Author Correction: Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia1. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications2. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments3. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids4, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway5. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid6, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases7,8. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency9. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

Dashing Growth Curves: a web application for rapid and interactive analysis of microbial growth curves.

BACKGROUND: Recording and analyzing microbial growth is a routine task in the life sciences. Microplate readers that record dozens to hundreds of growth curves simultaneously are increasingly used for this task raising the demand for their rapid and reliable analysis. RESULTS: Here, we present Dashing Growth Curves, an interactive web application ( http://dashing-growth-curves.ethz.ch/ ) that enables researchers to quickly visualize and analyze growth curves without the requirement for coding knowledge and independent of operating system. Growth curves can be fitted with parametric and non-parametric models or manually. The application extracts maximum growth rates as well as other features such as lag time, length of exponential growth phase and maximum population size among others. Furthermore, Dashing Growth Curves automatically groups replicate samples and generates downloadable summary plots for of all growth parameters. CONCLUSIONS: Dashing Growth Curves is an open-source web application that reduces the time required to analyze microbial growth curves from hours to minutes.

FCM marker importance for MRD assessment in T-cell acute lymphoblastic leukemia: An AIEOP-BFM-ALL-FLOW study group report.

T-lineage acute lymphoblastic leukemia (T-ALL) accounts for about 15% of pediatric and about 25% of adult ALL cases. Minimal/measurable residual disease (MRD) assessed by flow cytometry (FCM) is an important prognostic indicator for risk stratification. In order to assess the MRD a limited number of antibodies directed against the most discriminative antigens must be selected. We propose a pipeline for evaluating the influence of different markers for cell population classification in FCM data. We use linear support vector machine, fitted to each sample individually to avoid issues with patient and laboratory variations. The best separating hyperplane direction as well as the influence of omitting specific markers is considered. Ninety-one bone marrow samples of 43 pediatric T-ALL patients from five reference laboratories were analyzed by FCM regarding marker importance for blast cell identification using combinations of eight different markers. For all laboratories, CD48 and CD99 were among the top three markers with strongest contribution to the optimal hyperplane, measured by median separating hyperplane coefficient size for all samples per center and time point (diagnosis, Day 15, Day 33). Based on the available limited set tested (CD3, CD4, CD5, CD7, CD8, CD45, CD48, CD99), our findings prove that CD48 and CD99 are useful markers for MRD monitoring in T-ALL. The proposed pipeline can be applied for evaluation of other marker combinations in the future.

Association of Pain Location With Computed Tomography Abnormalities in Emergeny Department Patients With Abdominal Pain.

BACKGROUND: Over the last decade the usage of computed tomography (CT) imaging has risen dramatically in emergency department (ED) patients with abdominal pain. Recognizing the potential disadvantages of overuse of CT imaging, efforts are being made to reduce imaging. OBJECTIVE: We determined the operating characteristics for location of abdominal pain for the entities of acute appendicitis, diverticulitis, and intestinal obstruction. We hypothesized that patients with pain localized to the upper abdomen would be less likely to have CT abnormalities than those with lower abdominal pain. METHODS: This is a prospective, observational registry of ED patients with abdominal pain, performed in an academic, suburban ED with an annual census of 110,000. Presence of clinically significant CT abnormalities (e.g., appendicitis, diverticulitis, bowel obstruction) were recorded along with clinical variables including laboratory values, vital signs, reported location of pain, location of tenderness on examination, and physician pretest probability. RESULTS: A convenience sample of 1154 patients was enrolled. Of all patients, 273 cases (24%) had abnormal CT results, including appendicitis (n = 95), diverticulitis (n = 133), and bowel obstruction (n = 49). Right upper quadrant pain was negatively associated with abnormal CT (p = 0.02). Clinician gestalt was highly specific, but lacked sensitivity for the diagnosis of appendicitis, diverticulitis, and obstruction. Twenty-four percent of patients diagnosed with appendicitis had no right lower quadrant pain or tenderness, and 7% of patients with diverticulitis had no left lower quadrant pain or tenderness. CONCLUSIONS: Localization of abdominal pain by history or physical examination is not sufficient to accurately diagnose intra-abdominal pathology, especially cases of acute appendicitis, diverticulitis, or intestinal obstruction.

