Development and testing of a versatile genome editing application reporter (V-GEAR) system.

CRISPR-Cas9 and novel cas fusion proteins leveraging specific DNA targeting ability combined with deaminases or reverse transcriptases have revolutionized genome editing. However, their efficacy heavily relies upon protein variants, targeting single guide RNAs, and surrounding DNA sequence context within the targeted loci. This necessitates the need for efficient and rapid screening methods to evaluate these editing reagents and designs. Existing plasmid-based reporters lack flexibility, being fixed to specific DNA sequences, hindering direct comparisons between various editing approaches. To address this, we developed the versatile genome editing application reporter (V-GEAR) system. V-GEAR comprises genes detectable after desired editing via base editing, prime editing, or homology-directed repair within relevant genomic contexts. It employs a detectable synthetic cell surface protein (RQR8) followed by a customizable target sequence resembling genomic regions of interest. These genes allow for reliable identification of corrective editing and cell enrichment. We validated the V-GEAR system with base editors, prime editors, and Cas9-mediated homology-directed repair. Furthermore, the V-GEAR system offers versatility by allowing transient screening or stable integration at the AAVS1 safe harbor loci, rapidly achieved through immunomagnetic isolation. This innovative system enables direct comparisons among editing technologies, accelerating the development and testing of genome editing approaches.

Engineering Memory T Cells as a platform for Long-Term Enzyme Replacement Therapy in Lysosomal Storage Disorders.

Enzymopathy disorders are the result of missing or defective enzymes. Amongst these enzymopathies, mucopolysaccharidosis type I, is a rare genetic lysosomal storage disorder caused by mutations in the gene encoding alpha-L-iduronidase (IDUA), ultimately causes toxic build-up of glycosaminoglycans (GAGs). There is currently no cure and standard treatments provide insufficient relief to the skeletal structure and central nervous system (CNS). Human memory T cells (Tm) migrate throughout the body's tissues and can persist for years, making them an attractive approach for cellular-based, systemic enzyme replacement therapy. Here, we tested genetically engineered, IDUA-expressing Tm as a cellular therapy in an immunodeficient mouse model of MPS I. Our results demonstrate that a single dose of engineered Tm leads to detectable IDUA enzyme levels in the blood for up to 22 weeks and reduced urinary GAG excretion. Furthermore, engineered Tm take up residence in nearly all tested tissues, producing IDUA and leading to metabolic correction of GAG levels in the heart, lung, liver, spleen, kidney, bone marrow, and the CNS. Our study indicates that genetically engineered Tm holds great promise as a platform for cellular-based enzyme replacement therapy for the treatment of mucopolysaccharidosis type I and potentially many other enzymopathies and protein deficiencies.

Generative Adversarial Networks Accurately Reconstruct Pan-Cancer Histology from Pathologic, Genomic, and Radiographic Latent Features.

Artificial intelligence models have been increasingly used in the analysis of tumor histology to perform tasks ranging from routine classification to identification of novel molecular features. These approaches distill cancer histologic images into high-level features which are used in predictions, but understanding the biologic meaning of such features remains challenging. We present and validate a custom generative adversarial network - HistoXGAN - capable of reconstructing representative histology using feature vectors produced by common feature extractors. We evaluate HistoXGAN across 29 cancer subtypes and demonstrate that reconstructed images retain information regarding tumor grade, histologic subtype, and gene expression patterns. We leverage HistoXGAN to illustrate the underlying histologic features for deep learning models for actionable mutations, identify model reliance on histologic batch effect in predictions, and demonstrate accurate reconstruction of tumor histology from radiographic imaging for a 'virtual biopsy'.

Microstructural and thermodynamic characterization of wormlike micelles formed by polydisperse ionic surfactant solutions.

