A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria.

Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.

Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.

Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.

CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.

The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

Correlative montage parallel array cryo-tomography for in situ structural cell biology.

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here we present robust tools for montage parallel array cryo-tomography (MPACT) tailored for vitrified specimens. The combination of correlative cryo-fluorescence microscopy, focused-ion-beam milling, substrate micropatterning, and MPACT supports studies that contextually define the three-dimensional architecture of cells. To further extend the flexibility of MPACT, tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

Applied force reveals mechanistic and energetic details of transcription termination.

Transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences coding for a GC-rich RNA hairpin followed by a U-rich tract. We used single-molecule techniques to investigate the mechanism by which three representative terminators (his, t500, and tR2) destabilize the elongation complex (EC). For his and tR2 terminators, loads exerted to bias translocation did not affect termination efficiency (TE). However, the force-dependent kinetics of release and the force-dependent TE of a mutant imply a forward translocation mechanism for the t500 terminator. Tension on isolated U-tracts induced transcript release in a manner consistent with RNA:DNA hybrid shearing. We deduce that different mechanisms, involving hypertranslocation or shearing, operate at terminators with different U-tracts. Tension applied to RNA at terminators suggests that closure of the final 2-3 hairpin bases destabilizes the hybrid and that competing RNA structures modulate TE. We propose a quantitative, energetic model that predicts the behavior for these terminators and mutant variants.

A multisystem, dimensional interplay of assets versus adversities: Revised benevolent childhood experiences (BCEs) in the context of childhood maltreatment, threat, and deprivation.

This study expanded the Benevolent Childhood Experiences scale (termed the "BCEs-Original" scale) with 10 new multisystem items and identified a subset of items (termed the "BCEs-Revised" scale) that are systematically less commonly reported across samples. Total BCEs-Revised scores were tested against total BCEs-Original scores and three dimensions of childhood adversity (maltreatment, threat, and deprivation) as predictors of young adulthood mental health problems (depression, anxiety, and PTSD symptoms). Hypotheses expected stronger inverse associations of BCEs-Revised scores than BCEs-Original scores with all mental health problems. Participants were 1,746 U.S. young adults (M = 26.6 years, SD = 4.7, range = 19-35 years; 55.3% female, 42.4% male, 2.3% gender non-conforming; 67.0% White, 10.3% Asian, 8.6% Black, 8.4% Latine, 5.7% other) who completed a 20-item BCEs scale and well-validated instruments on childhood adversities and mental health problems. Compared to BCEs-Original scores, BCEs-Revised scores were significantly more strongly inversely associated with all mental health outcomes. Compared to childhood threat and deprivation, maltreatment was significantly more strongly associated with PTSD symptoms. After controlling for current depression symptoms, BCEs-Revised scores interacted with maltreatment to predict PTSD symptoms. Maltreatment and BCEs-Revised scores also influenced PTSD symptoms in person-oriented analyses. The BCEs-Revised scale has strong psychometric properties and unique strengths in research and practice. Implications for multisystem resilience are discussed.

CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy.

Cryo-correlative light and electron microscopy (CLEM) is a technique that uses the spatiotemporal cues from fluorescence light microscopy (FLM) to investigate the high-resolution ultrastructure of biological samples by cryo-electron microscopy (cryo-EM). Cryo-CLEM provides advantages for identifying and distinguishing fluorescently labeled proteins, macromolecular complexes, and organelles from the cellular environment. Challenges remain on how correlation workflows and software tools are implemented on different microscope platforms to support automated cryo-EM data acquisition. Here, we present CorRelator: an open-source desktop application that bridges between cryo-FLM and real-time cryo-EM/ET automated data collection. CorRelator implements a pixel-coordinate-to-stage-position transformation for flexible, high accuracy on-the-fly and post-acquisition correlation. CorRelator can be integrated into cryo-CLEM workflows and easily adapted to standard fluorescence and transmission electron microscope (TEM) system configurations. CorRelator was benchmarked under live-cell and cryogenic conditions using several FLM and TEM instruments, demonstrating that CorRelator reliably supports real-time, automated correlative cryo-EM/ET acquisition, through a combination of software-aided and interactive alignment. CorRelator is a cross-platform software package featuring an intuitive Graphical User Interface (GUI) that guides the user through the correlation process. CorRelator source code is available at: https://github.com/wright-cemrc-projects/corr.

Alignment-free filtering for cfNA fusion fragments.

