We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High ...cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H2O2 . Surprisingly, hcef2 was mapped to a nonsense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash induced thylakoid electric field suggest that these defect lead to accumulation of H2O2 in hcef2, which we have previously shown leads to activation of NDHrelated CEF. We observed similar increases in CEF and H2O2 accumulation in other translation defective mutants, suggesting that loss of coordination in plastid protein levels lead to imbalances in the photosynthetic energy balance that leads to increased CEF. These results, together with a large body of previous observations, support a general model in which processes that imbalances in chloroplast energetics result in the production of H2O2 , which activates CEF, either as a redox signal or by inducing deficits in ATP levels.
The boron-10 based multi-grid detector is being developed as an alternative to helium-3 based neutron detectors. At the European Spallation Source, the detector will be used for time-of-flight ...neutron spectroscopy at cold to thermal neutron energies. The objective of this work is to investigate fine time- and energy-resolved effects of the Multi-Grid detector, down to a few µeV, while comparing it to the performance of a typical helium-3 tube. Furthermore, it is to characterize differences between the detector technologies in terms of internal scattering, as well as the time reconstruction of ∼ µs short neutron pulses. The data were taken at the Helmholtz Zentrum Berlin, where the Multi-Grid detector and a helium-3 tube were installed at the ESS test beamline, V20. Using a Fermi-chopper, the neutron beam of the reactor was chopped into a few tens of µs wide pulses before reaching the detector, located a few tens of cm downstream. The data of the measurements show an agreement between the derived and calculated neutron detection efficiency curve. The data also provide fine details on the effect of internal scattering, and how it can be reduced. For the first time, the chopper resolution was comparable to the timing resolution of the Multi-Grid detector. This allowed a detailed study of time- and energy resolved effects, as well as a comparison with a typical helium-3 tube.
Cross-reactive immunity between SARS-CoV-2 and other related coronaviruses has been well-documented, and it may play a role in preventing severe COVID-19. Epidemiological studies early in the ...pandemic showed a geographical association between high influenza vaccination rates and lower incidence of SARS-CoV-2 infection. We, therefore, analyzed whether exposure to influenza A virus (IAV) antigens could influence the T cell repertoire in response to SARS-CoV-2, indicating a heterologous immune response between these 2 unrelated viruses. Using artificial antigen-presenting cells (aAPCs) combined with real-time reverse-transcription PCR (RT-qPCR), we developed a sensitive assay to quickly screen for antigen-specific T cell responses and detected a significant correlation between responses to SARS-CoV-2 epitopes and IAV dominant epitope (M158-66). Further analysis showed that some COVID-19 convalescent donors exhibited both T cell receptor (TCR) specificity and functional cytokine responses to multiple SARS-CoV-2 epitopes and M158-66. Utilizing an aAPC-based stimulation/expansion assay, we detected cross-reactive T cells with specificity to SARS-CoV-2 and IAV. In addition, TCR sequencing of the cross-reactive and IAV-specific T cells revealed similarities between the TCR repertoires of the two populations. These results indicate that heterologous immunity shaped by our exposure to other unrelated endemic viruses may affect our immune response to novel viruses such as SARS-CoV-2.
To determine the sensitivity and specificity of orbital ultrasonography in distinguishing papilledema from pseudopapilledema in adult patients.
The records of all adult patients referred to the ...neuro-ophthalmology service who underwent orbital ultrasonography for the evaluation of suspected papilledema were reviewed. The details of history, ophthalmologic examination, and results of ancillary testing including orbital ultrasonography, MRI, and lumbar puncture were recorded. Results of orbital ultrasonography were correlated with the final diagnosis of papilledema or pseudopapilledema on the basis of the clinical impression of the neuro-ophthalmologist. Ultrasound was considered positive when the optic nerve sheath diameter was ≥3.3 mm along with a positive 30° test.
The sensitivity of orbital ultrasonography for detection of papilledema was 90% (CI: 80.2-99.3%) and the specificity in detecting pseudopapilledema was 79% (CI: 67.7-90.7%).
Orbital ultrasonography is a rapid and noninvasive test that is highly sensitive, but less specific in differentiating papilledema from pseudopapilledema in adult patients, and can be useful in guiding further management of patients in whom the diagnosis is initially uncertain.
