CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation.

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

Prevented harm and cost avoidance with pharmacist intervention while utilizing a discharge medication reconciliation tool.

PURPOSE: The aim of this study was to determine prevented harm and cost avoidance following pharmacist intervention utilizing a discharge medication reconciliation tool. METHODS: A retrospective chart review was conducted to identify patients with pharmacist-initiated, provider-accepted discharge medication reconciliation interventions completed at a community teaching hospital in January 2021. Investigators assigned the discrepancies targeted for intervention a National Coordinating Council for Medication Error Reporting and Prevention (NCC MERP) category, probability of harm, and calculated cost avoidance. The primary endpoint was the total cost avoidance associated with discharge medication reconciliation. RESULTS: Pharmacists intervened 190 times in January 2021, avoiding an estimated $46,958 to $231,032 in cost. High-risk medications were associated with $33,920 to $147,203 in cost avoidance. The 3 high-risk therapeutic classes associated with the highest cost avoidance were insulin ($16,738-$70,793), antithrombotics ($13,884-$60,016), and opioids ($2,638-$11,834). CONCLUSION: Targeted pharmacist discharge medication reconciliation and related interventions avoid significant cost and patient harm.

Long COVID in long-term care: a rapid realist review.

OBJECTIVES: The goals of this rapid realist review were to ask: (a) what are the key mechanisms that drive successful interventions for long COVID in long-term care (LTC) and (b) what are the critical contexts that determine whether the mechanisms produce the intended outcomes? DESIGN: Rapid realist review. DATA SOURCES: Medline, CINAHL, Embase, PsycINFO and Web of Science for peer-reviewed literature and Google for grey literature were searched up to 23 February 2023. ELIGIBILITY CRITERIA: We included sources focused on interventions, persons in LTC, long COVID or post-acute phase at least 4 weeks following initial COVID-19 infection and ones that had a connection with source materials. DATA EXTRACTION AND SYNTHESIS: Three independent reviewers searched, screened and coded studies. Two independent moderators resolved conflicts. A data extraction tool organised relevant data into context-mechanism-outcome configurations using realist methodology. Twenty-one sources provided 51 intervention data excerpts used to develop our programme theory. Synthesised findings were presented to a reference group and expert panel for confirmatory purposes. RESULTS: Fifteen peer-reviewed articles and six grey literature sources were eligible for inclusion. Eleven context-mechanism-outcome configurations identify those contextual factors and underlying mechanisms associated with desired outcomes, such as clinical care processes and policies that ensure timely access to requisite resources for quality care delivery, and resident-centred assessments and care planning to address resident preferences and needs. The underlying mechanisms associated with enhanced outcomes for LTC long COVID survivors were: awareness, accountability, vigilance and empathetic listening. CONCLUSIONS: Although the LTC sector struggles with organisational capacity issues, they should be aware that comprehensively assessing and monitoring COVID-19 survivors and providing timely interventions to those with long COVID is imperative. This is due to the greater care needs of residents with long COVID, and coordinated efficient care is required to optimise their quality of life.

The Safety of Direct Oral Anticoagulants Compared to Warfarin in Patients Hospitalized With Acute Kidney Injury.

BACKGROUND: Direct oral anticoagulants (DOACs) are preferred over warfarin for many indications, though their safety has not been well established in patients with acute renal impairment. OBJECTIVE: The purpose of this study was to evaluate the frequency of bleeding complications associated with DOACs compared with warfarin in patients admitted to the hospital with acute kidney injury (AKI). METHODS: This was a retrospective cohort study evaluating patients admitted to the Penn Medicine Lancaster General Hospital with a diagnosis of AKI from October 2017 through September 2021 and receiving therapy with oral anticoagulants. Comparing DOACs with warfarin, the primary endpoint was the percent frequency of composite major and minor bleeding during the admission and within 30 days of discharge. RESULTS: There were 112 hospitalization encounters included in the study. Of these, 42 (37.5%) patients were receiving warfarin and 70 (62.5%) patients were receiving DOAC therapy before admission. There was a higher frequency of the primary endpoint of bleeding in patients receiving DOACs as compared with warfarin, though this was not statistically significant (18.5% vs. 11.9%, respectively, P = 0.432). There were no differences between groups in the frequency of major bleeding, minor bleeding, or transfusions. Patients receiving DOAC therapy were more likely to experience anticoagulation-related readmissions or emergency department visits compared with patients on warfarin therapy (11.4% vs. 0%, P = 0.024). CONCLUSION AND RELEVANCE: Direct oral anticoagulants and warfarin were associated with statistically similar rates of bleeding in patients presenting with AKI. Further research is necessary to elucidate if DOACs are safer than warfarin in this patient population.

Visual cortex is rescued from the effects of dark rearing by overexpression of BDNF.

Visual deprivation such as dark rearing (DR) prolongs the critical period for ocular dominance plasticity and retards the maturation of gamma-aminobutyric acid (GABA)ergic inhibition in visual cortex. The molecular signals that mediate the effects of DR on the development of visual cortex are not well defined. To test the role of brain-derived neurotrophic factor (BDNF), we examined the effects of DR in transgenic mice in which BDNF expression in visual cortex was uncoupled from visual experience and remained elevated during DR. In dark-reared transgenic mice, visual acuity, receptive field size of visual cortical neurons, critical period for ocular dominance plasticity, and intracortical inhibition were indistinguishable from those observed in light-reared mice. Therefore, BDNF overexpression is sufficient for the development of aspects of visual cortex in the absence of visual experience. These results suggest that reduced BDNF expression contributes to retarded maturation of GABAergic inhibition and delayed development of visual cortex during visual deprivation.

The serological response of patients infected with the agent of human granulocytic ehrlichiosis.

To characterize the serological response in humans to human granulocytic ehrlichiosis (HGE), we prospectively observed 152 patients for as long as 42 months. HGE was confirmed by detection of morulae in blood smears, polymerase chain reaction, blood culture, or a combination of these tests for 94 patients (62.3%), and 92 (97.8%) of the patients had specific serum antibodies thereafter. One hundred twenty-six (99.2%) of 127 patients tested at 1 month were seropositive (89 of 127 patients had seroconversion), and 150 (98.7%) of the 152 patients had become seropositive by 6 months. Eleven patients (7.3%) remained seropositive at 42 months. Neither antibiotic therapy initiated during the first week of illness nor preexisting immunosuppressive conditions abrogated a serological response. Indirect fluorescent antibody testing of acute-phase and convalescent-phase serum samples is a sensitive tool for laboratory confirmation of HGE.