Lactoferrin is a natural inhibitor of plasminogen activation.

The plasminogen system is essential for dissolution of fibrin clots, and in addition, it is involved in a wide variety of other physiological processes, including proteolytic activation of growth factors, cell migration, and removal of protein aggregates. On the other hand, uncontrolled plasminogen activation contributes to many pathological processes (e.g. tumor cells' invasion in cancer progression). Moreover, some virulent bacterial species (e.g. Streptococci or Borrelia) bind human plasminogen and hijack the host's plasminogen system to penetrate tissue barriers. Thus, the conversion of plasminogen to the active serine protease plasmin must be tightly regulated. Here, we show that human lactoferrin, an iron-binding milk glycoprotein, blocks plasminogen activation on the cell surface by direct binding to human plasminogen. We mapped the mutual binding sites to the N-terminal region of lactoferrin, encompassed also in the bioactive peptide lactoferricin, and kringle 5 of plasminogen. Finally, lactoferrin blocked tumor cell invasion in vitro and also plasminogen activation driven by Borrelia Our results explain many diverse biological properties of lactoferrin and also suggest that lactoferrin may be useful as a potential tool for therapeutic interventions to prevent both invasive malignant cells and virulent bacteria from penetrating host tissues.

Aneuploidy in children with relapsed B-cell precursor acute lymphoblastic leukaemia: clinical importance of detecting a hypodiploid origin of relapse.

Aneuploidy is common in paediatric B-cell precursor acute lymphoblastic leukaemia (ALL). Specific subgroups, such as high hyperdiploidy (>50 chromosomes or DNA Index ≥1·16) and hypodiploidy (<45 chromosomes), predict outcome of patients after primary treatment. Whether aneuploidy has a prognostic value for relapsed disease is yet to be determined. Using DNA index and centromere screening by multiplex ligation-dependent probe amplification, we investigated aneuploidy in 413 children treated for first relapse of B-cell precursor ALL according to the ALL-REZ BFM 2002 protocol. Ten-year event-free survival of patients with high hyperdiploid relapses approached 70%, whereas it was only 40% in low hyperdiploid relapses. Three patients with apparent hyperdiploid relapse had TP53 mutations. In these cases, array-based allelotyping revealed a hypodiploid origin with absence of the hypodiploid founder clone (masked hypodiploidy). Collectively, patients with evident or masked hypodiploid relapses showed an extremely low event-free survival rate of 9%. Importantly, the current relapse risk stratification did not identify cases with masked hypodiploidy as high-risk patients, due to their favourable clinical presentation. In multivariate analysis, hypodiploidy proved to be an independent prognostic factor. This finding supports stratification of relapses with hypodiploid origin into high-risk arms in future trials or allocation of patients to alternative treatment approaches.

Automated Flow Cytometric MRD Assessment in Childhood Acute B- Lymphoblastic Leukemia Using Supervised Machine Learning.

Minimal residual disease (MRD) as measured by multiparameter flow cytometry (FCM) is an independent and strong prognostic factor in B-cell acute lymphoblastic leukemia (B-ALL). However, reliable flow cytometric detection of MRD strongly depends on operator skills and expert knowledge. Hence, an objective, automated tool for reliable FCM-MRD quantification, able to overcome the technical diversity and analytical subjectivity, would be most helpful. We developed a supervised machine learning approach using a combination of multiple Gaussian Mixture Models (GMM) as a parametric density model. The approach was used for finding the weights of a linear combination of multiple GMMs to represent new, "unseen" samples by an interpolation of stored samples. The experimental data set contained FCM-MRD data of 337 bone marrow samples collected at day 15 of induction therapy in three different laboratories from pediatric patients with B-ALL for which accurate, expert-set gates existed. We compared MRD quantification by our proposed GMM approach to operator assessments, its performance on data from different laboratories, as well as to other state-of-the-art automated read-out methods. Our proposed GMM-combination approach proved superior over support vector machines, deep neural networks, and a single GMM approach in terms of precision and average F 1 -scores. A high correlation of expert operator-based and automated MRD assessment was achieved with reliable automated MRD quantification (F 1 -scores >0.5 in more than 95% of samples) in the clinically relevant range. Although best performance was found, if test and training samples were from the same system (i.e., flow cytometer and staining panel; lowest median F 1 -score 0.92), cross-system performance remained high with a median F 1 -score above 0.85 in all settings. In conclusion, our proposed automated approach could potentially be used to assess FCM-MRD in B-ALL in an objective and standardized manner across different laboratories. © 2019 International Society for Advancement of Cytometry.

