Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice

Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to Brca2-inactivation in a Kras-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research

Genetic screens identify a context-specific PI3K/p27Kip1 node driving extrahepatic biliary cancer

Biliary tract cancer ranks among the most lethal human malignancies, representing an unmet clinical need. Its abysmal prognosis is tied to an increasing incidence and a fundamental lack of mechanistic knowledge regarding the molecular basis of the disease. Here, we show that the Pdx1-positive extrahepatic biliary epithelium is highly susceptible toward transformation by activated PIK3CAH1047R but refractory to oncogenic KrasG12D. Using genome-wide transposon screens and genetic loss-of-function experiments, we discover context-dependent genetic interactions that drive extrahepatic cholangiocarcinoma (ECC) and show that PI3K signaling output strength and repression of the tumor suppressor p27Kip1 are critical context-specific determinants of tumor formation. This contrasts with the pancreas, where oncogenic Kras in concert with p53 loss is a key cancer driver. Notably, inactivation of p27Kip1 permits KrasG12D-driven ECC development. These studies provide a mechanistic link between PI3K signaling, tissue-specific tumor suppressor barriers, and ECC pathogenesis, and present a novel genetic model of autochthonous ECC and genes driving this highly lethal tumor subtype

Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes.

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest KrasMUTlevels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUTin driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfβ-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.The work was supported by the German Cancer Consortium Joint Funding Program, the Helmholtz Gemeinschaft (PCCC Consortium), the German Research Foundation (SFB1243; A13/A14) and the European Research Council (ERC CoG number 648521)

Neuroimaging and clinical outcomes of oral anticoagulant-associated intracerebral hemorrhage

Objective Methods Whether intracerebral hemorrhage (ICH) associated with non-vitamin K antagonist oral anticoagulants (NOAC-ICH) has a better outcome compared to ICH associated with vitamin K antagonists (VKA-ICH) is uncertain. We performed a systematic review and individual patient data meta-analysis of cohort studies comparing clinical and radiological outcomes between NOAC-ICH and VKA-ICH patients. The primary outcome measure was 30-day all-cause mortality. All outcomes were assessed in multivariate regression analyses adjusted for age, sex, ICH location, and intraventricular hemorrhage extension. Results Interpretation We included 7 eligible studies comprising 219 NOAC-ICH and 831 VKA-ICH patients (mean age = 77 years, 52.5% females). The 30-day mortality was similar between NOAC-ICH and VKA-ICH (24.3% vs 26.5%; hazard ratio = 0.94, 95% confidence interval [CI] = 0.67-1.31). However, in multivariate analyses adjusting for potential confounders, NOAC-ICH was associated with lower admission National Institutes of Health Stroke Scale (NIHSS) score (linear regression coefficient = -2.83, 95% CI = -5.28 to -0.38), lower likelihood of severe stroke (NIHSS > 10 points) on admission (odds ratio [OR] = 0.50, 95% CI = 0.30-0.84), and smaller baseline hematoma volume (linear regression coefficient = -0.24, 95% CI = -0.47 to -0.16). The two groups did not differ in the likelihood of baseline hematoma volume <30cm(3) (OR = 1.14, 95% CI = 0.81-1.62), hematoma expansion (OR = 0.97, 95% CI = 0.63-1.48), in-hospital mortality (OR = 0.73, 95% CI = 0.49-1.11), functional status at discharge (common OR = 0.78, 95% CI = 0.57-1.07), or functional status at 3 months (common OR = 1.03, 95% CI = 0.75-1.43). Although functional outcome at discharge, 1 month, or 3 months was comparable after NOAC-ICH and VKA-ICH, patients with NOAC-ICH had smaller baseline hematoma volumes and less severe acute stroke syndromes. Ann Neurol 2018;84:702-712Peer reviewe