For industrial applications of self-assembled wormlike micelles, measurement and characterization of a micellar material's microstructure and rheology are paramount for the development and deployment of new high-performing and cost-effective formulations. Within this workflow, there are significant bottlenecks associated with experimental delays and a lack of transferability of results from one chemistry to another. In this work, we outline a process to predict microscopic and thermodynamic characteristics of wormlike micelles directly from rheological data by combining a more robust and efficient fitting algorithm with a recently published constitutive model called the Toy Shuffling model [J. D. Peterson and M. E. Cates, J. Rheol. 64, 1465-1496 (2020) and J. D. Peterson and M. E. Cates, J. Rheol. 65, 633-662 (2021)]. To support this work, linear rheology measurements were taken for 143 samples comprising a common base formulation of commercial sodium lauryl ether sulfate, cocamidopropyl betaine, and salt (NaCl). The steady state zero shear viscosity evident in linear rheology was measured in duplicate via direct steady and oscillatory shear experiments. Fitting the collected data to the model, we found trends in the microstructural and thermodynamic characteristics that agree with molecular dynamics simulations. These trends validate our new perspective on the parameters that inform the study of the relationship between chemical formulation and rheology. This work, when implemented at scale, can potentially be used to inform and test strategies for predicting self-assembled micellar structures based on chemical formulation.

Novel computational biology modeling system can accurately forecast response to neoadjuvant therapy in early breast cancer.

BACKGROUND: Generalizable population-based studies are unable to account for individual tumor heterogeneity that contributes to variability in a patient's response to physician-chosen therapy. Although molecular characterization of tumors has advanced precision medicine, in early-stage and locally advanced breast cancer patients, predicting a patient's response to neoadjuvant therapy (NAT) remains a gap in current clinical practice. Here, we perform a study in an independent cohort of early-stage and locally advanced breast cancer patients to forecast tumor response to NAT and assess the stability of a previously validated biophysical simulation platform. METHODS: A single-blinded study was performed using a retrospective database from a single institution (9/2014-12/2020). Patients included: ≥ 18 years with breast cancer who completed NAT, with pre-treatment dynamic contrast enhanced magnetic resonance imaging. Demographics, chemotherapy, baseline (pre-treatment) MRI and pathologic data were input into the TumorScope Predict (TS) biophysical simulation platform to generate predictions. Primary outcomes included predictions of pathological complete response (pCR) versus residual disease (RD) and final volume for each tumor. For validation, post-NAT predicted pCR and tumor volumes were compared to actual pathological assessment and MRI-assessed volumes. Predicted pCR was pre-defined as residual tumor volume ≤ 0.01 cm3 (≥ 99.9% reduction). RESULTS: The cohort consisted of eighty patients; 36 Caucasian and 40 African American. Most tumors were high-grade (54.4% grade 3) invasive ductal carcinomas (90.0%). Receptor subtypes included hormone receptor positive (HR+)/human epidermal growth factor receptor 2 positive (HER2+, 30%), HR+/HER2- (35%), HR-/HER2+ (12.5%) and triple negative breast cancer (TNBC, 22.5%). Simulated tumor volume was significantly correlated with post-treatment radiographic MRI calculated volumes (r = 0.53, p = 1.3 × 10-7, mean absolute error of 6.57%). TS prediction of pCR compared favorably to pathological assessment (pCR: TS n = 28; Path n = 27; RD: TS n = 52; Path n = 53), for an overall accuracy of 91.2% (95% CI: 82.8% - 96.4%; Clopper-Pearson interval). Five-year risk of recurrence demonstrated similar prognostic performance between TS predictions (Hazard ratio (HR): - 1.99; 95% CI [- 3.96, - 0.02]; p = 0.043) and clinically assessed pCR (HR: - 1.76; 95% CI [- 3.75, 0.23]; p = 0.054). CONCLUSION: We demonstrated TS ability to simulate and model tumor in vivo conditions in silico and forecast volume response to NAT across breast tumor subtypes.

Next generation immuno-oncology tumor profiling using a rapid, non-invasive, computational biophysics biomarker in early-stage breast cancer.

