Rewiring cancer drivers to activate apoptosis.

Genes that drive the proliferation, survival, invasion and metastasis of malignant cells have been identified for many human cancers1-4. Independent studies have identified cell death pathways that eliminate cells for the good of the organism5,6. The coexistence of cell death pathways with driver mutations suggests that the cancer driver could be rewired to activate cell death using chemical inducers of proximity (CIPs). Here we describe a new class of molecules called transcriptional/epigenetic CIPs (TCIPs) that recruit the endogenous cancer driver, or a downstream transcription factor, to the promoters of cell death genes, thereby activating their expression. We focused on diffuse large B cell lymphoma, in which the transcription factor B cell lymphoma 6 (BCL6) is deregulated7. BCL6 binds to the promoters of cell death genes and epigenetically suppresses their expression8. We produced TCIPs by covalently linking small molecules that bind BCL6 to those that bind to transcriptional activators that contribute to the oncogenic program, such as BRD4. The most potent molecule, TCIP1, increases binding of BRD4 by 50% over genomic BCL6-binding sites to produce transcriptional elongation at pro-apoptotic target genes within 15 min, while reducing binding of BRD4 over enhancers by only 10%, reflecting a gain-of-function mechanism. TCIP1 kills diffuse large B cell lymphoma cell lines, including chemotherapy-resistant, TP53-mutant lines, at EC50 of 1-10 nM in 72 h and exhibits cell-specific and tissue-specific effects, capturing the combinatorial specificity inherent to transcription. The TCIP concept also has therapeutic applications in regulating the expression of genes for regenerative medicine and developmental disorders.

Defining synonymous codon compression schemes by genome recoding.

Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome.

Genomic targeting of epigenetic probes using a chemically tailored Cas9 system.

Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small-molecule bromodomain and extra-terminal family bromodomain inhibitor JQ1 or a peptide-based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials.

Information-optimal genome assembly via sparse read-overlap graphs.

MOTIVATION: In the context of third-generation long-read sequencing technologies, read-overlap-based approaches are expected to play a central role in the assembly step. A fundamental challenge in assembling from a read-overlap graph is that the true sequence corresponds to a Hamiltonian path on the graph, and, under most formulations, the assembly problem becomes NP-hard, restricting practical approaches to heuristics. In this work, we avoid this seemingly fundamental barrier by first setting the computational complexity issue aside, and seeking an algorithm that targets information limits In particular, we consider a basic feasibility question: when does the set of reads contain enough information to allow unambiguous reconstruction of the true sequence? RESULTS: Based on insights from this information feasibility question, we present an algorithm-the Not-So-Greedy algorithm-to construct a sparse read-overlap graph. Unlike most other assembly algorithms, Not-So-Greedy comes with a performance guarantee: whenever information feasibility conditions are satisfied, the algorithm reduces the assembly problem to an Eulerian path problem on the resulting graph, and can thus be solved in linear time. In practice, this theoretical guarantee translates into assemblies of higher quality. Evaluations on both simulated reads from real genomes and a PacBio Escherichia coli K12 dataset demonstrate that Not-So-Greedy compares favorably with standard string graph approaches in terms of accuracy of the resulting read-overlap graph and contig N50. AVAILABILITY: Available at github.com/samhykim/nsg CONTACT: courtade@eecs.berkeley.edu or dntse@stanford.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Simultaneous Single-Cell Profiling of the Transcriptome and Accessible Chromatin Using SHARE-seq.

The ability to analyze the transcriptomic and epigenomic states of individual single cells has in recent years transformed our ability to measure and understand biological processes. Recent advancements have focused on increasing sensitivity and throughput to provide richer and deeper biological insights at the cellular level. The next frontier is the development of multiomic methods capable of analyzing multiple features from the same cell, such as the simultaneous measurement of the transcriptome and the chromatin accessibility of candidate regulatory elements. In this chapter, we discuss and describe SHARE-seq (Simultaneous high-throughput ATAC, and RNA expression with sequencing) for carrying out simultaneous chromatin accessibility and transcriptome measurements in single cells, together with the experimental and analytical considerations for achieving optimal results.

CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping.

Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed CasKAS, a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein. We demonstrate this method in both in vitro and in vivo contexts.

Finding needles in a haystack: dissecting tumor heterogeneity with single-cell transcriptomic and chromatin accessibility profiling.

