Chromothripsis drives the evolution of gene amplification in cancer.

Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1-3, and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakage-fusion-bridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions.

The use of a tibial stem in primary total knee arthroplasty for patients with a frontal deformity of more than ten degrees reduces the rate of postoperative pain.

BACKGROUND: The use of a tibial stem for large deformities (> 10°) would reduce the incidence of pain. The aim of this study was to compare the effect of tibial stem on postoperative pain and aseptic loosening at the tibia in patients with a preoperative deformity > 10° in the frontal plane at 2 years follow-up. METHODS: This was a retrospective single-center case-control study. Ninety-eight patients with deformities greater than 10° in the frontal plane and a BMI > 30 kg/m2 who had undergone posterior-stabilized (PS) total knee arthroplasty (TKA) with a tibial stem were matched using a propensity score to 98 patients who had undergone PS TKA without a tibial stem. The primary endpoint was the pain rate at 2 years. The secondary endpoints were the rate of aseptic loosening of the tibia at 2 years post-operatively. RESULTS: A significant difference was found in the rate of postoperative pain at 2 years. It was higher in the group without tibial stem compared with the group with tibial stem (41.8% vs 17.3%, p = 0.0003). In the group without tibial stem, 24.4% of pain was mild, 61% moderate and no severe pain. In the tibial stem group, 47.1% of pain was mild, 41.2% moderate and no severe pain. A radiolucent line (RLL) was present at 2 years in 26.5% of prostheses in the without tibial stem group and in 9.2% of prostheses in the tibial stem group (p = 0.002). There was no difference between the two groups in terms of aseptic loosening. CONCLUSION: The use of a tibial stem in primary TKA in patients with frontal deformities greater than 10° reduces postoperative pain and the presence of radiolucent lines.

Rating norms should be calculated from cumulative link mixed effects models.

Studies which provide norms of Likert ratings typically report per-item summary statistics. Traditionally, these summary statistics comprise the mean and the standard deviation (SD) of the ratings, and the number of observations. Such summary statistics can preserve the rank order of items, but provide distorted estimates of the relative distances between items because of the ordinal nature of Likert ratings. Inter-item relations in such ordinal scales can be more appropriately modelled by cumulative link mixed effects models (CLMMs). In a series of simulations, and with a reanalysis of an existing rating norms dataset, we show that CLMMs can be used to more accurately norm items, and can provide summary statistics analogous to the traditionally reported means and SDs, but which are disentangled from participants' response biases. CLMMs can be applied to solve important statistical issues that exist for more traditional analyses of rating norms.

Expression, purification and characterization of the suppressor of copper sensitivity (Scs) B membrane protein from Proteus mirabilis.

Suppressor of copper sensitivity (Scs) proteins play a role in the bacterial response to copper stress in many Gram-negative bacteria, including in the human pathogen Proteus mirabilis. Recently, the ScsC protein from P. mirabilis (PmScsC) was characterized as a trimeric protein with isomerase activity that contributes to the ability of the bacterium to swarm in the presence of copper. The CXXC motif catalytic cysteines of PmScsC are maintained in their active reduced state by the action of its membrane-bound partner protein, the Proteus mirabilis ScsB (PmScsB). Thus, PmScsC and PmScsB form a redox relay in vivo. The predicted domain arrangement of PmScsB comprises a central transmembrane ß-domain and two soluble, periplasmic domains, the N-terminal α-domain and C-terminal γ-domain. Here, we provide a procedure for the recombinant expression and purification of the full-length PmScsB protein. Using Lemo21 (DE3) cells we expressed PmScsB and, after extraction and purification, we were able to achieve a yield of 3 mg of purified protein per 8 L of bacterial culture. Furthermore, using two orthogonal methods - AMS labelling of free thiols and a scrambled RNase A activity assay - PmScsB is shown to catalyze the reduction of PmScsC. Our results demonstrate that the PmScsC and PmScsB redox relay can be reconstituted in vitro using recombinant full-length PmScsB membrane protein. This finding provides a promising starting point for the in vitro biochemical and structural characterization of the P. mirabilis ScsC and ScsB interaction.

