Multiomics characterization of mouse hepatoblastoma identifies yes-associated protein 1 target genes.

BACKGROUND AND AIMS: Hepatoblastoma (HB) is the most common primary liver malignancy in childhood and lacks targeted therapeutic options. We previously engineered, to our knowledge, the first yes-associated protein 1 (YAP1) S127A -inducible mouse model of HB, demonstrating tumor regression and redifferentiation after YAP1 withdrawal through genome-wide enhancer modulation. Probing accessibility, transcription, and YAP1 binding at regulatory elements in HB tumors may provide more insight into YAP1-driven tumorigenesis and expose exploitable vulnerabilities in HB. APPROACH AND RESULTS: Using a multiomics approach, we integrated high-throughput transcriptome and chromatin profiling of our murine HB model to identify dynamic activity at candidate cis -regulatory elements (cCREs). We observed that 1301 of 305,596 cCREs exhibit "tumor-modified" (TM) accessibility in HB. We mapped 241 TM enhancers to corresponding genes using accessibility and histone H3K27Ac profiles. Anti-YAP1 cleavage under targets and tagmentation in tumors revealed 66 YAP1-bound TM cCRE/gene pairs, 31 of which decrease expression after YAP1 withdrawal. We validated the YAP1-dependent expression of a putative YAP1 target, Jun dimerization protein 2 (JDP2), in human HB cell lines using YAP1 and LATS1/2 small interfering RNA knockdown. We also confirmed YAP1-induced activity of the Jdp2 TM enhancer in vitro and discovered an analogous human enhancer in silico. Finally, we used transcription factor (TF) footprinting to identify putative YAP1 cofactors and characterize HB-specific TF activity genome wide. CONCLUSIONS: Our chromatin-profiling techniques define the regulatory frameworks underlying HB and identify YAP1-regulated gene/enhancer pairs. JDP2 is an extensively validated target with YAP1-dependent expression in human HB cell lines and hepatic malignancies.

A Large Segmental Mid-Diaphyseal Femoral Defect Sheep Model: Surgical Technique.

Background:There are numerous animal models available to study bone healing as well as test strategies to accelerate bone formation. Sheep are commonly used for evaluation of long bone fractures due to similar dimensions and weight bearing environments compared to patients. Large critical-size defects can be created in sheep to facilitate the study of implantable materials, osteogenic proteins, and stem cell treatments. Studies have been published using plates to stabilize large critical size defects in femoral, tibial, and metatarsal defects. External fixators have also been used to stabilize tibial defects in sheep.Methods: The purpose of the current paper is to detail the surgical technique for creation of a 42 mm mid-diaphyseal femoral defect stabilization with an intramedullary device in sheep. Additional surgical details are provided for dynamization, reverse dynamization, and device removal.Conclusion: The article provides multiple technical tips applicable to this and other ovine osteotomy models and concludes with a discussion comparing the use of each stabilization technique in clinically significant large critical-size bone defects.

Spontaneous Propionibacterium Acnes abscess with intraventricular rupture in an immunocompetent adult without prior neurosurgical intervention.

Previously viewed as a culture contaminant, Propionibacterium Acnes can cause infection following neurosurgical intervention. Its role in brain abscess in the immunocompetent, surgically naïve population has been infrequently reported. Herein, we describe an immunocompetent 55-year-old man with no risk factors found to have a thalamic abscess with intraventricular rupture.

Genome-wide detection of CRISPR editing in vivo using GUIDE-tag.

Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing.

YAP1 Withdrawal in Hepatoblastoma Drives Therapeutic Differentiation of Tumor Cells to Functional Hepatocyte-Like Cells.

