Engineering structural variants to interrogate genome function.

Structural variation, such as deletions, duplications, inversions and complex rearrangements, can have profound effects on gene expression, genome stability, phenotypic diversity and disease susceptibility. Structural variants can encompass up to millions of bases and have the potential to rearrange substantial segments of the genome. They contribute considerably more to genetic diversity in human populations and have larger effects on phenotypic traits than point mutations.

Base editing screens define the genetic landscape of cancer drug resistance mechanisms.

Drug resistance is a principal limitation to the long-term efficacy of cancer therapies. Cancer genome sequencing can retrospectively delineate the genetic basis of drug resistance, but this requires large numbers of post-treatment samples to nominate causal variants. Here we prospectively identify genetic mechanisms of resistance to ten oncology drugs from CRISPR base editing mutagenesis screens in four cancer cell lines using a guide RNA library predicted to install 32,476 variants in 11 cancer genes.

Misexpression of inactive genes in whole blood is associated with nearby rare structural variants.

Gene misexpression is the aberrant transcription of a gene in a context where it is usually inactive. Despite its known pathological consequences in specific rare diseases, we have a limited understanding of its wider prevalence and mechanisms in humans. To address this, we analyzed gene misexpression in 4,568 whole-blood bulk RNA sequencing samples from INTERVAL study blood donors.

Multiplex generation and single cell analysis of structural variants in a mammalian genome.

The functional consequences of structural variants (SVs) in mammalian genomes are challenging to study. This is due to several factors, including: 1) their numerical paucity relative to other forms of standing genetic variation such as single nucleotide variants (SNVs) and short insertions or deletions (indels); 2) the fact that a single SV can involve and potentially impact the function of more than one gene and/or regulatory element; and 3) the relative immaturity of methods to generate and map SVs, either randomly or in targeted fashion, in or model systems. Towards addressing these challenges, we developed , a straightforward method that enables the multiplex generation and mapping of several major forms of SVs (deletions, inversions, translocations) throughout a mammalian genome.

Predicting Mutations Generated by Cas9, Base Editing, and Prime Editing in Mammalian Cells.

The first fruits of the CRISPR-Cas revolution are starting to enter the clinic, with gene editing therapies offering solutions to previously incurable genetic diseases. The success of such applications hinges on control over the mutations that are generated, which are known to vary depending on the targeted locus. In this review, we present the current state of understanding and predicting CRISPR-Cas cutting, base editing, and prime editing outcomes in mammalian cells.

Prediction of prime editing insertion efficiencies using sequence features and DNA repair determinants.

Most short sequences can be precisely written into a selected genomic target using prime editing; however, it remains unclear what factors govern insertion. We design a library of 3,604 sequences of various lengths and measure the frequency of their insertion into four genomic sites in three human cell lines, using different prime editor systems in varying DNA repair contexts. We find that length, nucleotide composition and secondary structure of the insertion sequence all affect insertion rates.

Degradation of GSPT1 causes TP53-independent cell death in leukemia while sparing normal hematopoietic stem cells.

Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early-phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents.

BTB dimers as building blocks for reversible drug-induced protein oligomerization.

Here, we characterize the BTB domain of the transcription factor BCL6 (BTB) as a small-molecule-controlled, reversible oligomerization switch, which oligomerizes upon BI-3802 treatment and de-oligomerizes upon addition of BI-3812. We show that the magnitude of oligomerization can be controlled by BI-3802 concentration and exposure time. In cellular models, exposure to BI-3802/BI-3812 can drive multiple cycles of foci formation consisting of BTB fused to EGFP, which are not degraded due to the lack of a degron.

Predicting base editing outcomes using position-specific sequence determinants.

CRISPR/Cas base editors promise nucleotide-level control over DNA sequences, but the determinants of their activity remain incompletely understood. We measured base editing frequencies in two human cell lines for two cytosine and two adenine base editors at ∼14 000 target sequences and find that base editing activity is sequence-biased, with largest effects from nucleotides flanking the target base. Whether a base is edited depends strongly on the combination of its position in the target and the preceding base, acting to widen or narrow the effective editing window.

Recreational Marijuana Use, Adolescent Cognitive Development, and Schizophrenia Susceptibility.

Background: We investigated how low marijuana (MJ) use levels, the typical use pattern in most adolescent users, affect cognitive maturation and schizophrenia risk.

Methods: In two complementary adolescent samples where the majority reported minimal MJ use, we compared cognitive performances before and after MJ use initiation. The Iowa sample (40 first-degree relatives and 54 second-degree relatives of patients with schizophrenia and 117 control subjects with no schizophrenia family history) underwent a battery of standardized neuropsychological tests at 0, 18, and 36 months.

Characterization of a 200-nF unipolar, high current, low inductance capacitor switch assembly.

Capacitor switch assemblies (CSAs) are a viable alternative to the standard Linear Transformer Driver (LTD) brick-the prime power "building block" in LTD-based high current drivers. In a CSA, the geometrical advantages combined with the switch's placement within a CSA produce a significantly lower inductance structure compared to the LTD brick. Lower inductances enable higher peak currents and faster rise times when all other parameters are held constant, thus making low inductance a crucial feature in high current systems.

Small-molecule-induced polymerization triggers degradation of BCL6.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets, but a substantial subset of proteins are resistant to targeted degradation using existing approaches. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation.

Selection of Oncogenic Mutant Clones in Normal Human Skin Varies with Body Site.

Skin cancer risk varies substantially across the body, yet how this relates to the mutations found in normal skin is unknown. Here we mapped mutant clones in skin from high- and low-risk sites. The density of mutations varied by location.