Identification of New Purpuroine Analogues from the Arctic Echinodermata Pteraster militaris That Inhibit FLT3-ITD+ AML Cell Lines.

Isolation of bioactive products from the marine environment is considered a very promising approach to identify new compounds that can be used for further drug development. In this work we have isolated three new compounds from the purpuroine family by mass-guided preparative HPLC; purpuroine K-M. These compounds where screened for antibacterial- and antifungal activity, antibiofilm formation and anti-cell proliferation activity. Additionally, apoptosis-, cell cycle-, kinase binding- and docking studies were performed to evaluate the mechanism-of-action. None of the compounds showed activity in antibacterial-, antibiofilm- or antifungal assays. However, one of the isolated compounds, purpuroine K, showed activity against two cell lines, MV-4-11 and MOLM-13, two AML cell lines both carrying the FTL3-ITD mutation. In MV-4-11 cells, purpuroine K was found to increase apoptosis and arrest cells cycle in G1/G0, which is a common feature of FLT3 inhibitors. Interactions between purpuroine K and the FLT3 wild type or FLT3 ITD mutant proteins could however not be elucidated in our kinase binding and docking studies. In conclusion, we have isolated three novel molecules, purpuroine K-M, one of which (purpuroine K) shows a potent activity against FLT3-ITD mutated AML cell lines, however, the molecular target(s) of purpuroine K still need to be further investigated.

Bioinformatic and experimental evidence for suicidal and catalytic plant THI4s.

Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsistz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC-MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mechanism in planta. Unexpectedly, transcriptome data mining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in developing grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thiazole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain-filling period.

Real-time supersonic jet noise predictions from near-field sensors with a wavepacket model.

Parabolized stability equations (PSE) have been shown to model wavepackets and, consequently, the near-field of turbulent jets with reasonable accuracy. In this work, PSE were employed to obtain a reduced-order model that could estimate both the fluid-dynamic and the acoustic fields of a supersonic jet in a computationally efficient approximation for resolvent-based estimation based on a single input. From the unsteady pressure data at an input position, the time-domain pressure field was estimated using transfer functions obtained using PSE and a data-driven method based on a well-validated large-eddy simulation (LES). The prediction scheme employed is a single-input single-output, linear model. The unsteady pressure predicted by the PSE showed good agreement with the LES results, especially if the input position is outside the mixing layer, where the prediction capabilities of the PSE are comparable to those of the data-driven transfer functions. The good agreement indicates that PSE could not only be used to predict the sound generation but also to open up different potentialities to attenuate the noise by flow control. The exploration of the regions where the method displayed good agreement, which are presented in this work, can guide the positioning of the sensors for experimental implementation of closed-loop control in a jet.

A Guardian Angel Phosphatase for Mainline Carbon Metabolism.

It is increasingly clear that many metabolic enzymes mistakenly form minor but toxic side-products that must be eliminated to maintain normal fluxes. Collard et al. show that this is true of two iconic glycolytic enzymes, and that a hitherto somewhat mysterious phosphatase rescues central carbon metabolism from their mistakes.

Clathrin and AP1 are required for apical sorting of glycosyl phosphatidyl inositol-anchored proteins in biosynthetic and recycling routes in Madin-Darby canine kidney cells.

Recently, studies in animal models demonstrate potential roles for clathrin and AP1 in apical protein sorting in epithelial tissue. However, the precise functions of these proteins in apical protein transport remain unclear. Here, we reveal mistargeting of endogenous glycosyl phosphatidyl inositol-anchored proteins (GPI-APs) and soluble secretory proteins in Madin-Darby canine kidney (MDCK) cells upon clathrin heavy chain or AP1 subunit knockdown (KD). Using a novel directional endocytosis and recycling assay, we found that these KD cells are not only affected for apical sorting of GPI-APs in biosynthetic pathway but also for their apical recycling and basal-to-apical transcytosis routes. The apical distribution of the t-SNARE syntaxin 3, which is known to be responsible for selective targeting of various apical-destined cargo proteins in both biosynthetic and endocytic routes, is compromised suggesting a molecular explanation for the phenotype in KD cells. Our results demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI-APs in polarized MDCK cells.