Assessment of the American College of Radiology Thyroid Imaging Reporting and Data System for Thyroid Nodule Malignancy Risk Stratification in a Pediatric Population.

OBJECTIVE: The purpose of this study is to assess the diagnostic performance of the American College of Radiology (ACR) Thyroid Imaging Reporting and Data System (TIRADS) for malignancy risk in pediatric thyroid nodules. MATERIALS AND METHODS: Two radiologists reviewed ultrasound images of 74 tissue-proven thyroid nodules in 62 children. Points were given for individual features and then added to determine the ACR TI-RADS category, ranging from 1 (benign) to 5 (high suspicion). Kappa coefficients were generated to assess intra- and interobserver agreement. Generalized linear mixed-effects models were used to estimate the odds of malignancy with construction of a supplementary ROC curve. RESULTS: Fifty-four nodules were benign and 20 were malignant, with a median ACR TI-RADS category of 4 (interquartile range, 4-5). Nineteen of 20 (95.0%) malignant nodules were rated as TI-RADS category 4 or 5. There was substantial intraobserver agreement (κ = 0.69-0.77; p < 0.001) and moderate interobserver agreement (κ = 0.37; p = 0.002) for TIRADS category. Univariable analysis showed that, with every 1-unit increase of TI-RADS category, the likelihood of malignancy increased 2.63 times (95% CI, 1.08-6.41; p = 0.03). After adjusting for nodule size, TI-RADS category remained marginally associated with malignancy (adjusted odds ratio, 2.27; 95% CI, 0.93-5.54; p = 0.07). The AUC was 0.75 (95% CI, 0.64-0.86). An optimal cut point of TI-RADS category 5 was selected, with TI-RADS category 5 nodules 10.44 times (95% CI, 2.71-40.21; p < 0.0001) more likely than categories 1-4 nodules to be malignant. CONCLUSION: ACR TI-RADS discriminates well between malignant and benign nodules in a pediatric population, particularly at TI-RADS category 5.

Non-Destructive Testing of Ceramic Knee Implants Using Micro-Computed Tomography.

BACKGROUND: Because of the accumulated numbers and the increasing rate of knee replacement surgeries larger numbers of revision cases are likely. Although the success rate of knee arthroplasties is high, complications like the loosening of the implant necessitate subsequent treatments. Therefore, new concepts such as metal-free ceramic implants are necessary, for example, using zirconium dioxide (ZrO2). Several studies showed that the strength of ceramic ZrO2 implants is equivalent to cobalt-chromium components. METHODS: Non-destructive testing remains challenging due to the high density (6 g/cm³) of ZrO2. In this feasibility study, we investigated 8 tibial and 8 femoral implants respectively using an industrial X-ray micro-computed tomography (XCT) system at a voxel size of 100 µm. We established a non-destructive testing protocol for ceramic knee implants optimizing scanning parameters and sample orientation using CT simulations. Finally, we used an iterative artifact reduction procedure for beam hardening correction. RESULTS: The results show that corrected image data enable the non-destructive inspection of high-density components. In this sample, none of the investigated components show any internal defects like pores or cracks. In general, XCT is a major imaging method that is able to provide a 3-dimensional representation of higher dense objects that allows the inspection of metal or ceramic knee implants. Even though we established an optimized scanning routine for tibial and femoral ceramic components, it is not possible to completely eliminate scanning artifacts of XCT. CONCLUSION: Altogether, after visual inspection, none of the beam-hardening corrected XCT data sets for femoral and tibial implants showed any defects, that is, no inclusions, cracks, or pores were detected. XCT test is therefore an essential addition to the fatigue testing since it is the only non-destroying testing method.

