tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-associated non-protein-coding RNA (rancRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [3].

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the halophilic archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress-dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-associated non-coding RNA (rancRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [3].

Recommendations from GEC ESTRO Breast Cancer Working Group (I): Target definition and target delineation for accelerated or boost Partial Breast Irradiation using multicatheter interstitial brachytherapy after breast conserving closed cavity surgery

OBJECTIVE The aim was to develop a delineation guideline for target definition for APBI or boost by consensus of the Breast Working Group of GEC-ESTRO. PROPOSED RECOMMENDATIONS Appropriate delineation of CTV (PTV) with low inter- and intra-observer variability in clinical practice is complex and needs various steps as: (1) Detailed knowledge of primary surgical procedure, of all details of pathology, as well as of preoperative imaging. (2) Definition of tumour localization before breast conserving surgery inside the breast and translation of this information in the postoperative CT imaging data set. (3) Calculation of the size of total safety margins. The size should be at least 2 cm. (4) Definition of the target. (5) Delineation of the target according to defined rules. CONCLUSION Providing guidelines based on the consensus of a group of experts should make it possible to achieve a reproducible and consistent definition of CTV (PTV) for Accelerated Partial Breast Irradiation (APBI) or boost irradiation after breast conserving closed cavity surgery, and helps to define it after selected cases of oncoplastic surgery.

Interobserver variations of target volume delineation in multicatheter partial breast brachytherapy after open cavity surgery

PURPOSE To investigate interobserver variations of target volume delineations in accelerated partial breast irradiation with multicatheter brachytherapy (BT) and to assess the impact of guidelines on consistency of contouring. METHODS AND MATERIALS A contouring study with two phases in interstitial accelerated partial breast irradiation after open cavity surgery was conducted by the Groupe Européen de Curiethérapie-European Society for Radiotherapy and Oncology Breast Cancer Working Group. Contours of cavity and planning target volume (PTV) on preimplant and postimplant CT images were delineated. In Phase 1, nine radiation oncologists defined the target volumes of 5 patients, whereas in Phase 2, four observers draw the contours of 4 patients applying guidelines. In Phase 1, experience in breast BT after open cavity surgery was assessed. The delineations were compared between Phase 1 and Phase 2, the impact of guidelines was assessed, and cavity visualization score was related to consistency of delineations. RESULTS Significant interobserver variability in delineations of lumpectomy cavity and PTV was observed among the participants. Observers with BT experience after open cavity surgery outlined the cavity and PTV more consistently (conformity indexgen: 0.52 vs. 0.48 and 0.59 vs. 0.55 for preimplant and postimplant cavities). For all volumes, the mean Vmax/Vmin was 2.2 vs. 2.8. Having used guidelines all conformity indices increased significantly. For cavity, the increase was 14% and 11%, whereas for the PTV, 28% and 17% on the preimplant and postimplant CT images, respectively. A strong correlation was found between consistency of contours and cavity visualization score. CONCLUSIONS Simple guidelines on defining the lumpectomy cavity significantly increased the consistency of contouring. Reliable consistency of target volume definition can be expected only for good cavity visibility.

Limitation of seedling growth by potassium and magnesium supply for two ectomycorrhizal tree species of a Central African rain forest and its implication for their recruitment

In the ectomycorrhizal caesalpiniaceous groves of southern Korup National Park, the dominant tree species, Microberlinia bisulcata, displays very poor in situ recruitment compared with its codominant, Tetraberlinia bifoliolata. The reported ex situ experiment tested whether availabilities of soil potassium and magnesium play a role. Seedlings of the two species received applications of K and Mg fertilizer in potted native soil in a local shade house, and their responses in terms of growth and nutrient concentrations were recorded over 2 years. Amended soil concentrations were also determined. Microberlinia responded strongly and positively in its growth to Mg, but less to K; Tetraberlinia responded weakly to both. Added Mg led to strongly increased Mg concentration for Microberlinia while added K changed that concentration only slightly; Tetraberlinia strongly increased its concentration of K with added K, but only somewhat its Mg concentration with added Mg. Additions of Mg and K had small but important antagonistic effects. Microberlinia is Mg-demanding and apparently Mg-limited in Korup soil; Tetraberlinia, whilst K-demanding, appeared not to be K-limited (for growth). Added K enhanced plant P concentrations of both species. Extra applied Mg may also be alleviating soil aluminum toxicity, and hence improving growth indirectly and especially to the benefit of Microberlinia. Mg appears to be essential for Microberlinia seedling growth and its low soil availability in grove soils at Korup may be an important contributing factor to its poor recruitment. Microberlinia is highly shade-intolerant and strongly light-responding, whilst Tetraberlinia is more shade-tolerant and moderately light-responding, which affords an interesting contrast with respect to their differing responses to Mg supply. The study revealed novel aspects of functional traits and likely niche-partitioning among ectomycorrhizal caesalps in African rain forests. Identifying the direct and interacting indirect effects of essential elements on tropical tree seedling growth presents a considerable challenge due the complex nexus of causes involved.

