Focused ultrasound-mediated brain genome editing.

Gene editing in the brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches mainly rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain

Development and Validation of Electronic Quality Measures to Assess Care for Patients With Transient Ischemic Attack and Minor Ischemic Stroke

Background—Despite interest in using electronic health record (EHR) data to assess quality of care, the accuracy of such data is largely unknown. We sought to develop and validate transient ischemic attack and minor ischemic stroke electronic quality measures (eQMs) using EHR data. Methods and Results—A random sample of patients with transient ischemic attack or minor ischemic stroke, cared for in Veterans Health Administration facilities (fiscal year 2011), was identified. We constructed 31 eQMs based on existing quality measures. Chart review was the criterion standard for validating the eQMs. To evaluate eQMs in terms of eligibility, we calculated the proportion of patients who were genuinely not eligible to receive a process (based on chart review) and who were correctly identified as not eligible by the EHR data (specificity). To assess eQMs about classification of whether patients received a process, we calculated the proportion of patients who actually received the process (based on chart review) and who were classified correctly by the EHR data as passing (sensitivity). Seven hundred sixty-three patients were included. About eligibility, specificity varied from 25% (brain imaging; carotid imaging) to 99% (anticoagulation quality). About pass rates, sensitivity varied from 30% (antihypertensive class) to 100% (coronary risk assessment; international normalized ratio measured). The 16 eQMs with ≥70% specificity in eligibility and ≥70% sensitivity in pass rates included coronary risk assessment, international normalized ratio measured, HbA1c measurement, speech language pathology consultation, anticoagulation for atrial fibrillation, discharge on statin, lipid management, neurology consultation, Holter, deep vein thrombosis prophylaxis, oral hypoglycemic intensification, cholesterol medication intensification, antihypertensive intensification, antihypertensive class, carotid stenosis intervention, and substance abuse referral for alcohol. Conclusions—It is feasible to construct valid eQMs for processes of transient ischemic attack and minor ischemic stroke care. Healthcare systems with EHRs should consider using electronic data to evaluate care for their patients with transient ischemic attack and to complement and expand quality measurement programs currently focused on patients with stroke

Causes and Consequences of Programmed DNA Breaks and Translocations in B Cell Nuclei

Mammalian cells are regularly subjected to DNA damaging agents that create DNA double strand breaks (DSBs) within the genome which must be managed and repaired to avoid cancerous outcomes. In certain cell types, in particular B cells of the immune system, developmentally programmed DNA damage also occurs and must be correctly repaired. In this body of work, I investigated some of the causes and consequences of DNA repair failure as a result of programmed DSBs in B cell nuclei. The results of this research indicate that two proteins, recombination activating gene (RAG) and activation-induced cytidine deaminase (AID), which both mediate programmed DNA breaks in the immunoglobulin loci, have non-canonical activities. The novel non-canonical activities described in this work relate to both the timing of DNA cleavage and repair as well as the influence of the DNA target sequences in this process. An important consequence of genome instability created by RAG and AID is the formation of chromosomal translocations that lead to cancer development. However, the impact of translocation formation on the structure and function of affected chromosomes in the nucleus is not well understood. Here I used the translocations generated by unrepaired DNA breaks in progenitor B cells to examine the 3D relationships of translocated chromosomes in lymphoma nuclei and the effect of translocation on gene expression. My research demonstrated that despite gross changes in DNA sequence organization attributable to the translocation, there were minimal affects on the organization or expression of genes near the breakpoint. Taken together these studies reveal unappreciated non-canonical aspects of programmed DNA breakage and repair that occurs in B lymphocytes which influences both normal B cell development and cancer progression. In addition, these studies add a critical component to our understanding of the functional organization of the genome in the cell nucleus. Combined, these data can aid in both the identification of critical factors that may influence human lymphomagenesis and the development of potential therapeutics

A longitudinal analysis of the alignment between children’s early word-level reading trajectories, teachers’ reported concerns and supports provided

In this longitudinal study, the word-level reading trajectories of 118 childrenwere tracked alongside teachers’ reported concerns and types of support provided through Grades 1, 2 and 3. Results show a significant decline in composite scores relative to age norms over time, with children achieving significantly lower in phonemic decoding than word recognition at the subtest level. Five group trajectories were identified: children who achieved average or above average scores across all 3 years (n = 64), children who consistently bordered on average (n = 11), children who achieved below average in Grade 1 but who then achieved average or above in Grade 2 or Grade 3 (n = 7), children who achieved average or above in Grade 1 but then declined to below average in Grade 2 or Grade 3 (n = 10), and children who achieved below average across all 3 years (n = 26). Appropriately, teachers’ concerns were highest for students in the groups that improved, declined or remained persistently below average. However, analysis of the focus of teachers’ concerns and the supports they said were provided to the children in these three groups suggests that teachers are not always accurate in their interpretation of children’s presenting characteristics, resulting in the misalignment of support provision

Activation-Induced Cytidine Deaminase-Initiated Off-Target DNA Breaks Are Detected and Resolved during S Phase.