Postoperative CT of the Midfacial Skeleton After Trauma: Review of Normal Appearances and Common Complications.

OBJECTIVE: The objective of this article is to describe the CT appearance of the midfacial skeleton after surgical repair of posttraumatic Le Fort, nasoorbitoethmoidal (NOE), and frontal sinus fractures. Several of the more commonly encountered complications will also be described. CONCLUSION: Surgery after midfacial trauma is aimed at restoring both form and function. Knowledge of the principal tenets of Le Fort, NOE, and frontal sinus fracture repair is vital for radiologists to accurately assess the adequacy of treatment on postoperative CT and provide meaningful reports for the surgeon.

Postoperative CT of the Orbital Skeleton After Trauma: Review of Normal Appearances and Common Complications.

OBJECTIVE: The purpose of this article is to describe both the expected and unexpected imaging features of posttraumatic defects of the orbital skeleton after surgical repair. CONCLUSION: The goal of surgery is to restore the preinjury orbital anatomy to improve function and prevent enophthalmos. Radiologists need to be cognizant of the more frequently encountered operative procedures used for orbital fracture repair, the desired goals of treatment, and common complications. This will permit accurate interpretation of postoperative CT and provide surgeons with clinically useful results.

Effect of Low-Osmolar Intravenous Contrast on Renal Length.

OBJECTIVE: The purpose was to study the effect of low-osmolar nonionic contrast on renal length. MATERIAL AND METHODS: This study included 56 patients (4-phase renal computed tomography [CT] and 4-phase CT urogram [CTU], 19 patients each; split-phase CTU, 18 patients). Three radiologists created the best off-axis plane and renal lengths measured on a postprocessing workstation. Two-way analysis of variance with Bonferroni corrections was performed along with single-sample t tests. RESULTS: Four-phase renal CT and CTU average differences from unenhanced phases were 0.30/0.16 mm (corticomedullary), 0.88/1.33 mm (nephrographic), and 2.17/2.22 mm (delayed). The nephrographic and delayed phases were significantly different from their unenhanced phase (P < 0.01). Nonsignificant differences between the corticomedullary phase and the unenhanced phase were observed (P = 0.217, 4-phase renal CT; P = 0.232, 4-phase CTU). The split-phase CTU average difference in the enhanced phase was 1.36 mm (P < 0.001). CONCLUSION: Renal length increases 1 to 2 mm with low-osmolar nonionic contrast.

Molecular analysis of sunitinib resistant renal cell carcinoma cells after sequential treatment with RAD001 (everolimus) or sorafenib.

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 µM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 µM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short-term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross-resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR-inhibitor for second-line therapy could be the strategy of choice after first-line sunitinib failure.

Oligoarthritis caused by Borrelia bavariensis, Austria, 2014.

A case of Lyme oligoarthritis occurred in an 11-year-old boy in Vienna, Austria. DNA of Borrelia bavariensis was detected by PCR in 2 aspirates obtained from different joints. Complete recovery was achieved after a 4-week course with amoxicillin. Lyme arthritis must be considered in patients from Europe who have persisting joint effusions.

The management of the access tract after percutaneous nephrolithotomy.

PURPOSE: To describe the evolution of the current technique in percutaneous nephrolithotomy (PCNL) with a special focus on access tract closure techniques. METHODS: A systematic review of outcomes and complications of tubeless PCNL was conducted using the MEDLINE and Pubmed databases between 1976 and 2014. RESULTS: During the past decade, PCNL underwent fundamental modifications due to miniaturization of the instruments and advancements in technique. The routine use of the nephrostomy tube after PCNL has been subsequently questioned. Currently, the nephrostomy tube is increasingly omitted, and the access tract is usually sealed by haemostatic agents. An additionally ureteric stent is commonly inserted at the end of the procedure. However, the application of haemostatic sealants increases the immediate costs significantly. Still there are inconsistent data because of small study populations, lack of randomization, retrospective character and further more heterogeneous surgical techniques. CONCLUSION: The current body of literature does not provide high-level evidence for the preferred treatment of the access tract in PCNL. However, most authors agree that a tract sealing can be omitted without increasing the risk of complication in uncomplicated procedures.