BACKGROUND AND AIMS: Despite surgical and chemotherapeutic advances, the 5-year survival rate for stage IV hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. Yes-associated protein 1 (YAP1) and ß-catenin co-activation occurs in 80% of children's HB; however, a lack of conditional genetic models precludes tumor maintenance exploration. Thus, the need for a targeted therapy remains unmet. Given the predominance of YAP1 and ß-catenin activation in HB, we sought to evaluate YAP1 as a therapeutic target in HB. APPROACH AND RESULTS: We engineered the conditional HB murine model using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1S127A , constitutive ß-cateninDelN90 , and a luciferase reporter to murine liver. Tumor regression was evaluated using bioluminescent imaging, tumor landscape characterized using RNA and ATAC sequencing, and DNA footprinting. Here we show that YAP1S127A withdrawal mediates more than 90% tumor regression with survival for 230+ days in mice. YAP1S127A withdrawal promotes apoptosis in a subset of tumor cells, and in remaining cells induces a cell fate switch that drives therapeutic differentiation of HB tumors into Ki-67-negative hepatocyte-like HB cells ("HbHeps") with hepatocyte-like morphology and mature hepatocyte gene expression. YAP1S127A withdrawal drives the formation of hbHeps by modulating liver differentiation transcription factor occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and rescue liver damage in mice. CONCLUSIONS: YAP1S127A withdrawal, without silencing oncogenic ß-catenin, significantly regresses hepatoblastoma, providing in vivo data to support YAP1 as a therapeutic target for HB. YAP1S127A withdrawal alone sufficiently drives long-term regression in HB, as it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes.

A Fluorescence-Activated Single-Droplet Dispenser for High Accuracy Single-Droplet and Single-Cell Sorting and Dispensing.

The ability to sort and dispense droplets accurately is essential to droplet-based single-cell analysis. Here, we describe a fluorescence-activated single-droplet dispenser (FASD) that is analogous to a conventional fluorescence-activated cell sorter, but sorts droplets containing single cells within an oil emulsion. The FASD system uses cytometric detection and electrohydrodynamic actuation-based single-droplet manipulation, allowing droplet isolation and dispensing with high efficiency and accuracy. The system is compatible with multiwell plates and can be integrated with existing microfluidic devices and large-scale screening systems. By enabling sorting based on single-cell reactions such as PCR, this platform will help expand the basis of cell sorting from mainly protein biomarkers to nucleic acid sequences and secreted biomolecules.

CRISPR-SONIC: targeted somatic oncogene knock-in enables rapid in vivo cancer modeling.

CRISPR/Cas9 has revolutionized cancer mouse models. Although loss-of-function genetics by CRISPR/Cas9 is well-established, generating gain-of-function alleles in somatic cancer models is still challenging because of the low efficiency of gene knock-in. Here we developed CRISPR-based Somatic Oncogene kNock-In for Cancer Modeling (CRISPR-SONIC), a method for rapid in vivo cancer modeling using homology-independent repair to integrate oncogenes at a targeted genomic locus. Using a dual guide RNA strategy, we integrated a plasmid donor in the 3'-UTR of mouse ß-actin, allowing co-expression of reporter genes or oncogenes from the ß-actin promoter. We showed that knock-in of oncogenic Ras and loss of p53 efficiently induced intrahepatic cholangiocarcinoma in mice. Further, our strategy can generate bioluminescent liver cancer to facilitate tumor imaging. This method simplifies in vivo gain-of-function genetics by facilitating targeted integration of oncogenes.

Mesenchymal stem cell seeded, biomimetic 3D printed scaffolds induce complete bridging of femoral critical sized defects.

No current clinical treatments provide an ideal long-term solution for repair of long bone segment defects. Incomplete healing prevents patients from returning to preinjury activity and ultimately requires additional surgery to induce healing. Obtaining autologous graft material is costly, incurs morbidity, requires surgical time, and quality material is finite. In this pilot study, 3D printed biomimetic scaffolds were used to facilitate rapid bone bridging in critical sized defects in a sheep model. An inverse trabecular pattern based on micro-CT scans of sheep trabecular bone was printed in polybutylene terephthalate. Scaffolds were coated with micron-sized tricalcium phosphate particles to induce osteoconductivity. Mesenchymal stem cells (MSCs) were isolated from sheep inguinal and tail fat, in one group of sheep and scaffolds were infiltrated with MSCs in a bioreactor. Controls did not undergo surgery for cell extraction. Scaffolds were implanted into two experimental and two control adult sheep, and followed for either 3 or 6 months. Monthly radiographs and post explant micro-CT scanning demonstrated bone formation on the lateral, anterior, medial, and posterior-medial aspects along the entire length of the defect. Bone formation was absent on the posterior-lateral aspect where a muscle is generally attached to the bone. The 3-month time point showed 15.5% more cortical bone deposition around the scaffold circumference while the 6-month time point showed 40.9% more bone deposition within scaffold pores. Control sheep failed to unite. Serum collagen type-1C-terminus telopeptides (CTX-1) showed time-dependent levels of bone resorption, and calcein labeling demonstrated an increase in bone formation rate in treated animals compared with controls. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 242-252, 2019.

