The use of next generation sequencing technology to study the effect of radiation therapy on mitochondrial DNA mutation

The human mitochondrial genome has an exclusively maternal mode of inheritance. Mitochondrial DNA (mtDNA) is particularly vulnerable to environmental insults due in part to an underdeveloped DNA repair system, limited to base excision and homologous recombination repair. Radiation exposure to the ovaries may cause mtDNA mutations in oocytes, which may in turn be transmitted to offspring. We hypothesized that the children of female cancer survivors who received radiation therapy may have an increased rate of mtDNA heteroplasmy mutations, which conceivably could increase their risk of developing cancer and other diseases. We evaluated 44 DNA blood samples from 17 Danish and 1 Finnish families (18 mothers and 26 children). All mothers had been treated for cancer as children and radiation doses to their ovaries were determined based on medical records and computational models. DNA samples were sequenced for the entire mitochondrial genome using the Illumina GAII system. Mother's age at sample collection was positively correlated with mtDNA heteroplasmy mutations. There was evidence of heteroplasmy inheritance in that 9 of the 18 families had at least one child who inherited at least one heteroplasmy site from his or her mother. No significant difference in single nucleotide polymorphisms between mother and offspring, however, was observed. Radiation therapy dose to ovaries also was not significantly associated with the heteroplasmy mutation rate among mothers and children. No evidence was found that radiotherapy for pediatric cancer is associated with the mitochondrial genome mutation rate in female cancer survivors and their children.     

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution,new validation tools,and implications for the future

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) “Resolution Revolution” made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps. After describing the history of the PDB and the Worldwide Protein Data Bank (wwPDB) partnership, which jointly manages both the PDB and EMDB archives, this review examines the origins of the resolution revolution and analyzes its impact on structural biology viewed through the lens of PDB holdings. Six areas of focus exemplifying the impact of 3DEM across the biosciences are discussed in detail (icosahedral viruses, ribosomes, integral membrane proteins, SARS-CoV-2 spike proteins, cryogenic electron tomography, and integrative structure determination combining 3DEM with complementary biophysical measurement techniques), followed by a review of 3DEM structure validation by the wwPDB that underscores the importance of community engagement.     

Beta-agonist-associated reduction in RGS5 expression promotes airway smooth muscle hyper-responsiveness

Although short-acting and long-acting inhaled β(2)-adrenergic receptor agonists (SABA and LABA, respectively) relieve asthma symptoms, use of either agent alone without concomitant anti-inflammatory drugs (corticosteroids) may increase the risk of disease exacerbation in some patients. We found previously that pretreatment of human precision-cut lung slices (PCLS) with SABA impaired subsequent β(2)-agonist-induced bronchodilation, which occurred independently of changes in receptor quantities. Here we provide evidence that prolonged exposure of cultured human airway smooth muscle (HuASM) cells to β(2)-agonists directly augments procontractile signaling pathways elicited by several compounds including thrombin, bradykinin, and histamine. Such treatment did not increase surface receptor amounts or expression of G proteins and downstream effectors (phospholipase Cβ and myosin light chain). In contrast, β-agonists decreased expression of regulator of G protein signaling 5 (RGS5), which is an inhibitor of G-protein-coupled receptor (GPCR) activity. RGS5 knockdown in HuASM increased agonist-evoked intracellular calcium flux and myosin light chain (MLC) phosphorylation, which are prerequisites for contraction. PCLS from Rgs5(-/-) mice contracted more to carbachol than those from WT mice, indicating that RGS5 negatively regulates bronchial smooth muscle contraction. Repetitive β(2)-agonist use may not only lead to reduced bronchoprotection but also to sensitization of excitation-contraction signaling pathways as a result of reduced RGS5 expression.     

Lipid bilayer stabilization of the Na,K-ATPase reconstituted in DPPC/DPPE liposomes

Different subunit aggregates of the Na,K-ATPase may be formed depending on the method used to solubilize and purify the enzyme. We have studied the thermal unfolding of detergent-solubilized and dipalmitoylphosphatidylcholine/ dipalmitoylphosphatidylethanolamine liposome-reconstituted forms of the Na,K-ATPase by circular dichroism (CD) spectroscopy and p-nitrophenylphosphatase activity. The ellipticity at 222 nm of the solubilized and reconstituted forms showed a sigmoid decrease in the absolute value of the signal of 36 and 31% with T(50%) of 44 and 42 degrees C, respectively. The catalytic activity was reduced in two steps with T(50%) of 32 and 52 degrees C in the detergent-solubilized enzyme and T(50%) of 25 and 53 degrees C in the reconstituted enzyme. The reduction in catalytic activity of the detergent-solubilized enzyme was bi-exponential with t(1/2) of 8.3 and 67.9 min, resulting in the total loss of activity after 120 min. However, under the same conditions, the ATPase activity of the reconstituted enzyme was reduced by approx 35% with a t(1/2) of 145 min. The results suggest that the alpha- and beta-subunits present different thermal stability that may be modulated by the nature of the co-solvent (detergent or lipid) used in the preparations of the Na,K-ATPase. In addition, distinct processes of beta-subunit displacement and alpha-alpha-subunit aggregate formation may also contribute to the changes in both the CD spectra and the enzyme activity. Furthermore, we have demonstrated the protective role of the phospholipid bilayer in the reconstituted enzyme compared with the detergent-solubilized enzyme.     