CopywriteR: DNA copy number detection from off-target sequence data.

Current methods for detection of copy number variants (CNV) and aberrations (CNA) from targeted sequencing data are based on the depth of coverage of captured exons. Accurate CNA determination is complicated by uneven genomic distribution and non-uniform capture efficiency of targeted exons. Here we present CopywriteR, which eludes these problems by exploiting 'off-target' sequence reads. CopywriteR allows for extracting uniformly distributed copy number information, can be used without reference, and can be applied to sequencing data obtained from various techniques including chromatin immunoprecipitation and target enrichment on small gene panels. CopywriteR outperforms existing methods and constitutes a widely applicable alternative to available tools.

Radiochemical determination of 129I and 36Cl in MEGAPIE, a proton irradiated lead-bismuth eutectic spallation target

The concentrations of the long-lived nuclear reaction products 129I and 36Cl have been measured in samples from the MEGAPIE liquid metal spallation target. Samples from the bulk target material (lead-bismuth eutectic, LBE), from the interface of the metal free surface with the cover gas, from LBE/steel interfaces and from noble metal absorber foils installed in the cover gas system were analysed using Accelerator Mass Spectrometry at the Laboratory of Ion beam Physics at ETH Zürich. The major part of 129I and 36Cl was found accumulated on the interfaces, particularly at the interface of LBE and the steel walls of the target container, while bulk LBE samples contain only a minor fraction of these nuclides. Both nuclides were also detected on the absorber foils to a certain extent (≪ 1% of the total amount). The latter number is negligible concerning the radio-hazard of the irradiated target material; however it indicates a certain affinity of the absorber foils for halogens, thus proving the principle of using noble metal foils for catching these volatile radionuclides. The total amounts of 129I and 36Cl in the target were estimated from the analytical data by averaging within the different groups of samples and summing up these averages over the total target. This estimation could account for about half of the amount of 129I and 36Cl predicted to be produced using nuclear physics modelling codes for both nuclides. The significance of the results and the associated uncertainties are discussed.

Canopy gaps promote selective stem-cutting by small mammals of two dominant tree species in an African lowland forest: the importance of seedling chemistry

Small mammals can impede tree regeneration by injuring seedlings and saplings in several ways. One fatal way is by severing their stems, but apparently this type of predation is not well-studied in tropical rain forest. Here, we report on the incidence of 'stem-cutting' to new, wild seedlings of two locally dominant, canopy tree species monitored in 40 paired forest understorey and gap-habitat areas in Korup, Cameroon following a 2007 masting event. In gap areas, which are required for the upward growth and sapling recruitment of both species, 137 seedlings of the long-lived, light-demanding, fast-growing large tropical tree (Microberlinia bisulcata) were highly susceptible to stem-cutting (83% of deaths) - it killed 39% of all seedlings over a c. 2-y period. In stark contrast, seedlings of the more shade-tolerant, slower-growing tree species (Tetraberlinia bifoliolata) were hardly attacked (4.3%). In the understorey, however, stem-cutting was virtually absent. Across the gap areas, the incidence of stem-cutting of M. bisulcata seedlings showed significant spatial variation that could not be explained significantly by either canopy openness or Janzen-Connell type effects (proximity and basal area of conspecific adult trees). To examine physical and chemical traits that might explain the species difference to being cut, bark and wood tissues were collected from a separate sample of seedlings in gaps (i.e. not monitored for stem-cutting). These analyses suggested that, compared with T. bifoliolata, the lower stem density, higher Mg and K and fatty acid concentrations in bark, and fewer phenolic and terpene compounds in M. bisulcata seedlings made them more palatable and attractive to small-mammal predators, likely rodents. We conclude that selective stem-cutting is a potent countervailing force to the current local canopy dominance of the grove-forming M. bisulcata by limiting the recruitment and abundance of its saplings. Given the ubiquity of gaps and ground-dwelling rodents in pantropical forests, it would be surprising if this form of lethal browsing was restricted to Korup.

Expected impact of applying new 2013 AHA/ACC cholesterol guidelines criteria on the recommended lipid target achievement after acute coronary syndromes.