Understanding and repurposing CRISPR-mediated alternative splicing.

Two new studies refine our understanding of CRISPR-associated exon skipping and redefine its utility in engineering alternative splicing.

A Self-Digitization Dielectrophoretic (SD-DEP) Chip for High-Efficiency Single-Cell Capture, On-Demand Compartmentalization, and Downstream Nucleic Acid Analysis.

The design and fabrication of a self-digitization dielectrophoretic (SD-DEP) chip with simple components for single-cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single-cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single-cell research for precise medicine.

Self-digitization chip for single-cell genotyping of cancer-related mutations.

Cancer is a heterogeneous disease, and patient-level genetic assessments can guide therapy choice and impact prognosis. However, little is known about the impact of genetic variability within a tumor, intratumoral heterogeneity (ITH), on disease progression or outcome. Current approaches using bulk tumor specimens can suggest the presence of ITH, but only single-cell genetic methods have the resolution to describe the underlying clonal structures themselves. Current techniques tend to be labor and resource intensive and challenging to characterize with respect to sources of biological and technical variability. We have developed a platform using a microfluidic self-digitization chip to partition cells in stationary volumes for cell imaging and allele-specific PCR. Genotyping data from only confirmed single-cell volumes is obtained and subject to a variety of relevant quality control assessments such as allele dropout, false positive, and false negative rates. We demonstrate single-cell genotyping of the NPM1 type A mutation, an important prognostic indicator in acute myeloid leukemia, on single cells of the cell line OCI-AML3, describing a more complex zygosity distribution than would be predicted via bulk analysis.

Predictability of Pelvic Tilt During Total Hip Arthroplasty Using a Traction Table.

BACKGROUND: Pelvic positioning during total hip arthroplasty (THA) affects functional position of the acetabular component. We sought to evaluate whether preoperative pelvic tilt correlated with intraoperative pelvic tilt while positioned on a traction table for direct anterior THA and furthermore to evaluate whether there was a consistent and predictable effect on pelvic tilt while positioned for surgery. METHODS: We evaluated the sagittal spinopelvic preoperative standing and supine pelvic tilt radiographic measurements as compared with intraoperative measurements of 25 patients. Changes in pelvic tilt were analyzed for statistical significance and interobserver reliability. RESULTS: The mean standing pelvic tilt was 13.5° ± 5.7°. The mean supine pelvic tilt was 13.3° ± 6.1°. There was no statistically significant difference between standing and supine pelvic tilt (P = .866). The mean intraoperative pelvic tilt was 3.0° ± 6.2°. There was a statistically significant decrease in pelvic tilt between both standing to intraoperative comparison and supine to intraoperative comparison (P < .0001 for both). Difference in mean between these comparisons was 10.5° ± 4.6° (95% confidence interval, 8.7°-12.3°) and 10.3° ± 6.3° (95% confidence interval, 7.8°-12.8°), respectively. CONCLUSION: Patient positioning on a traction table for direct anterior THA has a reliable effect on pelvic tilt in the magnitude of approximately 10° decreased pelvic tilt. This effect on pelvic tilt correlates to approximately 7.4° and 3° altered anteversion and inclination, respectively. Taking into account this change in pelvic tilt at the time of surgery will allow the hip arthroplasty surgeon to more accurately place acetabular components in the desired functional position.

CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion.