MOTIVATION: Cell-free nucleic acid (cfNA) sequencing data require improvements to existing fusion detection methods along multiple axes: high depth of sequencing, low allele fractions, short fragment lengths and specialized barcodes, such as unique molecular identifiers. RESULTS: AF4 was developed to address these challenges. It uses a novel alignment-free kmer-based method to detect candidate fusion fragments with high sensitivity and orders of magnitude faster than existing tools. Candidate fragments are then filtered using a max-cover criterion that significantly reduces spurious matches while retaining authentic fusion fragments. This efficient first stage reduces the data sufficiently that commonly used criteria can process the remaining information, or sophisticated filtering policies that may not scale to the raw reads can be used. AF4 provides both targeted and de novo fusion detection modes. We demonstrate both modes in benchmark simulated and real RNA-seq data as well as clinical and cell-line cfNA data. AVAILABILITY AND IMPLEMENTATION: AF4 is open sourced, licensed under Apache License 2.0, and is available at: https://github.com/grailbio/bio/tree/master/fusion.

Single-molecule studies of RNA polymerase: one singular sensation, every little step it takes.

Transcription is the first of many biochemical steps that turn the genetic information found in DNA into the proteins responsible for driving cellular processes. In this review, we highlight certain advantages of single-molecule techniques in the study of prokaryotic and eukaryotic transcription, and the specific ways in which these techniques complement conventional, ensemble-based biochemistry. We focus on recent literature, highlighting examples where single-molecule methods have provided fresh insights into mechanism. We also present recent technological advances and outline future directions in the field.

The National Dental Practice-Based Research Network Adult Anterior Open Bite Study: Treatment recommendations and their association with patient and practitioner characteristics.

INTRODUCTION: This aim of this paper is to describe and identify the practitioner and patient characteristics that are associated with treatment recommendations for adult anterior open bite patients across the United States. METHODS: Practitioners and patients were recruited within the framework of the National Dental Practice-Based Research Network. Practitioners were asked about their demographic characteristics and their treatment recommendations for these patients. The practitioners also reported on their patients' dentofacial characteristics and provided initial cephalometric scans and intraoral photographs. Patients were asked about their demographic characteristics, previous orthodontic treatment, and goals for treatment. Four main treatment groups were evaluated: aligners, fixed appliances, temporary anchorage devices (TADs), and orthognathic surgery. Extractions were also investigated. Predictive multivariable models were created comparing various categories of treatment as well as extraction/nonextraction decisions. RESULTS: Ninety-one practitioners (mostly orthodontists) and 347 patients were recruited from October 2015 to December 2016. Increased aligner recommendations were associated with white and Asian patients, the presence of tongue habits, and female practitioners. TADs were recommended more often in academic settings. Recommendations for orthognathic surgery were associated with demographic factors, such as availability of insurance coverage and practitioner race/ethnicity, and dentofacial characteristics, such as anteroposterior discrepancies, more severe open bites, and steeper mandibular plane angles. Extraction recommendations were largely associated with severe crowding and incisor proclination. CONCLUSIONS: Both doctor and patient demographic factors, as well as dentofacial characteristics, were significantly associated with treatment recommendations for adult anterior open bite patients.

Catastrophic Antiphospholipid Syndrome Presenting with Genitourinary Manifestations.

Catastrophic antiphospholipid syndrome (CAPS) is a rare disorder characterized by acute, multi-system dysfunc- tion due to small-vessel thrombosis related to anti-phospholipid antibodies. Here we present an unusual case of CAPS presenting with genitourinary manifestations. A 72-year-old male developed a series of symptoms over the course of two weeks. His symptoms included testicular inflammation, scrotal edema, priapism, hematuria, penile eschar, elbow eschars, and acute kidney injury. He was found to have anti-phospholipid antibodies and treated with anticoagulation, high-dose steroids and plasma exchange. His symptoms resolved with minimal lasting effects. This case is unique to the literature because of the extensive genitourinary involvement.

Glucan Binding Protein C of Streptococcus mutans Mediates both Sucrose-Independent and Sucrose-Dependent Adherence.