Biological dosimetry is an essential tool for estimating radiation dose. The dicentric chromosome assay (DCA) is currently the tool of choice. Because the assay is labor-intensive and time-consuming, ...strategies are needed to increase throughput for use in radiation mass casualty incidents. One such strategy is to truncate metaphase spread analysis for triage dose estimates by scoring 50 or fewer metaphases, compared to a routine analysis of 500 to 1000 metaphases, and to increase throughput using a large group of scorers in a biodosimetry network. Previously, the National Institutes for Allergies and Infectious Diseases (NIAID) and the Armed Forces Radiobiology Research Institute (AFRRI) sponsored a double-blinded interlaboratory comparison among five established international cytogenetic biodosimetry laboratories to determine the variability in calibration curves and in dose measurements in unknown, irradiated samples. In the present study, we further analyzed the published data from this previous study to investigate how the number of metaphase spreads influences dose prediction accuracy and how this information could be of value in the triage and management of people at risk for the acute radiation syndrome (ARS). Although, as expected, accuracy decreased with lower numbers of metaphase spreads analyzed, predicted doses by the laboratories were in good agreement and were judged to be adequate to guide diagnosis and treatment of ARS. These results demonstrate that for rapid triage, a network of cytogenetic biodosimetry laboratories can accurately assess doses even with a lower number of scored metaphases.
Wilkins, R. C., Romm, H., Kao, T-C., Awa, A. A., Yoshida, M. A., Livingston, G. K., Jenkins, M. S., Oestreicher, U., Pellmar, T. C. and Prasanna, P. G. S. Interlaboratory Comparison of the Dicentric ...Chromosome Assay for Radiation Biodosimetry in Mass Casualty Events. Radiat. Res. 169, 551–560 (2008). This interlaboratory comparison validates the dicentric chromosome assay for assessing radiation dose in mass casualty accidents and identifies the advantages and limitations of an international biodosimetry network. The assay's validity and accuracy were determined among five laboratories following the International Organization for Standardization guidelines. Blood samples irradiated at the Armed Forces Radiobiology Research Institute were shipped to all laboratories, which constructed individual radiation calibration curves and assessed the dose to dose-blinded samples. Each laboratory constructed a dose–effect calibration curve for the yield of dicentrics for 60Co γ rays in the 0 to 5-Gy range, using the maximum likelihood linear-quadratic model, Y = c + αD + βD2. For all laboratories, the estimated coefficients of the fitted curves were within the 99.7% confidence intervals (CIs), but the observed dicentric yields differed. When each laboratory assessed radiation doses to four dose-blinded blood samples by comparing the observed dicentric yield with the laboratory's own calibration curve, the estimates were accurate in all laboratories at all doses. For all laboratories, actual doses were within the 99.75% CI for the assessed dose. Across the dose range, the error in the estimated doses, compared to the physical doses, ranged from 15% underestimation to 15% overestimation.
The analysis of dicentric chromosomes in human peripheral blood lymphocytes (PBLs) by Giemsa staining is the most established method for biological dosimetry. However, this method requires a ...well-trained person because of the difficulty in detecting aberrations rapidly and accurately. Here, we applied a fluorescence in situ hybridization (FISH) technique, using telomere and centromere peptide nucleic acid (PNA) probes, to solve the problem of biological dosimetry in radiation emergency medicine. A comparison by a well-trained observer found that FISH analysis of PBLs for the dose estimation was more accurate than the conventional Giemsa analysis, especially in samples irradiated at high doses. These results show that FISH analysis with centromeric/telomeric PNA probes could become the standard method for biological dosimetry in radiation emergency medicine.
Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug delivery vehicles for small RNAs (siRNA and miRNA) due to their natural role in intercellular RNA ...transport. However, the application of EVs for therapeutic RNA delivery may be limited by loading approaches that can induce cargo aggregation or degradation. Here, we report the use of sonication as a means to actively load functional small RNAs into EVs. Conditions under which EVs could be loaded with small RNAs with minimal detectable aggregation were identified, and EVs loaded with therapeutic siRNA
via
sonication were observed to be taken up by recipient cells and capable of target mRNA knockdown leading to reduced protein expression. This system was ultimately applied to reduce expression of HER2, an oncogenic receptor tyrosine kinase that critically mediates breast cancer development and progression, and could be extended to other therapeutic targets. These results define important parameters informing the application of sonication as a small RNA loading method for EVs and demonstrate the potential utility of this approach for versatile cancer therapy.
Microfluidic lumen-based systems are microscale models that recapitulate the anatomy and physiology of tubular organs. These technologies can mimic human pathophysiology and predict drug response, ...having profound implications for drug discovery and development. Herein, we review progress in the development of microfluidic lumen-based models from the 2000s to the present. The core of the review discusses models for mimicking blood vessels, the respiratory tract, the gastrointestinal tract, renal tubules, and liver sinusoids, and their application to modeling organ-specific diseases. We also highlight emerging application areas, such as the lymphatic system, and close the review discussing potential future directions.
PDF