Healthy aging delays the neural processing of face features relevant for behavior by 40 ms.

Fast and accurate face processing is critical for everyday social interactions, but it declines and becomes delayed with age, as measured by both neural and behavioral responses. Here, we addressed the critical challenge of understanding how aging changes neural information processing mechanisms to delay behavior. Young (20-36 years) and older (60-86 years) adults performed the basic social interaction task of detecting a face versus noise while we recorded their electroencephalogram (EEG). In each participant, using a new information theoretic framework we reconstructed the features supporting face detection behavior, and also where, when and how EEG activity represents them. We found that occipital-temporal pathway activity dynamically represents the eyes of the face images for behavior ~170 ms poststimulus, with a 40 ms delay in older adults that underlies their 200 ms behavioral deficit of slower reaction times. Our results therefore demonstrate how aging can change neural information processing mechanisms that underlie behavioral slow down.

Parts-Prospecting for a High-Efficiency Thiamin Thiazole Biosynthesis Pathway.

Plants synthesize the thiazole precursor of thiamin (cThz-P) via THIAMIN4 (THI4), a suicide enzyme that mediates one reaction cycle and must then be degraded and resynthesized. It has been estimated that this THI4 turnover consumes 2% to 12% of the maintenance energy budget and that installing an energy-efficient alternative pathway could substantially increase crop yield potential. Available data point to two natural alternatives to the suicidal THI4 pathway: (i) nonsuicidal prokaryotic THI4s that lack the active-site Cys residue on which suicide activity depends, and (ii) an uncharacterized thiazole synthesis pathway in flowers of the tropical arum lily Caladium bicolor that enables production and emission of large amounts of the cThz-P analog 4-methyl-5-vinylthiazole (MVT). We used functional complementation of an Escherichia coli ΔthiG strain to identify a nonsuicidal bacterial THI4 (from Thermovibrio ammonificans) that can function in conditions like those in plant cells. We explored whether C. bicolor synthesizes MVT de novo via a novel route, via a suicidal or a nonsuicidal THI4, or by catabolizing thiamin. Analysis of developmental changes in MVT emission, extractable MVT, thiamin level, and THI4 expression indicated that C. bicolor flowers make MVT de novo via a massively expressed THI4 and that thiamin is not involved. Functional complementation tests indicated that C. bicolor THI4, which has the active-site Cys needed to operate suicidally, may be capable of suicidal and - in hypoxic conditions - nonsuicidal operation. T. ammonificans and C. bicolor THI4s are thus candidate parts for rational redesign or directed evolution of efficient, nonsuicidal THI4s for use in crop improvement.

Viscerotropic disease and acute uveitis following yellow fever vaccination: a case report.

BACKGROUND: Yellow fever vaccine exists for over 80 years and is considered to be relatively safe. However, in rare cases it can produce serious neurotropic and viscerotropic complications. We report a case of a patient who presented both viscerotropic and neurological manifestations after yellow fever vaccination. CASE PRESENTATION: We describe the case of a 37 years old man who developed after the yellow fever vaccination a yellow fever vaccine-associated viscerotropic disease followed by acute uveitis. Prolonged detection of yellow fever RNA in blood and urine was consistent with yellow fever vaccine-associated adverse event. The final outcome was good, although with persistent fatigue over a few months. CONCLUSIONS: Even if the yellow fever vaccine is relatively safe, physicians should be aware of its possible serious adverse effects.

Is recipient's body mass index a determinant of short term complications in early renal transplantation?