Activation-induced cytidine deaminase (AID) initiates DNA double-strand breaks (DSBs) in the IgH gene (Igh) to stimulate isotype class switch recombination (CSR), and widespread breaks in non-Igh (off-target) loci throughout the genome. Because the DSBs that initiate class switching occur during the G(1) phase of the cell cycle, and are repaired via end joining, CSR is considered a predominantly G(1) reaction. By contrast, AID-induced non-Igh DSBs are repaired by homologous recombination. Although little is known about the connection between the cell cycle and either induction or resolution of AID-mediated non-Igh DSBs, their repair by homologous recombination implicates post-G(1) phases. Coordination of DNA breakage and repair during the cell cycle is critical to promote normal class switching and prevent genomic instability. To understand how AID-mediated events are regulated through the cell cycle, we have investigated G(1)-to-S control in AID-dependent genome-wide DSBs. We find that AID-mediated off-target DSBs, like those induced in the Igh locus, are generated during G(1). These data suggest that AID-mediated DSBs can evade G(1)/S checkpoint activation and persist beyond G(1), becoming resolved during S phase. Interestingly, DSB resolution during S phase can promote not only non-Igh break repair, but also Ig CSR. Our results reveal novel cell cycle dynamics in response to AID-initiated DSBs, and suggest that the regulation of the repair of these DSBs through the cell cycle may ensure proper class switching while preventing AID-induced genomic instability

Activation-induced cytidine deaminase-initiated off-target DNA breaks are detected and resolved during S phase

Activation-induced cytidine deaminase (AID) initiates DNA double-strand breaks (DSBs) in the IgH gene (Igh) to stimulate isotype class switch recombination (CSR), and widespread breaks in non-Igh (off-target) loci throughout the genome. Because the DSBs that initiate class switching occur during the G(1) phase of the cell cycle, and are repaired via end joining, CSR is considered a predominantly G(1) reaction. By contrast, AID-induced non-Igh DSBs are repaired by homologous recombination. Although little is known about the connection between the cell cycle and either induction or resolution of AID-mediated non-Igh DSBs, their repair by homologous recombination implicates post-G(1) phases. Coordination of DNA breakage and repair during the cell cycle is critical to promote normal class switching and prevent genomic instability. To understand how AID-mediated events are regulated through the cell cycle, we have investigated G(1)-to-S control in AID-dependent genome-wide DSBs. We find that AID-mediated off-target DSBs, like those induced in the Igh locus, are generated during G(1). These data suggest that AID-mediated DSBs can evade G(1)/S checkpoint activation and persist beyond G(1), becoming resolved during S phase. Interestingly, DSB resolution during S phase can promote not only non-Igh break repair, but also Ig CSR. Our results reveal novel cell cycle dynamics in response to AID-initiated DSBs, and suggest that the regulation of the repair of these DSBs through the cell cycle may ensure proper class switching while preventing AID-induced genomic instability

Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins.

Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. In vivo delivery systems for CRISPR-Cas proteins and single guide RNA (sgRNA) include both viral vectors and non-viral strategies, each presenting different advantages and disadvantages for clinical application. We developed non-viral and biodegradable PEGylated nanocapsules (NCs) that deliver preassembled Cas9-sgRNA ribonucleoproteins (RNPs). Here, we show that the RNP NCs led to robust genome editing in neurons following intracerebral injection into the healthy mouse striatum. Genome editing was predominantly observed in medium spiny neurons (\u3e80%), with occasional editing in cholinergic, calretinin, and parvalbumin interneurons. Glial activation was minimal and was localized along the needle tract. Our results demonstrate that the RNP NCs are capable of safe and efficient neuronal genome editing in vivo

Protest on the Fly

This article reexamines spontaneity as an important, albeit neglected, mechanism in collective action dynamics, and elaborates on its operation and effects in protest events and social movements. We do not presume that spontaneity is routinely at play in all collective actions. Rather, based on our grounded analysis of historical and ethnographic data, we contend that spontaneity is triggered by certain conditions: nonhierarchical organization; uncertain/ambiguous moments and events; behavioral/emotional priming; and certain ecological/spatial factors. We conclude by elaborating why the activation of spontaneous actions matters in shaping the course and character of protest events and movements, and we suggest that spontaneity be resuscitated in the study of collective action and everyday life more generally