BACKGROUND 2013 AHA/ACC guidelines on the treatment of cholesterol advised to tailor high-intensity statin after ACS, while previous ATP-III recommended titration of statin to reach low-density lipoprotein cholesterol (LDL-C) targets. We simulated the impact of this change of paradigm on the achievement of recommended targets. METHODS Among a prospective cohort study of consecutive patients hospitalized for ACS from 2009 to 2012 at four Swiss university hospitals, we analyzed 1602 patients who survived one year after recruitment. Targets based on the previous guidelines approach was defined as (1) achievement of LDL-C target < 1.8 mmol/l, (2) reduction of LDL-C ≥ 50% or (3) intensification of statin in patients who did not reach LDL-C targets. Targets based on the 2013 AHA/ACC guidelines approach was defined as the maximization of statin therapy at high-intensity in patients aged ≤75 years and moderate- or high-intensity statin in patients >75 years. RESULTS 1578 (99%) patients were prescribed statin at discharge, with 1120 (70%) at high-intensity. 1507 patients (94%) reported taking statin at one year, with 909 (57%) at high-intensity. Among 482 patients discharged with sub-maximal statin, intensification of statin was only observed in 109 patients (23%). 773 (47%) patients reached the previous LDL-C targets, while 1014 (63%) reached the 2013 AHA/ACC guidelines targetsone year after ACS (p value < 0.001). CONCLUSION The application of the new 2013 AHA/ACC guidelines criteria would substantially increase the proportion of patients achieving recommended lipid targets one year after ACS. Clinical trial number, NCT01075868.

Neural Regulation of Pancreatic Cancer: A Novel Target for Intervention

The tumor microenvironment is known to play a pivotal role in driving cancer progression and governing response to therapy. This is of significance in pancreatic cancer where the unique pancreatic tumor microenvironment, characterized by its pronounced desmoplasia and fibrosis, drives early stages of tumor progression and dissemination, and contributes to its associated low survival rates. Several molecular factors that regulate interactions between pancreatic tumors and their surrounding stroma are beginning to be identified. Yet broader physiological factors that influence these interactions remain unclear. Here, we discuss a series of preclinical and mechanistic studies that highlight the important role chronic stress plays as a physiological regulator of neural-tumor interactions in driving the progression of pancreatic cancer. These studies propose several approaches to target stress signaling via the β-adrenergic signaling pathway in order to slow pancreatic tumor growth and metastasis. They also provide evidence to support the use of β-blockers as a novel therapeutic intervention to complement current clinical strategies to improve cancer outcome in patients with pancreatic cancer.

Detecting single-target changes in multiple object tracking: The case of peripheral vision

In the current study it is investigated whether peripheral vision can be used to monitor multi-ple moving objects and to detect single-target changes. For this purpose, in Experiment 1, a modified MOT setup with a large projection and a constant-position centroid phase had to be checked first. Classical findings regarding the use of a virtual centroid to track multiple ob-jects and the dependency of tracking accuracy on target speed could be successfully replicat-ed. Thereafter, the main experimental variations regarding the manipulation of to-be-detected target changes could be introduced in Experiment 2. In addition to a button press used for the detection task, gaze behavior was assessed using an integrated eye-tracking system. The anal-ysis of saccadic reaction times in relation to the motor response shows that peripheral vision is naturally used to detect motion and form changes in MOT because the saccade to the target occurred after target-change offset. Furthermore, for changes of comparable task difficulties, motion changes are detected better by peripheral vision than form changes. Findings indicate that capabilities of the visual system (e.g., visual acuity) affect change detection rates and that covert-attention processes may be affected by vision-related aspects like spatial uncertainty. Moreover, it is argued that a centroid-MOT strategy might reduce the amount of saccade-related costs and that eye-tracking seems to be generally valuable to test predictions derived from theories on MOT. Finally, implications for testing covert attention in applied settings are proposed.

Measurements of neutrino oscillation in appearance and disappearance channels by the T2K experiment with 6.6×10²⁰ protons on target

We report on measurements of neutrino oscillation using data from the T2K long-baseline neutrino experiment collected between 2010 and 2013. In an analysis of muon neutrino disappearance alone, we find the following estimates and 68% confidence intervals for the two possible mass hierarchies: Normal Hierarchy: sin²θ₂₃= 0.514+0.055−0.056 and ∆m²_32 = (2.51 ± 0.10) × 10⁻³ eV²/c⁴ Inverted Hierarchy: sin²θ₂₃= 0.511 ± 0.055 and ∆m²_13 = (2.48 ± 0.10) × 10⁻³ eV²/c⁴ The analysis accounts for multi-nucleon mechanisms in neutrino interactions which were found to introduce negligible bias. We describe our first analyses that combine measurements of muon neutrino disappearance and electron neutrino appearance to estimate four oscillation parameters, |∆m^2|, sin²θ₂₃, sin²θ₁₃, δCP , and the mass hierarchy. Frequentist and Bayesian intervals are presented for combinations of these parameters, with and without including recent reactor measurements. At 90% confidence level and including reactor measurements, we exclude the region δCP = [0.15, 0.83]π for normal hierarchy and δCP = [−0.08, 1.09]π for inverted hierarchy. The T2K and reactor data weakly favor the normal hierarchy with a Bayes Factor of 2.2. The most probable values and 68% 1D credible intervals for the other oscillation parameters, when reactor data are included, are: sin²θ₂₃= 0.528+0.055−0.038 and |∆m²_32| = (2.51 ± 0.11) × 10⁻³ eV²/c⁴.

Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI) assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ), a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC50 of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors.

Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.