A high-throughput method for genotyping S-RNase alleles in apple

We present a new efficient screening tool for detection of S-alleles in apple. The protocol using general and multiplexed primers for PCR reaction and fragment detection on an automatized capillary DNA sequencer exposed a higher number of alleles than any previous studies. Analysis of alleles is made on basis of three individual fragment sizes making the allele interpretation highly accurate. The method was employed to genotype 432 Malus accessions and exposed 25 different S-alleles in a selection of Malus domestica cultivars of mainly Danish origin (402 accessions) as well as a selection of other Malus species (30 accessions). The allele S3 (28 %) was the most common among the Danish cultivars followed by S1 and S7 (both 27 %). The alleles S36 and S40 not previously reported from M. domestica were found in 6 and 17 cultivars, respectively. Complete allelic composition was found in 91 % of the 369 diploid accessions and in 86 % of the 63 triploids concerned. We further identified a relatively high frequency of S33 and S34, which has not been considered by most previous studies. The protocol presented here is easy to adopt and saves both time and work effort compared to previous methods. The robustness is illustrated by the great accuracy and a high number of S-alleles presented.     

Reduced rates of non-union with modified periacetabular osteotomy using peracetic-acid sterilized cancellous allografts

The objective of the present study was to analyze the clinical and radiological results of periacetabular osteotomies (PAO) using Kirschner wire fixation and an allogeneic cancellous bone graft. This retrospective cohort study included 73 patients (85 PAOs). The allografts were processed from distal femur of cadaveric donors, defatted, sterilized with a peracetic-acid ethanol solution and freeze-dried. The clinical outcome, as measured by the Harris Hip Scores (HHS), the complication rate and the acetabular correction, as measured by radiological parameters, were compared. The postoperative femoral head coverage and HSS were significantly improved. Major complications occurred in five cases (6 %), but in no case did we observe a non-union or a graft-associated adverse effect. Fixation of the acetabular fragment with Kirschner wires in combination with an allogeneic cancellous bone graft is a safe method, with a low complication rate, no loss of correction and can prevent the occurrence of non-union with a high degree of probability.     

YfaE, a ferredoxin involved in diferric-tyrosyl radical maintenance in Escherichia coli ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of a 1:1 complex of two homodimeric subunits: alpha and beta. beta contains the diferric-tyrosyl radical (Y*) cofactor essential for the reduction process. In vivo, the mechanism of Y* regeneration from the diferric-beta2 (met-beta2) or apo-beta2 is still unclear. Y* regenerations from met-beta2 and apo-beta2 have been designated the maintenance and biosynthetic pathways, respectively. To understand these two pathways, 181 genomes that contain nrdAnrdB (genes encoding alpha and beta) were examined. In 29% of the cases, an open reading frame annotated 2Fe2S ferredoxin (YfaE in Escherichia coli) is located next to nrdB. Thus, YfaE has been cloned, expressed, resolubilized, reconstituted anaerobically with Fe2+, Fe3+, and S2-, and characterized by M?ssbauer, EPR, and visible spectroscopies. Titration of met-beta2 with [2Fe2S]1+-YfaE anaerobically results in the formation of an equilibrium mixture of diferrous-beta2 and [2Fe2S]2+-YfaE with one Fe reduced/YfaE oxidized. At the end point of the titration, O2 is added to the mixture and the diferrous-beta2 rapidly undergoes reaction to form the diferric-Y* with a stoichiometry of 2Fe/Y* and a specific activity correlated to the amount of Y*. The reducing equivalent required for diferric-Y* cofactor biosynthesis is supplied by beta. Under anaerobic conditions, stopped flow kinetics have been used to monitor the disappearance of the diferric cluster and the formation of [2Fe2S]2+-YfaE. The titrations and kinetic studies provide the first evidence for a protein involved in the maintenance pathway and likely the biosynthetic pathway.     