Background: Immuno-oncology (IO) therapies targeting the PD-1/PD-L1 axis, such as immune checkpoint inhibitor (ICI) antibodies, have emerged as promising treatments for early-stage breast cancer (ESBC). Despite immunotherapy's clinical significance, the number of benefiting patients remains small, and the therapy can prompt severe immune-related events. Current pathologic and transcriptomic predictions of IO response are limited in terms of accuracy and rely on single-site biopsies, which cannot fully account for tumor heterogeneity. In addition, transcriptomic analyses are costly and time-consuming. We therefore constructed a computational biomarker coupling biophysical simulations and artificial intelligence-based tissue segmentation of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRIs), enabling IO response prediction across the entire tumor. Methods: By analyzing both single-cell and whole-tissue RNA-seq data from non-IO-treated ESBC patients, we associated gene expression levels of the PD-1/PD-L1 axis with local tumor biology. PD-L1 expression was then linked to biophysical features derived from DCE-MRIs to generate spatially- and temporally-resolved atlases (virtual tumors) of tumor biology, as well as the TumorIO biomarker of IO response. We quantified TumorIO within patient virtual tumors (n = 63) using integrative modeling to train and develop a corresponding TumorIO Score. Results: We validated the TumorIO biomarker and TumorIO Score in a small, independent cohort of IO-treated patients (n = 17) and correctly predicted pathologic complete response (pCR) in 15/17 individuals (88.2% accuracy), comprising 10/12 in triple negative breast cancer (TNBC) and 5/5 in HR+/HER2- tumors. We applied the TumorIO Score in a virtual clinical trial (n = 292) simulating ICI administration in an IO-naïve cohort that underwent standard chemotherapy. Using this approach, we predicted pCR rates of 67.1% for TNBC and 17.9% for HR+/HER2- tumors with addition of IO therapy; comparing favorably to empiric pCR rates derived from published trials utilizing ICI in both cancer subtypes. Conclusion: The TumorIO biomarker and TumorIO Score represent a next generation approach using integrative biophysical analysis to assess cancer responsiveness to immunotherapy. This computational biomarker performs as well as PD-L1 transcript levels in identifying a patient's likelihood of pCR following anti-PD-1 IO therapy. The TumorIO biomarker allows for rapid IO profiling of tumors and may confer high clinical decision impact to further enable personalized oncologic care.

Evaluation and management of paediatric vertigo.

PURPOSE OF REVIEW: This review summarizes the most current information on cause, evaluation and treatment of dizziness in children. RECENT FINDINGS: There has been an increased understanding of the multifactorial cause of dizziness in the paediatric population. Quantitative vestibular testing is increasingly used and valuable as a diagnostic adjunct. Vestibular rehabilitation, migraine hygiene, psychological therapies, pharmaceuticals and/or surgery can be used as well tolerated and effective treatments for vertigo in children and adolescents when tailored to cause. SUMMARY: Paediatric vertigo can be effectively evaluated through careful history taking and physical examination along with adjunctive tests, such as vestibular testing and audiometry, when appropriate. Options for treatment of vestibular disorders in children and adolescents have greatly expanded in recent years allowing for the effective management of nearly all cases of paediatric vertigo, though a multimodal and/or multidisciplinary approach is often needed.

Highly accurate response prediction in high-risk early breast cancer patients using a biophysical simulation platform.

PURPOSE: Pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) in early breast cancer (EBC) is largely dependent on breast cancer subtype, but no clinical-grade model exists to predict response and guide selection of treatment. A biophysical simulation of response to NAC has the potential to address this unmet need. METHODS: We conducted a retrospective evaluation of a biophysical simulation model as a predictor of pCR. Patients who received standard NAC at the University of Chicago for EBC between January 1st, 2010 and March 31st, 2020 were included. Response was predicted using baseline breast MRI, clinicopathologic features, and treatment regimen by investigators who were blinded to patient outcomes. RESULTS: A total of 144 tumors from 141 patients were included; 59 were triple-negative, 49 HER2-positive, and 36 hormone-receptor positive/HER2 negative. Lymph node disease was present in half of patients, and most were treated with an anthracycline-based regimen (58.3%). Sensitivity and specificity of the biophysical simulation for pCR were 88.0% (95% confidence interval [CI] 75.7 - 95.5) and 89.4% (95% CI 81.3 - 94.8), respectively, with robust results regardless of subtype. In patients with predicted pCR, 5-year event-free survival was 98%, versus 79% with predicted residual disease (log-rank p = 0.01, HR 4.57, 95% CI 1.36 - 15.34). At a median follow-up of 5.4 years, no patients with predicted pCR experienced disease recurrence. CONCLUSION: A biophysical simulation model accurately predicts pCR and long-term outcomes from baseline MRI and clinical data, and is a promising tool to guide escalation/de-escalation of NAC.