OBJECTIVES: Obesity prevalence has increased over the past 20 years in the general population and among kidney transplant recipients. General surgical belief is that obesity increases surgical difficulty. The aim of this study was to assess the impact of Body Mass Index (BMI) on perioperative complications. METHODS: All kidney transplantations performed in adults in our centre from 2006 to 2011 were analysed. Data on patients' characteristics, surgical protocol, intra and postoperative complications and renal function were collected. Patients were divided into 4 groups as follows: underweight (BMI<18.5kg/m2), normal weight (18.5kg/m2≤BMI<25kg/m2), overweight (25kg/m2≤BMI<30kg/m2) and obese (BMI≥30kg/m2). We also studied the impact of BMI on complications using it as a continuous variable to identify potential threshold values. RESULTS: Among 694 patients included, 52% had normal BMI, 7%, 31% and 9% were underweight, overweight and obese, respectively. In multivariate analysis, overweight was significantly associated with longer operative time compared to normal-weight patients (estimated mean difference of 10,4min, 95% confidence interval (CI) [4.0; 16.9]) and obesity was associated with an increased risk of wound dehiscence (odds ratio 3.1, 95%CI [1.3; 7.3] compared with normal-weight patients). Considering BMI as a continuous variable, the risk of parietal dehiscence significantly increased beyond a BMI of 26kg/m2, intraoperative blood loss and the risk of ureteral stenosis beyond 32kg/m2 and the risk of abdominal wall hematoma beyond a BMI of 34kg/m2. CONCLUSIONS: We found BMI thresholds above which intraoperative blood loss and the risk of parietal dehiscence, ureteral stenosis, and parietal hematoma significantly increased. LEVEL OF EVIDENCE: 3.

Modelling of jet noise: a perspective from large-eddy simulations.

In the last decade, many research groups have reported predictions of jet noise using high-fidelity large-eddy simulations (LES) of the turbulent jet flow and these methods are beginning to be used more broadly. A brief overview of the publications since the review by Bodony & Lele (2008, AIAA J. 56, 346-380) is undertaken to assess the progress and overall contributions of LES towards a better understanding of jet noise. In particular, we stress the meshing, numerical and modelling advances which enable detailed geometric representation of nozzle shape variations intended to impact the noise radiation, and sufficiently accurate capturing of the turbulent boundary layer at the nozzle exit. Examples of how LES is currently being used to complement experiments for challenging conditions (such as highly heated pressure-mismatched jets with afterburners) and guide jet modelling efforts are highlighted. Some of the physical insights gained from these numerical studies are discussed, in particular on crackle, screech and shock-associated noise, impingement tones, acoustic analogy models, wavepackets dynamics and resonant acoustic waves within the jet core. We close with some perspectives on the remaining challenges and upcoming opportunities for future applications. This article is part of the theme issue 'Frontiers of aeroacoustics research: theory, computation and experiment'.

An unusual diphosphatase from the PhnP family cleaves reactive FAD photoproducts.

Flavins are notoriously photolabile, but while the photoproducts derived from the iso-alloxazine ring are well known the other photoproducts are not. In the case of FAD, typically the main cellular flavin, the other photoproducts are predicted to include four- and five-carbon sugars linked to ADP. These FAD photoproducts were shown to be potent glycating agents, more so than ADP-ribose. Such toxic compounds would require disposal via an ADP-sugar diphosphatase or other route. Comparative analysis of bacterial genomes uncovered a candidate disposal gene that is chromosomally clustered with genes for FAD synthesis or transport and is predicted to encode a protein of the PhnP cyclic phosphodiesterase family. The representative PhnP family enzyme from Koribacter versatilis (here named Fpd, FAD photoproduct diphosphatase) was found to have high, Mn2+-dependent diphosphatase activity against FAD photoproducts, FAD, and ADP-ribose, but almost no phosphodiesterase activity against riboflavin 4',5'-cyclic phosphate, a chemical breakdown product of FAD. To provide a structural basis of the unique Fpd activity, the crystal structure of K. versatilis Fpd was determined. The results place Fpd in the broad metallo-ß-lactamase-like family of hydrolases, a diverse family commonly using two metals for hydrolytic catalysis. The active site of Fpd contains two Mn2+ ions and a bound phosphate, consistent with a diphosphatase mechanism. Our results characterize the first PhnP family member that is a diphosphatase rather than a cyclic phosphodiesterase and suggest its involvement in a cellular damage-control system that efficiently hydrolyzes the reactive, ADP-ribose-like products of FAD photodegradation.