Tumor evolution often results in a wealth of heterogeneous cancer cell types within a single tumor - heterogeneity that can include epigenetic and gene expression changes that are impossible to identify from histological features alone. The invasion of cancer cells into nearby healthy tissue, accompanied by the infiltration of responding immune cells, results in an even more complex architecture of tumor and non-tumor cells. However, bulk genomics-based methods can only assay the aggregate transcriptomic and epigenetic profiles across all of this rich cellular diversity. Such bulk averaging hides small subpopulations of tumor cells with unique phenotypes that might result in therapeutic resistance or metastatic progression. The advent of single-cell-based genomics assays for measuring transcription and chromatin accessibility - particularly scRNA-seq and scATAC-seq - has enabled the dissection of cell-types within tumors at a scale and resolution capable of unraveling the epigenetic and gene expression programs of rare and unique cellular subpopulations. This Review focuses on recent advances in scRNA-seq and scATAC-seq technologies and their application to cancer biology in the context of furthering our understanding of tumor heterogeneity.

SEQUIN Multiscale Imaging of Mammalian Central Synapses Reveals Loss of Synaptic Connectivity Resulting from Diffuse Traumatic Brain Injury.

The brain's complex microconnectivity underlies its computational abilities and vulnerability to injury and disease. It has been challenging to illuminate the features of this synaptic network due to the small size and dense packing of its elements. Here, we describe a rapid, accessible super-resolution imaging and analysis workflow-SEQUIN-that quantifies central synapses in human tissue and animal models, characterizes their nanostructural and molecular features, and enables volumetric imaging of mesoscale synaptic networks without the production of large histological arrays. Using SEQUIN, we identify cortical synapse loss resulting from diffuse traumatic brain injury, a highly prevalent connectional disorder. Similar synapse loss is observed in three murine models of Alzheimer-related neurodegeneration, where SEQUIN mesoscale mapping identifies regional synaptic vulnerability. These results establish an easily implemented and robust nano-to-mesoscale synapse quantification and characterization method. They furthermore identify a shared mechanism-synaptopathy-between Alzheimer neurodegeneration and its best-established epigenetic risk factor, brain trauma.

Isolated proximal greater saphenous vein thrombosis and the risk of propagation to deep vein thrombosis and pulmonary embolism.

OBJECTIVES: Greater saphenous vein (GSV) thrombosis is concerning due to its close proximity to the deep femoral vein. This study sought to identify the risk of propagation to deep vein thrombosis (DVT) or pulmonary embolism (PE) among patients with isolated proximal GSV superficial thrombosis and describe provider practice patterns related to treatment. MATERIALS AND METHODS: This is an Institutional Review Board-approved retrospective multi-center study. Patients presented to one of three possible emergency departments in a large health system. About 21,716 patients were queried through the electronic medical record. Ninety-five patients or 0.4% of study subjects met inclusion criteria of isolated proximal GSV thrombosis. Forty-five patients were excluded, leaving a final data set of 40 patients. Investigators recorded radiology impressions, patient demographics, past medical history, DVT/PE risk factors, and treatment plans. Propagation of GSV thrombosis to DVT/PE was also noted. Follow-up methods included chart review, primary care physician follow-up, and direct, scripted patient follow-up phone calls. Descriptive statistics were applied to study subjects using SAS for Windows, version 9.3. RESULTS: Three patients (7.5%) had progression of GSV thrombosis to DVT/PE. Twenty percent of patients without malignancy were treated with anticoagulation compared to 14% of those with preexisting malignancy upon initial diagnosis of isolated GSV thrombosis. Forty-five percent of patients were prescribed some type of supportive therapy to aid in the treatment of GSV thrombosis. CONCLUSION: Isolated proximal GSV thrombosis, while uncommon, may frequently progress to DVT or PE. Our work suggests clinicians should consider anticoagulation for isolated GSV thrombosis.

The diagnosis of acute appendicitis in a pediatric population: to CT or not to CT.

PURPOSE: The aim of this study was to determine if focused appendiceal computed tomography with colon contrast (FACT-CC) increases the accuracy of the preoperative diagnosis of acute appendicitis in children. METHODS: A 5-year retrospective review was conducted of a university hospital database of 283 patients (age 0.8 to 19.3 years; mean, 11.3 years) treated with appendectomy for presumed acute appendicitis. RESULTS: Of the 283 patients in whom appendectomies were performed, 268 were confirmed by pathologic analysis of the specimen to have acute appendicitis for a diagnostic accuracy in our institution of 94.7%. Ninety-six patients (34%) underwent FACT-CC scans as part of their preoperative evaluation. The sensitivity of the computed tomography (CT) scan was 94.6%, and the positive predictive value was 95.6%. In girls older than 10 years, CT imaging was not significantly more accurate in predicting appendicitis than examination alone (93.9% v. 87.5%; P =.46). CONCLUSIONS: Preoperative FACT-CC did not increase the accuracy in diagnosing appendicitis when compared with patients diagnosed by history, physical examination and laboratory studies. If there was a strong suspicion of appendicitis, a negative CT scan did not exclude the diagnosis of appendicitis. However, focused appendiceal CT scan is a sensitive test with a high positive predictive value and may be useful in a patient with an atypical history or examination.