CRISPR is widely used to disrupt gene function by inducing small insertions and deletions. Here, we show that some single-guide RNAs (sgRNAs) can induce exon skipping or large genomic deletions that delete exons. For example, CRISPR-mediated editing of ß-catenin exon 3, which encodes an autoinhibitory domain, induces partial skipping of the in-frame exon and nuclear accumulation of ß-catenin. A single sgRNA can induce small insertions or deletions that partially alter splicing or unexpected larger deletions that remove exons. Exon skipping adds to the unexpected outcomes that must be accounted for, and perhaps taken advantage of, in CRISPR experiments.

Genetic disruption of oncogenic Kras sensitizes lung cancer cells to Fas receptor-mediated apoptosis.

Genetic lesions that activate KRAS account for ∼30% of the 1.6 million annual cases of lung cancer. Despite clinical need, KRAS is still undruggable using traditional small-molecule drugs/inhibitors. When oncogenic Kras is suppressed by RNA interference, tumors initially regress but eventually recur and proliferate despite suppression of Kras Here, we show that tumor cells can survive knockout of oncogenic Kras, indicating the existence of Kras-independent survival pathways. Thus, even if clinical KRAS inhibitors were available, resistance would remain an obstacle to treatment. Kras-independent cancer cells exhibit decreased colony formation in vitro but retain the ability to form tumors in mice. Comparing the transcriptomes of oncogenic Kras cells and Kras knockout cells, we identified 603 genes that were specifically up-regulated in Kras knockout cells, including the Fas gene, which encodes a cell surface death receptor involved in physiological regulation of apoptosis. Antibodies recognizing Fas receptor efficiently induced apoptosis of Kras knockout cells but not oncogenic Kras-expressing cells. Increased Fas expression in Kras knockout cells was attributed to decreased association of repressive epigenetic marks at the Fas promoter. Concordant with this observation, treating oncogenic Kras cells with histone deacetylase inhibitor and Fas-activating antibody efficiently induced apoptosis, thus bypassing the need to inhibit Kras. Our results suggest that activation of Fas could be exploited as an Achilles' heel in tumors initiated by oncogenic Kras.

Prothrombin Complex Concentrates: An Alternative to Fresh Frozen Plasma.

Insufficiency fractures are a common cause of morbidity among geriatric patients worldwide. Improved outcomes are known to result from decreased delay to definitive operative fixation and mobilization. Use of warfarin is an important potential cause of delay. The ideal mode of warfarin reversal is currently unknown. Prothrombin complex concentrates (PCCs) offer rapid correction with small infusion volume, both of which are important for elderly patients with multiple comorbidities. The authors present 2 cases of insufficiency fractures occurring in geriatric patients receiving warfarin therapy reversed with a 3-factor PCC. Both patients were independent, community ambulators without significant functional disability and returned to their prior level of functioning. There were no significant bleeding or venous thromboembolic complications. To the authors' knowledge, no previous reports have described the use of PCC in geriatric patients with fractures. Nonetheless, its potential is well documented in emergency and trauma surgery literature. The use of PCC could potentially allow surgeons and hospitals to avoid complications related to immobility and the associated costs of treatment, extended hospital stay, and readmission. The authors' limited experience suggests 3-factor PCC preparations may provide adequate correction to allow expeditious surgical treatment. [Orthopedics. 2017; 40(2):e367-e369.].

One-step immortalization of primary human airway epithelial cells capable of oncogenic transformation.

BACKGROUND: The ability to transform normal human cells into cancer cells with the introduction of defined genetic alterations is a valuable method for understanding the mechanisms of oncogenesis. Easy establishment of immortalized but non-transformed human cells from various tissues would facilitate these genetic analyses. RESULTS: We report here a simple, one-step immortalization method that involves retroviral vector mediated co-expression of the human telomerase protein and a shRNA targeting the CDKN2A gene locus. We demonstrate that this method could successfully immortalize human small airway epithelial cells while maintaining their chromosomal stability. We further showed that these cells retain p53 activity and can be transformed by the KRAS oncogene. CONCLUSIONS: Our method simplifies the immortalization process and is broadly applicable for establishing immortalized epithelial cell lines from primary human tissues for cancer research.