Maximizing the Area of Overlap of two Unions of Disks under Rigid Motion ∗

Let A and B be two sets of n resp. m disjoint unit disks in the plane, with m ≥ n. We consider the problem of finding a translation or rigid motion of A that maximizes the total area of overlap with B. The function describing the area of overlap is quite complex, even for combinatorially equivalent translations and, hence, we turn our attention to approximation algorithms. We give deterministic (1 − ɛ)-approximation algorithms for translations and for rigid motions, which run in O((nm/ɛ 2) log(m/ɛ)) and O((n 2 m 2 /ɛ 3) log m)) time, respectively. For rigid motions, we can also compute a (1 − ɛ)-approximation in O((m 2 n 4/3 ∆ 1/3 /ɛ 3) log n log m) time, where ∆ is the diameter of set A. Under the condition that the maximum area of overlap is at least a constant fraction of the area of A, we give a probabilistic (1 − ɛ)-approximation algorithm for rigid motions that runs in O((m 2 /ɛ 4) log 2 (m/ɛ) log m) time and succeeds with high probability. Our results generalize to the case where A and B consist of possibly intersecting disks of different radii, provided that (i) the ratio of the radii of any two disks in A ∪ B is bounded, and (ii) within each set, the maximum number of disks with a non-empty intersection is bounded

Systematic investigation of insulin fibrillation on a chip

A microfluidic protein aggregation device (microPAD) that allows the user to perform a series of protein incubations with various concentrations of two reagents is demonstrated. The microfluidic device consists of 64 incubation chambers to perform individual incubations of the protein at 64 specific conditions. Parallel processes of metering reagents, stepwise concentration gradient generation, and mixing are achieved simultaneously by pneumatic valves. Fibrillation of bovine insulin was selected to test the device. The effect of insulin and sodium chloride (NaCl) concentration on the formation of fibrillar structures was studied by observing the growth rate of partially folded protein, using the fluorescent marker Thioflavin-T. Moreover, dual gradients of different NaCl and hydrochloric acid (HCl) concentrations were formed, to investigate their interactive roles in the formation of insulin fibrils and spherulites. The chip-system provides a bird’s eye view on protein aggregation, including an overview of the factors that affect the process and their interactions. This microfluidic platform is potentially useful for rapid analysis of the fibrillation of proteins associated with many misfolding-based diseases, such as quantitative and qualitative studies on amyloid growth.BT/Bioprocess Engineerin

Improved detection of silent atrial fibrillation using 72-hour Holter ECG in patients with ischemic stroke:a prospective multicenter cohort study

Background and Purpose— Adequate diagnosis of atrial fibrillation (AF), including paroxysmal AF, is an important part of stroke workup. Prolonged ECG monitoring may improve the detection of paroxysmal, previously undiagnosed AF (unknown AF). Therefore, we evaluated systematic 72-hour Holter ECG monitoring to detect unknown AF for the workup of patients with stroke. Methods— Unselected survivors of a stroke or transient ischemic attack (TIA) without known AF were enrolled in a prospective, multicenter cohort study of 72-hour Holter ECG monitoring in 9 German secondary and tertiary stroke centers between May 2010 and January 2011. In addition to standardized workup of stroke pathogenesis according to the German Stroke Unit protocol, all patients underwent 72-hour Holter ECG monitoring directly after admission. All ECGs were centrally analyzed by 2 independent observers. We determined the proportion of unknown AF and compared the detection rates of 72- and 24-hour monitoring. Results— A total of 1135 patients were enrolled (mean age, 67 years [SD, 13.1 years], 45% women, 29% TIA). Unknown AF was detected in 49 out of 1135 patients (4.3%, [95% confidence interval, 3.4–5.2%]) by 72-hour ECG monitoring. Unknown AF was diagnosed in 29 patients (2.6%) within the first 24 hours of ECG monitoring, and in 20 more patients only by 72 hours of ECG monitoring. The number needed to screen by 72-hour ECG was 55 patients (95% confidence interval [35–123]) for each additional AF diagnosis. Patients with unknown AF were significantly older and had more often a history of previous stroke. Patients with unknown AF were equally distributed within categories of pathogenesis according to Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification. Conclusions— In unselected survivors of stroke or TIA, 72-hour ECG monitoring is feasible and improves the detection rate of silent paroxysmal AF. </jats:sec