AurF from Streptomyces thioluteus and a possible new family of manganese/iron oxygenases

We recently reported that the R2 subunit of class Ic ribonucleotide reductase from Chlamydia trachomatis contains a heterodinuclear Mn/Fe redox cofactor [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. The N-oxygenase, AurF, from Streptomyces thioluteus catalyzes the six-electron oxidation of p-aminobenzoate to p-nitrobenzoate and contains the EX2HX60-180EX2H sequence motif previously used to identify proteins with non-heme diiron clusters. Two research groups independently obtained evidence for the presence of iron and manganese in preparations of AurF. The electron paramagnetic resonance (EPR) spectrum of purified, resting AurF presented in one of these studies is markedly similar to the spectrum of the MnIII/FeIII form of C. trachomatis R2. We propose that S. thioluteus AurF also may harbor a heterodinuclear Mn/Fe cofactor, which it may use to activate O2 for oxidation of the aryl amine to the nitro compound. Hypothetical proteins encoded in the genomes of several other bacteria have similar sequences and may also be members of this nascent family of oxygen-activating Mn/Fe proteins.     

Cofilin1 oxidation links oxidative distress to mitochondrial demise and neuronal cell death

Many cell death pathways, including apoptosis, regulated necrosis, and ferroptosis, are relevant for neuronal cell death and share common mechanisms such as the formation of reactive oxygen species (ROS) and mitochondrial damage. Here, we present the role of the actin-regulating protein cofilin1 in regulating mitochondrial pathways in oxidative neuronal death. Cofilin1 deletion in neuronal HT22 cells exerted increased mitochondrial resilience, assessed by quantification of mitochondrial ROS production, mitochondrial membrane potential, and ATP levels. Further, cofilin1-deficient cells met their energy demand through enhanced glycolysis, whereas control cells were metabolically impaired when challenged by ferroptosis. Further, cofilin1 was confirmed as a key player in glutamate-mediated excitotoxicity and associated mitochondrial damage in primary cortical neurons. Using isolated mitochondria and recombinant cofilin1, we provide a further link to toxicity-related mitochondrial impairment mediated by oxidized cofilin1. Our data revealed that the detrimental impact of cofilin1 on mitochondria depends on the oxidation of cysteine residues at positions 139 and 147. Overall, our findings show that cofilin1 acts as a redox sensor in oxidative cell death pathways of ferroptosis, and also promotes glutamate excitotoxicity. Protective effects by cofilin1 inhibition are particularly attributed to preserved mitochondrial integrity and function. Thus, interfering with the oxidation and pathological activation of cofilin1 may offer an effective therapeutic strategy in neurodegenerative diseases.Subject terms: Apoptosis, Cell death in the nervous system, Neurodegeneration     

1α,25-Dihydroxyvitamin D3 receptor as a mediator of transrepression of retinoid signaling

The receptors for retinoic acid (RA) and for 1α,25-dihydroxyvitamin D₃ (VD), RAR, RXR, and VDR are ligand-inducible members of the nuclear receptor superfamily. These receptors mediate their regulatory effects by binding as dimeric complexes to response elements located in regulatory regions of hormone target genes. Sequence scanning of the tumor necrosis factor-α type I receptor (TNFαRI) gene identified a 3′ enhancer region composed of two directly repeated hexameric core motifs spaced by 2 nucleotides (DR2). On this novel DR2-type sequence, but not on a DR5-type RA response element, VD was shown to act through its receptor, the vitamin D receptor (VDR), as a repressor of retinoid signalling. The repression appears to be mediated by competitive protein–protein interactions between VDR, RAR, RXR, and possibly their cofactors. This VDR-mediated transrepression of retinoid signaling suggests a novel mechanism for the complex regulatory interaction between retinoids and VD. J. Cell. Biochem. 67:287–296, 1997. © 1997 Wiley-Liss, Inc.     