Oncogenesis driven by the Ras/Raf pathway requires the SUMO E2 ligase Ubc9.

The small GTPase KRAS is frequently mutated in human cancer and currently there are no targeted therapies for KRAS mutant tumors. Here, we show that the small ubiquitin-like modifier (SUMO) pathway is required for KRAS-driven transformation. RNAi depletion of the SUMO E2 ligase Ubc9 suppresses 3D growth of KRAS mutant colorectal cancer cells in vitro and attenuates tumor growth in vivo. In KRAS mutant cells, a subset of proteins exhibit elevated levels of SUMOylation. Among these proteins, KAP1, CHD1, and EIF3L collectively support anchorage-independent growth, and the SUMOylation of KAP1 is necessary for its activity in this context. Thus, the SUMO pathway critically contributes to the transformed phenotype of KRAS mutant cells and Ubc9 presents a potential target for the treatment of KRAS mutant colorectal cancer.

Evidence-based Comprehensive Approach to Forearm Arterial Laceration.

INTRODUCTION: Penetrating injury to the forearm may cause an isolated radial or ulnar artery injury, or a complex injury involving other structures including veins, tendons and nerves. The management of forearm laceration with arterial injury involves both operative and nonoperative strategies. An evolution in management has emerged especially at urban trauma centers, where the multidisciplinary resource of trauma and hand subspecialties may invoke controversy pertaining to the optimal management of such injuries. The objective of this review was to provide an evidence-based, systematic, operative and nonoperative approach to the management of isolated and complex forearm lacerations. A comprehensive search of MedLine, Cochrane Library, Embase and the National Guideline Clearinghouse did not yield evidence-based management guidelines for forearm arterial laceration injury. No professional or societal consensus guidelines or best practice guidelines exist to our knowledge. DISCUSSION: The optimal methods for achieving hemostasis are by a combination approach utilizing direct digital pressure, temporary tourniquet pressure, compressive dressings followed by wound closure. While surgical hemostasis may provide an expedited route for control of hemorrhage, this aggressive approach is often not needed (with a few exceptions) to achieve hemostasis for most forearm lacerations. Conservative methods mentioned above will attain the same result. Further, routine emergent or urgent operative exploration of forearm laceration injuries are not warranted and not cost-beneficial. It has been widely accepted with ample evidence in the literature that neither injury to forearm artery, nerve or tendon requires immediate surgical repair. Attention should be directed instead to control of bleeding, and perform a complete physical examination of the hand to document the presence or absence of other associated injuries. Critical ischemia will require expeditious surgical restoration of arterial perfusion. In a well-perfused hand, however, the presence of one intact artery is adequate to sustain viability without long-term functional disability, provided the palmar arch circulation is intact. Early consultation with a hand specialist should be pursued, and follow-up arrangement made for delayed primary repair in cases of complex injury. CONCLUSION: Management in accordance with well-established clinical principles will maximize treatment efficacy and functional outcome while minimizing the cost of medical care.

A high-throughput assay for small molecule destabilizers of the KRAS oncoprotein.

Mutations in the Ras family of small GTPases, particularly KRAS, occur at high frequencies in cancer and represent a major unmet therapeutic need due to the lack of effective targeted therapies. Past efforts directed at inhibiting the activity of the Ras oncoprotein have proved difficult. We propose an alternative approach to target Ras by eliminating Ras protein from cells with pharmacological means. In this study, we developed a cell-based, high-content screening platform to identify small molecules that could promote the degradation of the KRAS oncoprotein. We generated an EGFP-KRASG12V fluorescence reporter system and implemented it for automated screening in 1536-well plates using high-throughput cellular imaging. We screened a library of clinically relevant compounds at wide dose range and identified Ponatinib and AMG-47a as two candidate compounds that selectively reduced the levels of EGFP-KRASG12V protein but did not affect EGFP protein in cells. This proof-of-principle study demonstrates that it is feasible to use a high-throughput screen to identify compounds that promote the degradation of the Ras oncoprotein as a new approach to target Ras.