Effect of sulfasalazine on human neuroblastoma: Analysis of sepiapterin reductase (SPR) as a new therapeutic target

Background: Neuroblastoma (NB) is an aggressive childhood malignancy in children up to 5 years of age. High-stage tumors frequently relapse even after aggressive multimodal treatment, and then show therapy resistance, typically resulting in patient death. New molecular-targeted compounds that effectively suppress tumor growth and prevent relapse with more efficacy are urgently needed. We and others previously showed that polyamines (PA) like spermidine and spermine are essential for NB tumorigenesis and that DFMO, an inhibitor of the key PA synthesis gene product ODC, is effective both in vitro and in vivo, securing its evaluation in NB clinical trials. To find additional compounds interfering with PA biosynthesis, we tested sulfasalazine (SSZ), an FDA-approved salicylate-based anti-inflammatory and immune-modulatory drug, recently identified to inhibit sepiapterin reductase (SPR). We earlier presented evidence for a physical interaction between ODC and SPR and we showed that RNAi-mediated knockdown of SPR expression significantly reduced native ODC enzyme activity and impeded NB cell proliferation. Methods: Human NB mRNA expression datasets in the public domain were analyzed using the R2 platform. Cell viability, isobologram, and combination index analyses as a result of SSZ treatment with our without DFMO were carried out in NB cell cultures. Molecular protein-ligand docking was achieved using the GRAMM algorithm. Statistical analyses were performed with the Kruskal-Wallis test, 2log Pearson test, and Student's t test. Results: In this study, we show the clinical relevance of SPR in human NB tumors. We found that high SPR expression is significantly correlated to unfavorable NB characteristics like high age at diagnosis, MYCN amplification, and high INSS stage. SSZ inhibits the growth of NB cells in vitro, presumably due to the inhibition of SPR as predicted by computational docking of SSZ into SPR. Importantly, the combination of SSZ with DFMO produces synergistic antiproliferative effects in vitro. Conclusions: The results suggest the use of SSZ in combination with DFMO for further experiments, and possible prioritization as a novel therapy for the treatment of NB patients

Dynein structure and power stroke

Dynein ATPases are microtubule motors that are critical to diverse processes such as vesicle transport and the beating of sperm tails; however, their mechanism of force generation is unknown. Each dynein comprises a head, from which a stalk and a stem emerge. Here we use electron microscopy and image processing to reveal new structural details of dynein c, an isoform from Chlamydomonas reinhardtii flagella, at the start and end of its power stroke. Both stem and stalk are flexible, and the stem connects to the head by means of a linker approximately 10 nm long that we propose lies across the head. With both ADP and vanadate bound, the stem and stalk emerge from the head 10 nm apart. However, without nucleotide they emerge much closer together owing to a change in linker orientation, and the coiled-coil stalk becomes stiffer. The net result is a shortening of the molecule coupled to an approximately 15-nm displacement of the tip of the stalk. These changes indicate a mechanism for the dynein power stroke

Evolutionary Insights from a Genetically Divergent Hantavirus Harbored by the European Common Mole (Talpa europaea)

BACKGROUND:The discovery of genetically distinct hantaviruses in shrews (Order Soricomorpha, Family Soricidae) from widely separated geographic regions challenges the hypothesis that rodents (Order Rodentia, Family Muridae and Cricetidae) are the primordial reservoir hosts of hantaviruses and also predicts that other soricomorphs harbor hantaviruses. Recently, novel hantavirus genomes have been detected in moles of the Family Talpidae, including the Japanese shrew mole (Urotrichus talpoides) and American shrew mole (Neurotrichus gibbsii). We present new insights into the evolutionary history of hantaviruses gained from a highly divergent hantavirus, designated Nova virus (NVAV), identified in the European common mole (Talpa europaea) captured in Hungary. METHODOLOGY/PRINCIPAL FINDINGS:Pair-wise alignment and comparison of the full-length S- and L-genomic segments indicated moderately low sequence similarity of 54-65% and 46-63% at the nucleotide and amino acid levels, respectively, between NVAV and representative rodent- and soricid-borne hantaviruses. Despite the high degree of sequence divergence, the predicted secondary structure of the NVAV nucleocapsid protein exhibited the characteristic coiled-coil domains at the amino-terminal end, and the L-segment motifs, typically found in hantaviruses, were well conserved. Phylogenetic analyses, using maximum-likelihood and Bayesian methods, showed that NVAV formed a distinct clade that was evolutionarily distant from all other hantaviruses. CONCLUSIONS:Newly identified hantaviruses harbored by shrews and moles support long-standing virus-host relationships and suggest that ancestral soricomorphs, rather than rodents, may have been the early or original mammalian hosts

Expression and genomic analysis of midasin, a novel and highly conserved AAA protein distantly related to dynein