Multiplanar Reformations in the Measurement of Renal Length on CT: Is It Plain Which Plane to Use?

OBJECTIVE: The objective of this study was to determine the accuracy of renal measurement on CT in multiple imaging planes. MATERIALS AND METHODS: In this study, three board-certified radiologists retrospectively measured 110 kidneys on CT in 55 consecutive patients. Five measurement methods were used: axial, coronal single image, coronal multiimage, sagittal single image, and sagittal multiimage. The coronal database was sent to a postprocessing workstation, and each radiologist performed a maximum renal measurement using a best off-axis plane that was our reference standard. An ANOVA test with repeated measures and posthoc Bonferroni corrected t tests were performed. RESULTS: The mean differences (± standard error) compared with the reference standard method were as follows: axial, 7.7 ± 0.7 mm; coronal single image, 13.1 ± 1.4 mm; coronal multiimage, 6.4 ± 0.8 mm; sagittal single image, 6.4 ± 0.6 mm; and sagittal multiimage, 2.8 ± 0.3 mm. The reference standard measurement was larger (p < 0.001), whereas the coronal single-image measurement (p ≤ 0.006) was smaller than all other methods. The sagittal multiimage (p ≤ 0.005) was statistically significantly different from all other methods. There were no statistically significant differences among the axial, coronal multiimage, and sagittal single-image methods (p ≥ 0.088). CONCLUSION: The single-image coronal method is the least accurate, with an error of approximately 13 mm. The axial, multiimage coronal, and single-image sagittal methods underestimate renal size by approximately 6-8 mm. Multiimage sagittal is the most accurate method for measuring kidneys with an error of approximately 3 mm.

Surgical management of retrorectal lesions: what the radiologist needs to know.

OBJECTIVE. The purpose of this article is to highlight the most salient imaging features of retrorectal masses with regard to surgical planning, preoperative biopsy, and identification of nonneoplastic mimickers of malignancy. CONCLUSION. Retrorectal tumors are associated with high morbidity. CT and MRI aid in preoperative planning because surgical resection is the treatment of choice for both benign and malignant entities. Radiologists need to understand the operative techniques currently used for retrorectal tumors because the first attempt at excision is the best chance for complete resection and optimal outcome.

Postoperative imaging of the orbital contents.

Ophthalmologists perform a wide array of interventions on the orbital contents. The surgical treatment of glaucoma, cataracts, retinal detachment, and ocular trauma or malignancy results in alteration of the standard anatomy, which is often readily evident at radiologic examinations. The ability to accurately recognize the various imaging manifestations after orbital surgery is critical for radiologists to avoid misdiagnosis. Of particular importance is familiarity with the numerous types of implanted devices, such as glaucoma drainage devices, orbital implants, and eyelid weights. Although knowledge of patients' surgical history is helpful, this information is often not available at the time of interpretation. Fortunately, there are characteristic posttreatment findings that enable diagnosis. The imaging features of the most commonly performed ophthalmologic procedures are highlighted, with emphasis on computed tomography and magnetic resonance (MR) imaging, because they are currently the primary modalities involved in evaluating the orbits. Glaucoma drainage devices and orbital implants after enucleation are two of the more pertinent implanted devices because their composition has substantially evolved over the past 2 decades, which affects their imaging appearance. Some devices, such as the Baerveldt Glaucoma Implant and platinum-weighted eyelid implants, may distort radiologic images. The MR imaging safety profiles of numerous implanted devices are also reported.

Subtrochanteric femur fractures: review of the complete pathologic spectrum with emphasis on distinguishing imaging features.

Subtrochanteric femur fractures result from a variety of mechanisms of injury and underlying pathologies. Radiologists can play a pivotal role by differentiating the etiology of a subtrochanteric femur fracture, which assists the orthopedic surgeon in choosing the correct method of treatment. To provide this valuable service, one must be familiar with the characteristic radiologic features of the numerous causative etiologies, ranging from Paget disease to underlying bone lesions to iatrogenic fractures.