The potential for signal integration and processing in interacting MAP kinase cascades

The cellular response to environmental stimuli requires biochemical information processing through which sensory inputs and cellular status are integrated and translated into appropriate responses by way of interacting networks of enzymes. One such network, the mitogen-activated protein (MAP) kinase cascade is a highly conserved signal transduction module that propagates signals from cell surface receptors to various cytosolic and nuclear targets by way of a phosphorylation cascade. We have investigated the potential for signal processing within a network of interacting feed-forward kinase cascades typified by the MAP kinase cascade. A genetic algorithm was used to search for sets of kinetic parameters demonstrating representative key input-output patterns of interest. We discuss two of the networks identified in our study, one implementing the exclusive-or function (XOR) and another implementing what we refer to as an in-band detector (IBD) or two-sided threshold. These examples confirm the potential for logic and amplitude-dependent signal processing in interacting MAP kinase cascades demonstrating limited cross-talk. Specifically, the XOR function allows the network to respond to either one, but not both signals simultaneously, while the IBD permits the network to respond exclusively to signals within a given range of strength, and to suppress signals below as well as above this range. The solution to the XOR problem is interesting in that it requires only two interacting pathways, crosstalk at only one layer, and no feedback or explicit inhibition. These types of responses are not only biologically relevant but constitute signal processing modules that can be combined to create other logical functions and that, in contrast to amplification, cannot be achieved with a single cascade or with two non-interacting cascades. Our computational results revealed surprising similarities between experimental data describing the JNK/MKK4/MKK7 pathway and the solution for the IBD that evolved from the genetic algorithm. The evolved IBD not only exhibited the required non-monotonic signal strength-response, but also demonstrated transient and sustained responses that properly reflected the input signal strength, dependence on both of the MAPKKs for signaling, phosphorylation site preferences by each of the MAPKKs, and both activation and inhibition resulting from the overexpression of one of the MAPKKs.     

Characterization of mutations in the GTP-binding domain of IF2 resulting in cold-sensitive growth of Escherichia coli

The infB gene encodes translation initiation factor IF2. We have determined the entire sequence of infB from two cold-sensitive Escherichia coli strains IQ489 and IQ490. These two strains have been isolated as suppressor strains for the temperature-sensitive secretion mutation secY24. The mutations causing the suppression phenotype are located within infB. The only variations from the wild-type (wt) infB found in the two mutant strains are a replacement of Asp409 with Glu in strain IQ489 and an insertion of Gly between Ala421 and Gly422 in strain IQ490. Both positions are located in the GTP-binding G-domain of IF2. A model of the G-domain of E.coli IF2 is presented in. Physiological quantities of the recombinant mutant proteins were expressed in vivo in E.coli strains from which the chromosomal infB gene has been inactivated. At 42 degrees C, the mutants sustained normal cell growth, whereas a significant decrease in growth rate was found at 25 degrees C for both mutants as compared to wt IF2 expressed in the control strain. Circular dichroism spectra were recorded of the wt and the two mutant proteins to investigate the structural properties of the proteins. The spectra are characteristic of alpha-helix dominated structure, and reveal a significant different behavior between the wt and mutant IF2s with respect to temperature-induced conformational changes. The temperature-induced conformational change of the wt IF2 is a two-state process. In a ribosome-dependent GTPase assay in vitro the two mutants showed practically no activity at temperatures below 10 degrees C and a reduced activity at all temperatures up to 45 degrees C, as compared to wt IF2. The results indicate that the amino acid residues, Asp409 and Gly422, are located in important regions of the IF2 G-domain and demonstrate the importance of GTP hydrolysis in translation initiation for optimal cell growth.     

The anammox case-a new experimental manifesto for microbiological eco-physiology

The anaerobic ammonium oxidation process is a new process for ammonia removal from wastewater. It is also a new microbial physiology that was previously believed to be impossible. The identification of Candidatus Brocadia anammoxidans and its relatives as the responsible bacteria was only possible with the development of a new experimental approach. That approach is the focus of this paper. The approach is a modernisation of the Winogradsky/Beyerinck strategy of selective enrichment and is based on the introduction of the molecular toolbox and modern bioreactor engineering to microbial ecology. It consists of five steps: (1) postulation of an ecological niche based on thermodynamic considerations and macro-ecological field data; (2) engineering of this niche into a laboratory bioreactor for enrichment culture; (3) black-box physiological characterisation of the enrichment culture as a whole; (4) phylogenetic characterisation of the enriched community using molecular tools; (5) physical separation of the dominant members of the enrichment culture using gradient centrifugation and the identification of the species of interest in accordance with Koch's postulates; (6) verification of the in situ importance of these species in the actual ecosystems. The power of this approach is illustrated with a case study: the identification of the planctomycetes responsible for anaerobic ammonium oxidation. We argue that this was impossible using molecular ecology or conventional ‘cultivation based techniques’ alone. We suggest that the approach might also be used for the microbiological study of many interesting microbes such as anaerobic methane oxidisers. This revised version was published online in August 2006 with corrections to the Cover Date.