BACKGROUND: The largest open reading frame in the Saccharomyces genome encodes midasin (MDN1p, YLR106p), an AAA ATPase of 560 kDa that is essential for cell viability. Orthologs of midasin have been identified in the genome projects for Drosophila, Arabidopsis, and Schizosaccharomyces pombe. RESULTS: Midasin is present as a single-copy gene encoding a well-conserved protein of ~600 kDa in all eukaryotes for which data are available. In humans, the gene maps to 6q15 and encodes a predicted protein of 5596 residues (632 kDa). Sequence alignments of midasin from humans, yeast, Giardia and Encephalitozoon indicate that its domain structure comprises an N-terminal domain (35 kDa), followed by an AAA domain containing six tandem AAA protomers (~30 kDa each), a linker domain (260 kDa), an acidic domain (~70 kDa) containing 35–40% aspartate and glutamate, and a carboxy-terminal M-domain (30 kDa) that possesses MIDAS sequence motifs and is homologous to the I-domain of integrins. Expression of hemagglutamin-tagged midasin in yeast demonstrates a polypeptide of the anticipated size that is localized principally in the nucleus. CONCLUSIONS: The highly conserved structure of midasin in eukaryotes, taken in conjunction with its nuclear localization in yeast, suggests that midasin may function as a nuclear chaperone and be involved in the assembly/disassembly of macromolecular complexes in the nucleus. The AAA domain of midasin is evolutionarily related to that of dynein, but it appears to lack a microtubule-binding site

Remnant radio-loud AGN in the Herschel-ATLAS field

Only a small fraction of observed active galactic nuclei (AGN) display large-scale radio emission associated with jets, yet these radio-loud AGN have become increasingly important in models of galaxy evolution. In determining the dynamics and energetics of the radio sources over cosmic time, a key question concerns what happens when their jets switch off. The resulting ‘remnant' radio-loud AGN have been surprisingly evasive in past radio surveys, and therefore statistical information on the population of radio-loud AGN in their dying phase is limited. In this paper, with the recent developments of Low-Frequency Array (LOFAR) and the Very Large Array, we are able to provide a systematically selected sample of remnant radio-loud AGN in the Herschel-ATLAS field. Using a simple core-detection method, we constrain the upper limit on the fraction of remnants in our radio-loud AGN sample to 9 per cent, implying that the extended lobe emission fades rapidly once the core/jets turn off. We also find that our remnant sample has a wide range of spectral indices (−1.5 ⩽ α1400150 ⩽ −0.5), confirming that the lobes of some remnants may possess flat spectra at low frequencies just as active sources do. We suggest that, even with the unprecedented sensitivity of LOFAR, our sample may still only contain the youngest of the remnant population

Radio-loud active galactic nuclei at high redshifts and the cosmic microwave background

The interaction between the emitting electrons and the cosmic microwave background (CMB) affects the observable properties of radio-loud active galactic nuclei (AGN) at early epochs. At high redshifts z, the CMB energy density [UCMB 1d (1 + z)4] can exceed the magnetic one (UB) in the lobes of radio-loud AGN. In this case, the relativistic electrons cool preferentially by scattering off CMB photons, rather than by synchrotron emission. This makes more distant sources less luminous in radio and more luminous in X-rays than their closer counterparts. In contrast, in the inner jet and the hotspots, where UB > UCMB, synchrotron radiation is unaffected by the presence of the CMB. The decrease in radio luminosity is thus more severe in misaligned (with respect to our line of sight) high-z sources, whose radio flux is dominated by the extended isotropic component. These sources can fail detection in current flux-limited radio surveys, where they are possibly underrepresented. As the cooling time is longer for lower energy electrons, the radio luminosity deficit due to the CMB is less important at low radio frequencies. Therefore, objects not detected so far at a few GHz could be picked up by low-frequency deep surveys, such as Low-Frequency Array and Square Kilometre Array. Until then, we can estimate the number of high-z radio-loud AGN through the census of their aligned proxies, i.e. blazars, since their observed radio emission arises in the inner and strongly magnetized compact core of the jet and it is not affected by inverse Compton scattering off CMB photons

Direct observation shows superposition and large scale flexibility within cytoplasmic dynein motors moving along microtubules

Cytoplasmic dynein is a dimeric AAA+ motor protein that performs critical roles in eukaryotic cells by moving along microtubules using ATP. Here using cryo-electron microscopy we directly observe the structure of Dictyostelium discoideum dynein dimers on microtubules at near-physiological ATP concentrations. They display remarkable flexibility at a hinge close to the microtubule binding domain (the stalkhead) producing a wide range of head positions. About half the molecules have the two heads separated from one another, with both leading and trailing motors attached to the microtubule. The other half have the two heads and stalks closely superposed in a front-to-back arrangement of the AAA+ rings, suggesting specific contact between the heads. All stalks point towards the microtubule minus end. Mean stalk angles depend on the separation between their stalkheads, which allows estimation of inter-head tension. These findings provide a structural framework for understanding dynein’s directionality and unusual stepping behaviour