A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.

Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.

Impact of Question Bank Use for In-Training Examination Preparation by OBGYN Residents - A Multicenter Study.

OBJECTIVE: To examine the impact of access to and utilization of a commercially available question bank (TrueLearn) for in-training examination (ITE) preparation in Obstetrics and Gynecology (OBGYN). DESIGN: This was a retrospective cohort study examining the impact of TrueLearn usage on ITE examination performance outcomes. Produced by the educational arm of the American College of Obstetricians and Gynecologists, the Council on Resident Education in Obstetrics and Gynecology (CREOG) exam is a multiple-choice test given to all residents annually. Residency programs participating in this study provided residency program mean CREOG scores from the year prior (2015), and the first (2016) and second (2017) years of TrueLearn usage. Programs also contributed resident-specific CREOG scores for each resident for 2016 and 2017. This data was combined with each resident's TrueLearn usage data that was provided by TrueLearn with residency program consent. The CREOG scores consisted of the CREOG score standardized to all program years, the CREOG score standardized to the same program year (PGY) and the total percent (%) correct. TrueLearn usage data included number of practice questions completed, number of practice tests taken, average number of days between successive tests, and percent correct of answered practice questions. SETTING: OBGYN Residency Training Programs. PARTICIPANTS: OBGYN residency programs that purchased and utilized TrueLearn for the 2016 CREOG examination were eligible for participation (n = 14). Ten residency programs participated, which consisted of 212 residents in 2016 and 218 residents in 2017. RESULTS: TrueLearn was used by 78.8% (167/212) of the residents in 2016 and 84.9% (185/218) of the residents in 2017. No significant difference was seen in the average CREOG scores available on a per- program level before versus after the first year of implementation either using the CREOG score standardized to all PGYs (mean difference 1.0; p = 0.58) or standardized to the same PGY (mean difference 3.1; p = 0.25). Using resident-level data, there was no significant difference in mean CREOG score standardized to all PGYs between users and non-users of TrueLearn in 2016 (mean, 199.4 vs 196.7; p = 0.41) or 2017 (mean, 198.2 vs 203.4; p = 0.19). The percent of practice questions answered correctly on TrueLearn was positively correlated with the CREOG score standardized to all PGYs (r = 0.47 for 2016 and r = 0.60 for 2017), as well as with the CREOG total percent correct (r = 0.47 for 2016 and r = 0.61 for 2017). Based on a simple linear regression, for every 500 practice questions completed, the CREOG score significantly increased for PGY-2 residents by an average (±SE) of 7.3 ± 2.8 points (p = 0.013); the average increase was 0.7 ± 2.5 (p = 0.79) for PGY-3 residents and 5.8 ± 3.3 points (p = 0.09) for PGY-4 residents. CONCLUSIONS: Adoption of an online question bank did not result in higher mean CREOG scores at participating institutions. However, performance on the TrueLearn questions correlated with ITE performance, supporting predictive validity and the use of this question bank as a formative assessment for resident education and exam preparation.

Thyroidectomy practice in pediatric population: a national perspective.