Fin56-induced ferroptosis is supported by autophagy-mediated GPX4 degradation and functions synergistically with mTOR inhibition to kill bladder cancer cells

Ferroptosis is a form of regulated cell death that emerges to be relevant for therapy-resistant and dedifferentiating cancers. Although several lines of evidence suggest that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms remain unclear. Fin56, a type 3 ferroptosis inducer, triggers ferroptosis by promoting glutathione peroxidase 4 (GPX4) protein degradation via a not fully understood pathway. Here, we determined that Fin56 induces ferroptosis and autophagy in bladder cancer cells and that Fin56-triggered ferroptosis mechanistically depends on the autophagic machinery. Furthermore, we found that autophagy inhibition at different stages attenuates Fin56-induced oxidative stress and GPX4 degradation. Moreover, we investigated the effects of Fin56 in combination with Torin 2, a potent mTOR inhibitor used to activate autophagy, on cell viability. We found that Fin56 synergizes with Torin 2 in cytotoxicity against bladder cancer cells. Collectively, our findings not only support the concept that ferroptosis is a type of autophagy-dependent cell death but imply that the combined application of ferroptosis inducers and mTOR inhibitors is a promising approach to improve therapeutic options in the treatment of bladder cancer.Subject terms: Macroautophagy, Macroautophagy     

The RicAFT (YmcA‐YlbF‐YaaT) complex carries two [4Fe‐4S]2+ clusters and may respond to redox changes

During times of environmental insult, Bacillus subtilis undergoes developmental changes leading to biofilm formation, sporulation and competence. Each of these states is regulated in part by the phosphorylated form of the master response regulator Spo0A (Spo0A～P). The phosphorylation state of Spo0A is controlled by a multi‐component phosphorelay. RicA, RicF and RicT (previously YmcA, YlbF and YaaT) have been shown to be important regulatory proteins for multiple developmental fates. These proteins directly interact and form a stable complex, which has been proposed to accelerate the phosphorelay. Indeed, this complex is sufficient to stimulate the rate of phosphotransfer amongst the phosphorelay proteins in vitro. In this study, we demonstrate that two [4Fe‐4S]²⁺ clusters can be assembled on the complex. As with other iron‐sulfur cluster‐binding proteins, the complex was also found to bind FAD, hinting that these cofactors may be involved in sensing the cellular redox state. This work provides the first comprehensive characterization of an iron‐sulfur protein complex that regulates Spo0A～P levels. Phylogenetic and genetic evidence suggests that the complex plays a broader role beyond stimulation of the phosphorelay.     

Does the colonization of new biogeographic regions influence the diversification and accumulation of clade richness among the Corvides (Aves: Passeriformes)?

Regional variation in clade richness can be vast, reflecting differences in the dynamics of historical dispersal and diversification among lineages. Although it has been proposed that dispersal into new biogeographic regions may facilitate diversification, to date there has been limited assessment of the importance of this process in the generation, and maintenance, of broad‐scale biodiversity gradients. To address this issue, we analytically derive biogeographic regions for a global radiation of passerine birds (the Corvides, c. 790 species) that are highly variable in the geographic and taxonomic distribution of species. Subsequently, we determine rates of historical dispersal between regions, the dynamics of diversification following regional colonization, and spatial variation in the distribution of species that differ in their rates of lineage diversification. The results of these analyses reveal spatiotemporal differences in the build‐up of lineages across regions. The number of regions occupied and the rate of transition between regions both predict family richness well, indicating that the accumulation of high clade richness is associated with repeated expansion into new geographic areas. However, only the largest family (the Corvidae) had significantly heightened rates of both speciation and regional transition, implying that repeated regional colonization is not a general mechanism promoting lineage diversification among the Corvides.     

Intermediates and Generic Convergence to Equilibria

Known graphical conditions for the generic and global convergence to equilibria of the dynamical system arising from a reaction network are shown to be invariant under the so-called successive removal of intermediates, a systematic procedure to simplify the network, making the graphical conditions considerably easier to check.