, there is only a partial understanding of the fundamental physics behind electron spin relaxation in organic semiconductors. Mechanisms based on hyperfine interaction have been demonstrated, but the role of the spin-orbit interaction remains elusive. Here, we report muon spin spectroscopy and time-resolved photoluminescence measurements on two series of molecular semiconductors in which the strength of the spin-orbit interaction has been systematically modified with a targeted chemical substitution of different atoms at a particular molecular site. We find that the spin-orbit interaction is a significant source of electron spin relaxation in these materials

Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells

The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6-RACK1-ERK1/2-FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked

General practitioners’ perspectives on campaigns to promote rapid help-seeking behaviour at the onset of rheumatoid arthritis

Objective. To explore general practitioners’ (GPs’ ) perspectives on public health campaigns to encourage people with the early symptoms of rheumatoid arthritis (RA) to seek medical help rapidly. Design. Nineteen GPs participated in four semistructured focus groups. Focus groups were audio-recorded, transcribed verbatim, and analysed using thematic analysis. Results. GPs recognised the need for the early treatment of RA and identified that facilitating appropriate access to care was important. However, not all held the view that a delay in help seeking was a clinically significant issue. Furthermore, many were concerned that the early symptoms of RA were often non-specific, and that current knowledge about the nature of symptoms at disease onset was inadequate to inform the content of a help-seeking campaign. They argued that a campaign might not be able to specifically target those who need to present urgently. Poorly designed campaigns were suggested to have a negative impact on GPs’ workloads, and would “clog up” the referral pathway for genuine cases of RA. Conclusions. GPs were supportive of strategies to improve access to Rheumatological care and increase public awareness of RA symptoms. However, they have identified important issues that need to be considered in developing a public health campaign that forms part of an overall strategy to reduce time to treatment for patients with new onset RA. This study highlights the value of gaining GPs’ perspectives before launching health promotion campaigns

Gene expression drives the evolution of dominance.

Dominance is a fundamental concept in molecular genetics and has implications for understanding patterns of genetic variation, evolution, and complex traits. However, despite its importance, the degree of dominance in natural populations is poorly quantified. Here, we leverage multiple mating systems in natural populations of Arabidopsis to co-estimate the distribution of fitness effects and dominance coefficients of new amino acid changing mutations. We find that more deleterious mutations are more likely to be recessive than less deleterious mutations. Further, this pattern holds across gene categories, but varies with the connectivity and expression patterns of genes. Our work argues that dominance arises as a consequence of the functional importance of genes and their optimal expression levels

Plain language summary of the TRANSFORM study primary analysis results:liso-cell as a second treatment regimen for large B-cell lymphoma following failure of the first treatment regimen

What is this summary about?People diagnosed with a disease called large B-cell lymphoma (LBCL) may experience return, or early relapse, of their disease within the first year after receiving and responding to their first (first-line) treatment regimen. Others may have primary refractory disease, meaning that the disease either did not respond to first-line treatment at all or only responded for a very brief period. Second (second-line) treatment includes immunotherapy followed by high-dose chemotherapy and ASCT, which has the potential to cure LBCL. However, if the disease does not respond to immunotherapy, people cannot receive ASCT, and less than 30% of people are cured.Therefore, new second-line treatment options are required, such as CAR T cell therapy, which uses a person's own genetically engineered lymphocytes, also called T cells, to fight their lymphoma. In this article, we summarize the key results of the phase 3 TRANSFORM clinical study that tested if liso-cel, a CAR T cell treatment, can safely and effectively be used as a second-line treatment for people with early relapsed or primary refractory (relapsed/refractory) LBCL.A total of 184 adults with relapsed/refractory LBCL who were able to receive ASCT were randomly treated with either liso-cel or standard of care (SOC) as second-line treatment. SOC included immunochemotherapy followed by high-dose chemotherapy and ASCT.What were the key takeaways?Almost all (97%) people in the liso-cel group completed treatment, whereas 53% of people in the SOC group did not complete treatment, mostly due to their disease not responding or relapsing, and therefore they were not able to receive ASCT. People who received liso-cel as a second-line treatment lived longer without the occurrence of an unfavorable medical event or worsening of the disease and had a better response to treatment than those who received SOC as second-line treatment. People who received liso-cel reported side effects that researchers considered to be manageable, and that were known to occur with CAR T cell treatment.What were the main conclusions reported by the researchers?Results from the TRANSFORM study support the use of liso-cel as a more effective second-line treatment compared with SOC that is safe for people with relapsed/refractory LBCL