PURPOSE: To examine presentations and outcomes of pediatric patients underoing thyroidectomy. MATERIALS AND METHODS: A retrospective cross-sectional analysis of the Nationwide Readmissions Database, 2010-2014, was performed. Study population included pediatric (<18 years) inpatients undergoing thyroidectomy. RESULTS: A total of 361 patients were included. Mean age was 13.5 ± 0.2 years, and 79.8% were female. Thyroid diseases included: (i) 19.0% thyroid cancer, (ii) 5.4% Multiple Endocrine Neoplasia type II, (iii) 33.6% toxic nodular disease, and (iv) 42.0% non-toxic benign disease. Total thyroidectomy was performed in 67.7% of the patients, and 3.2% of the patients who had initial lobectomy were readmitted within 3 months for completion thyroidectomy. Postoperative complications were reported in 14.2% of the sample, and hypocalcemia was the most common complication (98.2%). Risk of hypocalcemia was significantly higher in patients who had thyroid cancer (risk = 20.9%, p = 0.011) or toxic thyroid diseases (risk = 19.8%, p = 0.033). Of the study population, 25.6% were managed exclusively in children's hospitals. Management in children's hospitals was not associated with improved outcomes or shorter hospital stay; however, it was associated with a significantly higher cost of health services [US $19,4575.0 ± 195.49 vs. US $13,788.00 ± 238.51, p < 0.001]. CONCLUSIONS: This study reports a national perspective on thyroidectomy in the pediatric population. Most thyroid surgeries performed in the pediatric population are performed for benign conditions. Most pediatric thyroidectomies are performed at low-volume centers. Surgeries performed in children's hospitals are significantly higher in cost without any associated improvement in outcomes or length of hospital stay.

Efficient and flexible methods for simulating models of time since infection.

Epidemic models are useful tools in the fight against infectious diseases, as they allow policy makers to test and compare various strategies to limit disease transmission while mitigating collateral damage on the economy. Epidemic models that are more faithful to the microscopic details of disease transmission can offer more reliable projections, which in turn can lead to more reliable control strategies. For example, many epidemic models describe disease progression via a series of artificial stages or compartments (e.g., exposed, activated, infectious, etc.) but an epidemic model that explicitly tracks time since infection (TSI) can provide a more precise description. At present, epidemic models with compartments are more common than TSI models, largely due to the higher computational cost and complexity typically associated with TSI models. Here, however, we show that with the right discretization scheme a TSI model is not much more difficult to solve than a compartment model with three or four stages for the infected class. We also provide a perspective for adding stages to a TSI model in a way that decouples the disease transmission dynamics from the residence time distributions at each stage. These results are also generalized for age-structured TSI models in an Appendix. Finally, as proof of principle for the efficiency of the proposed numerical methods, we provide calculations for optimal epidemic control by nonpharmaceutical intervention. Many of the tools described in this paper are available through the software package pyross.

Efficient Bayesian inference of fully stochastic epidemiological models with applications to COVID-19.

Epidemiological forecasts are beset by uncertainties about the underlying epidemiological processes, and the surveillance process through which data are acquired. We present a Bayesian inference methodology that quantifies these uncertainties, for epidemics that are modelled by (possibly) non-stationary, continuous-time, Markov population processes. The efficiency of the method derives from a functional central limit theorem approximation of the likelihood, valid for large populations. We demonstrate the methodology by analysing the early stages of the COVID-19 pandemic in the UK, based on age-structured data for the number of deaths. This includes maximum a posteriori estimates, Markov chain Monte Carlo sampling of the posterior, computation of the model evidence, and the determination of parameter sensitivities via the Fisher information matrix. Our methodology is implemented in PyRoss, an open-source platform for analysis of epidemiological compartment models.

Bite force estimates in juvenile Tyrannosaurus rex based on simulated puncture marks.

BACKGROUND: Bite marks attributed to adult Tyrannosaurus rex have been subject to numerous studies. However, few bite marks attributed to T. rex have been traced to juveniles, leaving considerable gaps in understanding ontogenetic changes in bite mechanics and force, and the paleoecological role of juvenile tyrannosaurs in the late Cretaceous. METHODS: Here we present bite force estimates for a juvenile Tyrannosaurus rex based on mechanical tests designed to replicate bite marks previously attributed to a T. rex of approximately 13 years old. A maxillary tooth of the juvenile Tyrannosaurus specimen BMR P2002.4.1 was digitized, replicated in dental grade cobalt chromium alloy, and mounted to an electromechanical testing system. The tooth was then pressed into bovine long bones in various locations with differing cortical bone thicknesses at varying speeds for a total of 17 trials. Forces required to replicate punctures were recorded and puncture dimensions were measured. RESULTS: Our experimentally derived linear models suggest bite forces up to 5,641.19 N from cortical bone thickness estimated from puncture marks on an Edmontosaurus and a juvenile Tyrannosaurus. These findings are slightly higher than previously estimated bite forces for a juvenile Tyrannosaurus rex of approximately the same size as BMR P2002.4.1 but fall within the expected range when compared to estimates of adult T. rex. DISCUSSION: The results of this study offer further insight into the role of juvenile tyrannosaurs in late Cretaceous ecosystems. Furthermore, we discuss the implications for feeding mechanisms, feeding behaviors, and ontogenetic niche partitioning.

