Explore the vast range of titles available.

Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and transcription of the RNA genome constitute essential processes performed by the virally encoded multi-domain RNA-dependent RNA polymerase. Here, we describe the complete high-resolution cryo-EM structure of La Crosse virus polymerase. It reveals the presence of key protruding C-terminal domains, notably the cap-binding domain, which undergoes large movements related to its role in transcription initiation, and a zinc-binding domain that displays a fold not previously observed. We capture the polymerase structure at pre-initiation and elongation states, uncovering the coordinated movement of the priming loop, mid-thumb ring linker and lid domain required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. These structural details and the observed dynamics of key functional elements will be instrumental for structure-based development of polymerase inhibitors. RNA-dependent RNA polymerases from segmented negative stranded RNA viruses catalyze genome replication and viral transcription. Here, the authors present the cryo-EM structure of full-length La Crosse virus polymerase and structurally characterize the pre-initiation and elongation states, which is of interest for the development of polymerase inhibitors.

Segmented negative strand RNA viruses of the arena-, bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism. This involves cleavage of host mRNAs close to their capped 5' end by an endonuclease (EN) domain located in the N-terminal region of the viral polymerase. We present the structure of the cap-snatching EN of Hantaan virus, a bunyavirus belonging to hantavirus genus. Hantaan EN has an active site configuration, including a metal co-ordinating histidine, and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs (orthobunyavirus and orthomyxovirus respectively), but is more active in cleaving a double stranded RNA substrate. In contrast, Lassa arenavirus EN has only acidic metal co-ordinating residues. We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan, La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive. The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase.

The nuclear RNA exosome complex mediates the processing of structured RNAs and the decay of aberrant non-coding RNAs, an important function particularly in human cells. Most mechanistic studies to date have focused on the yeast system. Here, we reconstituted and studied the properties of a recombinant 14-subunit human nuclear exosome complex. In biochemical assays, the human exosome embeds a longer RNA channel than its yeast counterpart. The 3.8 Å resolution cryo-EM structure of the core complex bound to a single-stranded RNA reveals that the RNA channel path is formed by two distinct features of the hDIS3 exoribonuclease: an open conformation and a domain organization more similar to bacterial RNase II than to yeast Rrp44. The cryo-EM structure of the holo-complex shows how obligate nuclear cofactors position the hMTR4 helicase at the entrance of the core complex, suggesting a striking structural conservation from lower to higher eukaryotes.

The RNA exosome is a versatile ribonuclease. In the nucleoplasm of mammalian cells, it is assisted by its adaptors the Nuclear EXosome Targeting (NEXT) complex and the PolyA eXosome Targeting (PAXT) connection. Via its association with the ARS2 and ZC3H18 proteins, NEXT/exosome is recruited to capped and short unadenylated transcripts. Conversely, PAXT/exosome was considered to target longer and adenylated substrates via their poly(A) tails. Here, mutational analysis of the core PAXT component ZFC3H1 uncovers a separate branch of the PAXT pathway, which targets short adenylated RNAs and relies on a direct ARS2-ZFC3H1 interaction. We further demonstrate that similar acidic-rich short linear motifs of ZFC3H1 and ZC3H18 compete for a common ARS2 epitope. Consequently, while promoting NEXT function, ZC3H18 antagonizes PAXT activity. We suggest that this unprecedented organization of RNA decay complexes provides co-activation of NEXT and PAXT at loci with abundant production of short exosome substrates.

Bunyavirales is an order of segmented negative stranded RNA viruses comprising several life-threatening pathogens such as Lassa fever virus (Arenaviridae), Rift Valley Fever virus (Phenuiviridae) and La Crosse virus (LACV, Peribunyaviridae) against which neither specific treatment nor licenced vaccine is available. Replication and transcription of Bunyavirales genome constitute essential reactions of their viral cycle that are catalysed by the virally encoded RNA-dependent RNA polymerase or L protein. Here we describe the complete high-resolution cryo-EM structure of the full-length (FL) LACV-L protein. It reveals the presence of key C-terminal domains, notably the cap-binding domain that undergoes large movements related to its role in transcription initiation and a zinc-binding domain that displays a fold not previously observed. We capture the structure of LACV-L FL in two functionally relevant states, pre-initiation and elongation, that reveal large conformational changes inherent to its function. We uncover the coordinated movement of the polymerase priming loop, lid domain and C-terminal region required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. The revealed structural details and dynamics of functional elements will be instrumental for structure-based development of compounds that inhibit RNA synthesis by the polymerase.