Strain-dependent host transcriptional responses to toxoplasma infection are largely conserved in mammalian and avian hosts

Toxoplasma gondii has a remarkable ability to infect an enormous variety of mammalian and avian species. Given this, it is surprising that three strains (Types I/II/III) account for the majority of isolates from Europe/North America. The selective pressures that have driven the emergence of these particular strains, however, remain enigmatic. We hypothesized that strain selection might be partially driven by adaptation of strains for mammalian versus avian hosts. To test this, we examine in vitro, strain-dependent host responses in fibroblasts of a representative avian host, the chicken (Gallus gallus). Using gene expression profiling of infected chicken embryonic fibroblasts and pathway analysis to assess host response, we show here that chicken cells respond with distinct transcriptional profiles upon infection with Type II versus III strains that are reminiscent of profiles observed in mammalian cells. To identify the parasite drivers of these differences, chicken fibroblasts were infected with individual F1 progeny of a Type II x III cross and host gene expression was assessed for each by microarray. QTL mapping of transcriptional differences suggested, and deletion strains confirmed, that, as in mammalian cells, the polymorphic rhoptry kinase ROP16 is the major driver of strain-specific responses. We originally hypothesized that comparing avian versus mammalian host response might reveal an inversion in parasite strain-dependent phenotypes; specifically, for polymorphic effectors like ROP16, we hypothesized that the allele with most activity in mammalian cells might be less active in avian cells. Instead, we found that activity of ROP16 alleles appears to be conserved across host species; moreover, additional parasite loci that were previously mapped for strain-specific effects on mammalian response showed similar strain-specific effects in chicken cells. These results indicate that if different hosts select for different parasite genotypes, the selection operates downstream of the signaling occurring during the beginning of the host's immune response. © 2011 Ong et al

Hyperfibrinogenemia is associated with lymphatic as well as hematogenous metastasis and worse clinical outcome in T2 gastric cancer

BACKGROUND: Abnormal hemostasis in cancer patients has previously been described, however the correlation between the plasma fibrinogen level and cancer metastasis and prognosis has not been reported in a large-scale clinical study. METHODS: Preoperative plasma fibrinogen levels were retrospectively examined in 405 patients who underwent surgery for advanced gastric cancer. The association of fibrinogen levels with clinical/pathological findings and clinical outcome was evaluated. RESULTS: There was a positive correlation between plasma fibrinogen levels and the depth of invasion (p < 0.05). Hyperfibrinogenemia (>310 mg/dl) was independently associated with lymph node (Odds Ratio; 2.342, P = 0.0032) and liver (Odds Ratio; 2.933, P = 0.0147) metastasis, not with peritoneal metastasis in this series. Patients with hyperfibrinogenemia showed worse clinical outcome in T2 gastric cancer, however, there was no correlation of plasma fibrinogen level with prognosis in T3/T4 gastric cancer. CONCLUSION: Our results might support the idea that hyperfibrinogenemia can augment lymphatic and hematogeneous metastasis of advanced gastric cancer, which is major determinant of the prognosis in T2 gastric cancer. Therefore, in the situation without peritoneal involvement, hyperfibrinogenemia is a useful biomarker to predict the possible metastasis and worse clinical outcome in T2 gastric cancer