Rigid Video Laryngoscopy for Intubation in Severe Pierre Robin Sequence: A Retrospective Review.

OBJECTIVES/HYPOTHESIS: The anatomy of children with severe Pierre Robin sequence can present a challenge for direct laryngoscopy and intubation. Advanced techniques including flexible fiberoptic laryngoscopic intubation have been described but require highly specialized skill and equipment. Rigid video laryngoscopy is more accessible but has not been described in this population. STUDY DESIGN: Retrospective cohort study. METHODS: A retrospective review was completed at a tertiary care center of all children between January 2016 and March 2020 with Pierre Robin sequence who underwent a mandibular distraction osteogenesis procedure. Intubation events were collected, and a descriptive analysis was performed. A univariate logistic regression model was applied to direct laryngoscopy and flexible fiberoptic laryngoscopy with rigid video laryngoscopy as a reference. RESULTS: Twenty-five patients were identified with a total of 56 endotracheal events. All patients were successfully intubated. Direct laryngoscopy was successful at first intubation attempt in 47.3% (9/19) of events. Six direct laryngoscopy events required switching to another device. Rigid video laryngoscopy was successful at first intubation attempt in 80.5% (29/36) of events. Two cases required switching to another device. Flexible fiberoptic laryngoscopy was found successful at first intubation attempt in 88.9% (8/9) of events. Direct laryngoscopy was 4 times more likely to fail first intubation attempt when compared to rigid video laryngoscopy (P < .05). There was no significant difference between rigid video laryngoscopy and flexible fiberoptic laryngoscopy for intubation. CONCLUSIONS: For children with Pierre Robin sequence rigid video laryngoscopy should be considered as a first attempt intubation device both in the operating room and for emergent situations. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:1647-1651, 2021.

Neonatal Lateral Epiglottic Defects.

INTRODUCTION: Multiple congenital abnormalities of the epiglottis have been reported and iatrogenic injuries to the larynx and subglottis are well known. We present a new pattern of defect not previously reported in the literature. METHODS: Epiglottic abnormalities at two institutions are reviewed. Cases of defects involving the lateral aspect of the epiglottis and aryepiglottic fold are identified. A literature review of known epiglottic defects is performed. RESULTS: Two children possessing lateral notch injuries at the aryepiglottic attachment to the epiglottis are described. Both children have a history of multiple laryngeal instrumentation attempts and prolonged intubation. Both have swallowing difficulties and are gastrostomy dependent. Congenital epiglottic defects include aplasia and midline bifidity, however, no lateral congenital epiglottic defects have been reported. CONCLUSION: Epiglottic defects, while rare, should be part of the differential for children with aspiration and feeding difficulties. A new pattern of defect is described and iatrogenic etiology proposed.

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9.

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function-to produce large amounts of antibodies-can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Implication of ZNF217 in Accelerating Tumor Development and Therapeutically Targeting ZNF217-Induced PI3K-AKT Signaling for the Treatment of Metastatic Osteosarcoma.