Recently introduced high-efficiency (HE) SPECT cameras with solid-state CZT detectors have been shown to decrease imaging time and reduce radiation exposure to patients. An automated, computer-derived quantification of HE MPI has been shown to correlate well with coronary angiography on one HE SPECT camera system (D-SPECT), but has not been compared to visual interpretation on any of the HE SPECT platforms. Patients undergoing a clinically indicated Tc-99m sestamibi HE SPECT (GE Discovery 530c with supine and prone imaging) study over a 1-year period followed by a coronary angiogram within 2 months were included. Only patients with a history of CABG surgery were excluded. Both MPI studies and coronary angiograms were reinterpreted by blinded readers. One hundred and twenty two very low (risk of CAD < 5%) or low (risk of CAD < 10%) likelihood subjects with normal myocardial perfusion were used to create normal reference limits. Computer-derived quantification of the total perfusion deficit at stress and rest was obtained with QPS software. The visual and automated MPI quantification were compared to coronary angiography (≥70% luminal stenosis) by receiver operating curve (ROC) analysis. Of the 3,111 patients who underwent HE SPECT over a 1-year period, 160 patients qualified for the correlation study (66% male, 52% with a history of CAD). The ROC area under the curve (AUC) was similar for both the automated and the visual interpretations using both supine only and combined supine and prone images (0.69-0.74). Using thresholds determined from sensitivity and specificity curves, the automated reads showed higher specificity (59%-67% vs 27%-60%) and lower sensitivity (71%-72% vs 79%-93%) than the visual reads. By including prone images sensitivity decreased slightly but specificity increased for both. By excluding patients with known CAD and cardiomyopathies, AUC and specificity increased for both techniques (0.72-0.82). The use of a difference score to evaluate ischemic burden resulted in lower sensitivities but higher specificities for both automated and visual quantification. There was good agreement between the visual interpretation and automated quantification in the entire cohort of 160 unselected consecutive patients (r = 0.70-0.81, P < .0001). Automated and visual quantification of high-efficiency SPECT MPI with the GE Discovery camera provides similar overall diagnostic accuracy when compared to coronary angiography. There was good correlation between the two methods of assessment. Combined supine and prone stress imaging provided the best diagnostic accuracy.

While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Supported through the European Union’s Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers, and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e. performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system and assessment services for those research units that successfully implement the quality system and seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use.

Background Current guidelines of Food and Drug Administration for the evaluation of SPECT myocardial perfusion imaging (MPI) in clinical trials recommend independent visual interpretation by multiple experts. Few studies have addressed whether quantitative SPECT MPI assessment would be more reproducible for this application. Methods and Results We studied 31 patients (age 68 ± 13, 25 male) with abnormal stress MPI who underwent repeat exercise (n = 11) or adenosine (n = 20) MPI within 9-22 months (mean 14.9 ± 3.8 months) and had no interval revascularization or myocardial infarction and no change in symptoms, stress type, rest or stress ECG, or clinical response to stress on the second study. Visual interpretation per FDA Guidance used 17-segment, 5-point scoring by two independent expert readers with overread of discordance by a third expert, and percent myocardium abnormal was derived from normalized summed scores. The quantitative magnitude of perfusion abnormality was assessed by the total perfusion deficit (TPD), expressing stress, rest, and ischemic perfusion abnormality. High linear correlations were observed between visual and quantitative size of stress, rest, and ischemic defects ( R  = 0.94, 0.92, 0.84). Correlations of two tests were higher by quantitative than by visual methods for stress ( R  = 0.97 vs R  = 0.91, P  = 0.03) and rest defects ( R  = 0.94 vs R  = 0.82, P  = 0.03), respectively, and statistically similar for ischemic defects ( R  = 0.84 vs R  = 0.70, P  = ns). Conclusions In stable patients having serial SPECT MPI, quantification is more reproducible than visual for magnitude of perfusion abnormality, suggesting its superiority for use in randomized clinical trials and monitoring the effects of therapy in an individual patient.

Background Attenuation corrected myocardial perfusion SPECT (AC-MPS) has been demonstrated to improve the specificity of detecting coronary artery disease (CAD) by visual analysis which utilizes both non-corrected (NC) and AC data. However, the combined automated quantification of NC and AC-MPS has not been previously described. We aimed to develop a combined quantitative analysis from AC and NC data to improve the accuracy of automated detection of CAD from AC-MPS. Methods Stress total perfusion deficit (TPD) values were generated by standard analysis for NC (NC-TPD), AC (AC-TPD) and by combined NC-AC analysis (NA-TPD), in which the hypoperfusion severity in each polar map location was defined as the average of AC and NC severity computed by comparison with separate AC and NC normal limits. Ischemic TPD was also calculated as the difference between stress TPD and rest TPD for each measure. Stress/rest Tc-99m sestamibi MPS studies in 650 patients with correlating coronary angiography and in 345 patients with a low-likelihood (LLk) of CAD were used to assess diagnostic performance of combined NC-AC analysis. Results NA-TPD had a higher receiver-operator-characteristic area under the curve (ROC-AUC) (0.87) than NC-TPD (0.85; P  < .01) or AC-TPD (0.85; P  < .01) for detection of stenosis ≥70% in angiographic group. It also had higher specificity (75%) vs NC-TPD (65%; P  < .0001), or AC-TPD (70%; P  = .016). In LLk group, the normalcy rate of NA-TPD (95%) was higher than for NC-TPD (90%; P  < .01) and similar to AC-TPD (94%; P  = NS). NA-TPD had higher ROC-AUC than that for 17-segment expert visual scoring of stress scans in angiographic group (0.84; P  = .01), comparable accuracy (81%) and similar normalcy rates (95% vs 97%; P  = NS). Ischemic TPD by combined NC-AC analysis had higher ROC-AUC than that for any ischemic measure. Similar to stress NA-TPD, it also obtained the similar performance results as compared with ischemic TPD based on NC or AC and higher sensitivity (89% vs 85%; P  = .0295) as compared with ischemic visual score in angiographic group. Conclusion Combined NC-AC MPS quantification using either stress or ischemic TPD shows significant improvements for ROC-AUC and specificity of MPS in the detection of CAD compared with standard NC-MPS or AC-MPS and comparable performance to expert visual scoring. This technique may lead to an enhancement in a fully automated quantification for the perfusion analysis by AC-MPS.