The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K.

Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously.

Impulsive decision making, brain cortical thickness and familial schizophrenia risk.

Background: Schizophrenia (SZ) patients and their biological relatives are more impulsive than controls. Although greater impulsivity in SZ has been associated with dysfunction in prefrontal neural circuits implicated in reward processing, little is known regarding brain structural correlates of heightened impulsivity in unaffected adolescent relatives of SZ patients.

Methods: Impulsive decision-making was assessed using the delay discounting task in 174 adolescents: 36 first-degree relatives (FDR) and 50 second-degree relatives (SDR) of SZ patients, and 88 healthy controls with no SZ family history (NSFH).

Loss of Neurological Disease HSAN-I-Associated Gene SPTLC2 Impairs CD8 T Cell Responses to Infection by Inhibiting T Cell Metabolic Fitness.

Patients with the neurological disorder HSAN-I suffer frequent infections, attributed to a lack of pain sensation and failure to seek care for minor injuries. Whether protective CD8 T cells are affected in HSAN-I patients remains unknown. Here, we report that HSAN-I-associated mutations in serine palmitoyltransferase subunit SPTLC2 dampened human T cell responses.

The Most Common Causes of Eye Pain at 2 Tertiary Ophthalmology and Neurology Clinics.

Background: Eye pain is a common complaint, but no previous studies have determined the most common causes of this presenting symptom. Our objective was to determine the most common causes of eye pain in 2 ophthalmology and neurology departments at academic medical centers.

Methods: This was a retrospective cross-sectional analysis and chart review at the departments of ophthalmology and neurology at the University Hospital Zurich (USZ), University of Zürich, Switzerland, and the University of Utah (UU), USA.

Impulsivity in unaffected adolescent biological relatives of schizophrenia patients.

Objective: Although schizophrenia is not a prototypic impulse-control disorder, patients report more impulsive behaviors, have higher rates of substance use, and show dysfunction in brain circuits that underlie impulsivity. We investigate impulsivity in unaffected biological relatives of schizophrenia patients to further understand the relationships between schizophrenia risk and impulse control during adolescence.

Method: Group differences in impulsivity (UPPS-P Impulsive Behavior Scale and delay discounting) were tested in 210 adolescents contrasting 39 first- and 53 second-degree biological relatives of schizophrenia patients, and 118 subjects with no schizophrenia family history (NSFH).

Cerebral white matter correlates of delay discounting in adolescents.

The adolescent brain undergoes extensive structural white matter (WM) changes. Adolescence is also a critical time period during which cognitive, emotional and social maturation occurs in transition into adulthood. Compared to adults, adolescents are generally more impulsive with increased risk-taking behaviors.

Preliminary Investigation of Cerebral Blood Flow and Amyloid Burden in Veterans With and Without Combat-Related Traumatic Brain Injury.

This study aimed to examine global and regional cerebral blood flow and amyloid burden in combat veterans with and without traumatic brain injury (TBI). Cerebral blood flow (in milliliters per minute per 100 mL) was measured by quantitative [(15)O]water, and amyloid burden was measured by [(11)C]PIB imaging. Mean global cerebral blood flow was significantly lower in veterans with TBI compared with non-TBI veterans.

Dolphin shows and interaction programs: benefits for conservation education?

Dolphin shows and dolphin interaction programs are two types of education programs within zoological institutions used to educate visitors about dolphins and the marine environment. The current study examined the short- and long-term effects of these programs on visitors' conservation-related knowledge, attitude, and behavior. Participants of both dolphin shows and interaction programs demonstrated a significant short-term increase in knowledge, attitudes, and behavioral intentions.

Effects of smoking marijuana on focal attention and brain blood flow.

Using an attention task to control cognitive state, we previously found that smoking marijuana changes regional cerebral blood flow (rCBF). The present study measured rCBF during tasks requiring attention to left and right ears in different conditions. Twelve occasional marijuana users (mean age 23.

Effect of acute marijuana on cardiovascular function and central nervous system pharmacokinetics of [(15)O]water: effect in occasional and chronic users.

The objective of this study was to evaluate the effect of the acute administration of marijuana (MJ) on cardiovascular (CV) function and CNS pharmacokinetics (PK) of [(15)O]water in occasional (O) versus chronic (C) MJ users. Each subject received four injections of [(15)O]water (one prior and three postsmoking) on two occasions in which they received active or placebo MJ. For each injection, measures of CV function and CNS PK [(15)O]water were made.

Marijuana alters the human cerebellar clock.

The effects of marijuana on brain perfusion and internal timing were assessed using [15O] water PET in occasional and chronic users. Twelve volunteers who smoked marijuana recreationally about once weekly, and 12 volunteers who smoked daily for a number of years performed a self-paced counting task during PET imaging, before and after smoking marijuana and placebo cigarettes. Smoking marijuana increased rCBF in the ventral forebrain and cerebellar cortex in both groups, but resulted in significantly less frontal lobe activation in chronic users.

Effects of smoking marijuana on brain perfusion and cognition.

The effects of smoking marijuana on regional cerebral blood flow (rCBF) and cognitive performance were assessed in 12 recreational users in a double-blinded, placebo-controlled study. PET with [(15)Oxygen]-labeled water ([(15)O]H(2)O) was used to measure rCBF before and after smoking of marijuana and placebo cigarettes, as subjects repeatedly performed an auditory attention task. Smoking marijuana resulted in intoxication, as assessed by a behavioral rating scale, but did not significantly alter mean behavioral performance on the attention task.