We previously identified ZNF217 as an oncogenic driver of a subset of osteosarcomas using the Sleeping Beauty (SB) transposon system. Here, we followed up by investigating the genetic role of ZNF217 in osteosarcoma initiation and progression through the establishment of a novel genetically engineered mouse model, in vitro assays, orthotopic mouse studies, and paired these findings with preclinical studies using a small-molecule inhibitor. Throughout, we demonstrate that ZNF217 is coupled to numerous facets of osteosarcoma transformation, including proliferation, cell motility, and anchorage independent growth, and ultimately promoting osteosarcoma growth, progression, and metastasis in part through positive modulation of PI3K-AKT survival signaling. Pharmacologic blockade of AKT signaling with nucleoside analogue triciribine in ZNF217+ orthotopically injected osteosarcoma cell lines reduced tumor growth and metastasis. Our data demonstrate that triciribine treatment may be a relevant and efficacious therapeutic strategy for patients with osteosarcoma with ZNF217+ and p-AKT rich tumors. With the recent revitalization of triciribine for clinical studies in other solid cancers, our study provides a rationale for further evaluation preclinically with the purpose of clinical evaluation in patients with incurable, ZNF217+ osteosarcoma.

PLX3397 treatment inhibits constitutive CSF1R-induced oncogenic ERK signaling, reduces tumor growth, and metastatic burden in osteosarcoma.

Osteosarcoma (OSA) is a heterogeneous and aggressive solid tumor of the bone. We recently identified the colony stimulating factor 1 receptor (Csf1r) gene as a novel driver of osteosarcomagenesis in mice using the Sleeping Beauty (SB) transposon mutagenesis system. Here, we report that a CSF1R-CSF1 autocrine/paracrine signaling mechanism is constitutively activated in a subset of human OSA cases and is critical for promoting tumor growth and contributes to metastasis. We examined CSF1R and CSF1 expression in OSAs. We utilized gain-of-function and loss-of-function studies (GOF/LOF) to evaluate properties of cellular transformation, downstream signaling, and mechanisms of CSF1R-CSF1 action. Genetic perturbation of CSF1R in immortalized osteoblasts and human OSA cell lines significantly altered oncogenic properties, which were dependent on the CSF1R-CSF1 autocrine/paracrine signaling. These functional alterations were associated with changes in the known CSF1R downstream ERK effector pathway and mitotic cell cycle arrest. We evaluated the recently FDA-approved CSF1R inhibitor Pexidartinib (PLX3397) in OSA cell lines in vitro and in vivo in cell line and patient-derived xenografts. Pharmacological inhibition of CSF1R signaling recapitulated the in vitro genetic alterations. Moreover, in orthotopic OSA cell line and subcutaneous patient-derived xenograft (PDX)-injected mouse models, PLX3397 treatment significantly inhibited local OSA tumor growth and lessened metastatic burden. In summary, CSF1R is utilized by OSA cells to promote tumorigenesis and may represent a new molecular target for therapy.

An in-silico human cell model reveals the influence of spatial organization on RNA splicing.

Spatial organization is a characteristic of all cells, achieved in eukaryotic cells by utilizing both membrane-bound and membrane-less organelles. One of the key processes in eukaryotes is RNA splicing, which readies mRNA for translation. This complex and highly dynamical chemical process involves assembly and disassembly of many molecules in multiple cellular compartments and their transport among compartments. Our goal is to model the effect of spatial organization of membrane-less organelles (specifically nuclear speckles) and of organelle heterogeneity on splicing particle biogenesis in mammalian cells. Based on multiple sources of complementary experimental data, we constructed a spatial model of a HeLa cell to capture intracellular crowding effects. We then developed chemical reaction networks to describe the formation of RNA splicing machinery complexes and splicing processes within nuclear speckles (specific type of non-membrane-bound organelles). We incorporated these networks into our spatially-resolved human cell model and performed stochastic simulations for up to 15 minutes of biological time, the longest thus far for a eukaryotic cell. We find that an increase (decrease) in the number of nuclear pore complexes increases (decreases) the number of assembled splicing particles; and that compartmentalization is critical for the yield of correctly-assembled particles. We also show that a slight increase of splicing particle localization into nuclear speckles leads to a disproportionate enhancement of mRNA splicing and a reduction in the noise of generated mRNA. Our model also predicts that the distance between genes and speckles has a considerable effect on the mRNA production rate, with genes located closer to speckles producing mRNA at higher levels, emphasizing the importance of genome organization around speckles. The HeLa cell model, including organelles and sub-compartments, provides a flexible foundation to study other cellular processes that are strongly modulated by spatiotemporal heterogeneity.