The high-resolution structure of glucan binding protein C (GbpC) at 1.14 Å, a sucrose-dependent virulence factor of the dental caries pathogen Streptococcus mutans, has been determined. GbpC shares not only structural similarities with the V regions of AgI/II and SspB but also functional adherence to salivary agglutinin (SAG) and its scavenger receptor cysteine-rich domains (SRCRs). This is not only a newly identified function for GbpC but also an additional fail-safe binding mechanism for S. mutans Despite the structural similarities with S. mutans antigen I/II (AgI/II) and SspB of Streptococcus gordonii, GbpC remains unique among these surface proteins in its propensity to adhere to dextran/glucans. The complex crystal structure of GbpC with dextrose (ß-d-glucose; Protein Data Bank ligand BGC) highlights exclusive structural features that facilitate this interaction with dextran. Targeted deletion mutant studies on GbpC's divergent loop region in the vicinity of a highly conserved calcium binding site confirm its role in biofilm formation. Finally, we present a model for adherence to dextran. The structure of GbpC highlights how artfully microbes have engineered the lectin-like folds to broaden their functional adherence repertoire.

Endostatin: A novel inhibitor of androgen receptor function in prostate cancer.

Acquired resistance to androgen receptor (AR)-targeted therapies compels the development of novel treatment strategies for castration-resistant prostate cancer (CRPC). Here, we report a profound effect of endostatin on prostate cancer cells by efficient intracellular trafficking, direct interaction with AR, reduction of nuclear AR level, and down-regulation of AR-target gene transcription. Structural modeling followed by functional analyses further revealed that phenylalanine-rich α1-helix in endostatin-which shares structural similarity with noncanonical nuclear receptor box in AR-antagonizes AR transcriptional activity by occupying the activation function (AF)-2 binding interface for coactivators and N-terminal AR AF-1. Together, our data suggest that endostatin can be recognized as an endogenous AR inhibitor that impairs receptor function through protein-protein interaction. These findings provide new insights into endostatin whose antitumor effect is not limited to inhibiting angiogenesis, but can be translated to suppressing AR-mediated disease progression in CRPC.

With or Without You? Contextualizing the Impact of Romantic Relationship Breakup on Crime Among Serious Adolescent Offenders.

The decline and delay of marriage has prolonged adolescence and the transition to adulthood, and consequently fostered greater romantic relationship fluidity during a stage of the life course that is pivotal for both development and offending. Yet, despite a growing literature of the consequences of romantic relationships breakup, little is known about its connection with crime, especially among youth enmeshed in the criminal justice system. This article addresses this gap by examining the effects of relationship breakup on crime among justice-involved youth-a key policy-relevant group. We refer to data from the Pathways to Desistance Study, a longitudinal study of 1354 (14% female) adjudicated youth from the juvenile and adult court systems in Phoenix and Philadelphia, to assess the nature and complexity of this association. In general, our results support prior evidence of breakup's criminogenic influence. Specifically, they suggest that relationship breakup's effect on crime is particularly acute among this at-risk sample, contingent upon post-breakup relationship transitions, and more pronounced for relationships that involve cohabitation. Our results also extend prior work by demonstrating that breakup is attenuated by changes in psychosocial characteristics and peer associations/exposure. We close with a discussion of our findings, their policy implications, and what they mean for research on relationships and crime among serious adolescent offenders moving forward.

Predictors of emotional and physical dating violence in a sample of serious juvenile offenders.

AIM: We estimate group-based dating violence trajectories and identify the adolescent risk factors that explain membership in each trajectory group. METHOD: Using longitudinal data from the Pathways to Desistance Study, which follows a sample of 1354 serious juvenile offenders from Philadelphia, Pennsylvania and Phoenix, Arizona between mid-adolescence and early adulthood, we estimate group-based trajectory models of both emotional dating violence and physical dating violence over a span of five years in young adulthood. We then estimate multinomial logistic regression models to identify theoretically motivated risk factors that predict membership in these groups. RESULTS: We identified three developmental patterns of emotional dating violence: none (33%), low-level (59%) and high-level decreasing (8%). The best-fitting model for physical dating violence also had three groups: none (73%), low-level (24%) and high-level (3%). Race/ethnicity, family and psychosocial variables were among the strongest predictors of both emotional and physical dating violence. In addition, delinquency history variables predicted emotional dating violence and relationship variables predicted physical dating violence. CONCLUSIONS: Dating violence is quite prevalent in young adulthood among serious juvenile offenders. Numerous predictors distinguish between chronic dating violence perpetrators and other groups. These may suggest points of intervention for reducing future violence. Copyright © 2016 John Wiley & Sons, Ltd.