Three-dimensional Frankfort horizontal plane for 3D cephalometry: a comparative assessment of conventional versus novel landmarks and horizontal planes.

Background: To assess the reproducibility of landmarks in three dimensions that determine the Frankfort horizontal plane (FH) as well as two new landmarks, and to evaluate the angular differences of newly introduced planes to the FH. Methods: Three-dimensional (3D) surface models were created from CBCT scans of 26 dry human skulls. Porion (Po), orbitale (Or), internal acoustic foramen (IAF), and zygomatico-maxillary suture (ZyMS) were indicated in the software by three observers twice with a 4-week interval. Angles between two FHs (FH 1: Or-R, Or-L, mid-Po; FH 2: Po-R, Po-L, mid-Or) and between FHs and new planes (Plane 1-6) were measured. Coordinates were exported to a spreadsheet. A statistical analysis was performed to define the landmark reproducibility and 3D angles. Results: Intra- and inter-observer landmark reproducibility showed mean difference more than 1 mm for x-coordinates of all landmarks except IAF. IAF showed significantly better reproducibility than other landmarks (P < 0.0018). The mean angular difference between FH 1 and FH 2 was 0.7 degrees. Plane 3, connecting Or-R, Or-L and mid-IAF, and Plane 4, connecting Po-R, Po-L and mid-ZyMS, both showed an angular difference of less than 1 degree when compared to FHs. Conclusions: This study revealed poor reproducibility of the traditional FH landmarks on the x-axis and good reproducibility of a new landmark tested to replace Po, the IAF. Yet, Or showed superior results compared to ZyMS. The potential of using new horizontal planes was demonstrated. Future studies should focus on identification of a valid alternative for Or and ZyMS and on clinical implementation of the findings.

[Partial nephrectomy for renal masses >7cm: Morbidity, oncological and functional outcomes (UroCCR-7 study)]. / Néphrectomie partielle pour tumeur de plus de 7cm : morbidité, résultats oncologiques et fonctionnels (UroCCR-7 study).

OBJECTIVES: To describe the morbidity, mortality, oncological and functional results of Partial nephrectomy (PN) for the treatment of renal tumors of more than 7cm. MATERIAL AND METHODS: Thirty-seven partial nephrectomies for tumors larger than 7cm operated in a single center between 1987 and 2016 were analyzed retrospectively. The pre, per and postoperative clinico-biological data were collected within the UroCCR database. The GFR was assessed at day 5, 1 month and last follow-up. Intraoperative and postoperative surgical complications, the recurrence rate and the overall and specific mortality were collected. RESULTS: The mean age of the patients was 57 years (44-68). The preoperative GFR and the median tumor size were 80mL/min and 8cm, respectively. The indication for surgery was elective in 21 cases (60%) and 19 tumors (54%) were malignant. Postoperative complications occurred in 24,3 cases (24.3%). The median post-operative GFR was respectively 77mL/min, 80mL/min and 77mL/min at day 5, 1month and at last follow-up. With a median follow up of 31 months [1-168], 5 patients (26,3%) had metastatic progression of whom 1 (5.3%) had a concomitant local recurrence and 3 (15.8%) had died from cancer. CONCLUSION: This study confirms the feasibility of PN for large tumors with acceptable morbidity, limited risk of local recurrence and excellent functional results. LEVEL OF EVIDENCE: 4.

Beyond differences in means: robust graphical methods to compare two groups in neuroscience.

If many changes are necessary to improve the quality of neuroscience research, one relatively simple step could have great pay-offs: to promote the adoption of detailed graphical methods, combined with robust inferential statistics. Here, we illustrate how such methods can lead to a much more detailed understanding of group differences than bar graphs and t-tests on means. To complement the neuroscientist's toolbox, we present two powerful tools that can help us understand how groups of observations differ: the shift function and the difference asymmetry function. These tools can be combined with detailed visualisations to provide complementary perspectives about the data. We provide implementations in R and MATLAB of the graphical tools, and all the examples in the article can be reproduced using R scripts.