Heterologous survey of 130 DNA transposons in human cells highlights their functional divergence and expands the genome engineering toolbox.

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

More action needed: Psychotropic prescribing in Australian residential aged care.

OBJECTIVE: For at least two decades, concerns have been raised about inappropriate psychotropic prescribing in Australian residential aged care facilities, due to their modest therapeutic benefit and increased risk of falls and mortality. To date, the majority of prevalence data has been collected in Sydney exclusively and it is not known if recent initiatives to promote appropriate psychotropic prescribing have impacted utilisation. Thus, we aimed to comprehensively analyse psychotropic use in a large national sample of residential aged care facility residents. METHOD: A cross-sectional, retrospective cohort study of residents from 150 residential aged care facilities distributed nationally during April 2014-October 2015. Antipsychotic, anxiolytic/hypnotic and antidepressant utilisation was assessed, along with anticonvulsant and anti-dementia drug use. Negative binomial regression analysis was used to examine variation in psychotropic use. RESULTS: Full psychotropic prescribing data was available from 11,368 residents. Nearly two-thirds (61%) were taking psychotropic agents regularly, with over 41% prescribed antidepressants, 22% antipsychotics and 22% of residents taking benzodiazepines. Over 30% and 11% were charted for 'prn' (as required) benzodiazepines and antipsychotics, respectively. More than 16% of the residents were taking sedating antidepressants, predominantly mirtazapine. South Australian residents were more likely to be taking benzodiazepines ( p < 0.05) and residents from New South Wales/Australian Capital Territory less likely to be taking them ( p < 0.01), after adjustment for rurality and size of residential aged care facility. Residents located in New South Wales/Australian Capital Territory were also significantly less likely to take antidepressants ( p < 0.01), as were residents from outer regional residential aged care facilities ( p < 0.01). Antipsychotic use was not associated with State, rurality or residential aged care facility size. CONCLUSION: Regular antipsychotic use appears to have decreased in residential aged care facilities but benzodiazepine prevalence is higher, particularly in South Australian residential aged care facilities. Sedating antidepressant and 'prn' psychotropic prescribing is widespread. Effective interventions to reduce the continued reliance on psychotropic management, in conjunction with active promotion of non-pharmacological strategies, are urgently required.

Efficient mRNA delivery system utilizing chimeric VSVG-L7Ae virus-like particles.

The delivery of mRNA is advantageous over DNA delivery as it is transient and does not carry the risk of genomic DNA integration. However, there are currently few efficient mRNA delivery options available, especially for hard-to-transfect cell types, and thus new delivery methods are needed. To this end, we have established a novel mRNA delivery system utilizing chimeric virus-like particles (VLPs). We generated a novel VLP by fusing protein G of Vesicular stomatitis virus (VSV-G) with a ribosomal protein L7Ae of Archeoglobus fulgidus. This system allowed the efficient delivery of EGFP mRNA which was independent from the presence of BoxC/D motif in the mRNA sequence. Our VSVG-L7Ae VLP system demonstrated high transduction efficacy in hard-to-transfect cell lines, such as human induced pluripotent stem cells (iPS cells) and monocytes. In summary, this platform may serve as an efficient and transient transgene delivery tool for an mRNA of interest.

RedUSe: reducing antipsychotic and benzodiazepine prescribing in residential aged care facilities.

OBJECTIVE: To assess the impact of a multi-strategic, interdisciplinary intervention on antipsychotic and benzodiazepine prescribing in residential aged care facilities (RACFs). Design, setting: Prospective, longitudinal intervention in Australian RACFs, April 2014 - March 2016. PARTICIPANTS: 150 RACFs (with 12 157 residents) comprised the main participant group; two further groups were consultant pharmacists (staff education) and community pharmacies (prescribing data). Data for all RACF residents, excluding residents receiving respite or end-stage palliative care, were included. INTERVENTION: A multi-strategic program comprising psychotropic medication audit and feedback, staff education, and interdisciplinary case review at baseline and 3 months; final audit at 6 months. MAIN OUTCOME MEASURE: Mean prevalence of regular antipsychotic and benzodiazepine prescribing at baseline, and at 3 and 6 months. Secondary measures: chlorpromazine and diazepam equivalent doses/day/resident; proportions of residents for whom drug was ceased or the dose reduced; prevalence of antidepressant and prn (as required) psychotropic prescribing (to detect any substitution practice). RESULTS: During the 6-month intervention, the proportion of residents prescribed antipsychotics declined by 13% (from 21.6% [95% CI, 20.4-22.9%] to 18.9% [95% CI, 17.7-20.1%]), and that of residents regularly prescribed benzodiazepines by 21% (from 22.2% [95% CI, 21.0-23.5%] to 17.6% [95% CI, 16.5-18.7]; each, P < 0.001). Mean chlorpromazine equivalent dose declined from 22.9 mg/resident/day (95% CI, 19.8-26.0) to 20.2 mg/resident/day (95% CI, 17.5-22.9; P < 0.001); mean diazepam equivalent dose declined from 1.4 mg/resident/day (95% CI, 1.3-1.5) to 1.1 mg/resident/day (95% CI, 0.9-1.2; P < 0.001). For 39% of residents prescribed antipsychotics and benzodiazepines at baseline, these agents had been ceased or their doses reduced by 6 months. There was no substitution by sedating antidepressants or prn prescribing of other psychotropic agents. CONCLUSIONS: The RedUSe program achieved significant reductions in the proportions of RACF residents prescribed antipsychotics and benzodiazepines. TRIAL REGISTRATION: Australian New Zealand Clinical Trials, ACTRN12617001257358.

Patients' Experience of Living with Cancer-associated thrombosis in Spain (PELICANOS).

INTRODUCTION: The previously reported Patients' Experience of LIving with CANcer-associated thrombosis (PELICAN) identified several areas of unmet clinical and support need for cancer patients diagnosed with venous thromboembolism (VTE) in the UK. It is not known whether such experiences are restricted to one particular country's healthcare system and culture. We therefore undertook an evaluation of patients' experience of cancer-associated thrombosis (CAT) within a Spanish setting. METHODS: Twenty consecutive Spanish patients with CAT were interviewed about their experiences of living with CAT as per the previous PELICAN study. Where possible, spouses were interviewed in conjunction. Semi-structured interviews were audio recorded, transcribed and translated into English. Transcripts were coded using Nvivo software and data were analysed using framework analysis. A pragmatic approach was undertaken to allow explication of the potential cultural and operational differences that were not apparent in the UK dataset. RESULTS: Several commonalities between the UK and Spanish patients were identified including the traumatic nature of the experience, the need for information and adaptive behaviors through ritualisation. Within the major themes lay new themes as follows. (1) The traumatic experience of CAT impacted on the family dynamic with respect to discussions within the family unit and support needs of individuals other than the patient. It also had a profound impact on the patient's concept of self with increased awareness of their mortality and seriousness of the cancer. (2) The need for information extended to the family as well as the patients. This was needed at the point of CAT diagnosis as well as an opportunity to later address unanswered questions. (3) Adaptive behaviors were common with similar ritualisations seen in the UK patients. CONCLUSION: The distress experienced by patients with CAT is not isolated to the UK alone but is similar in Spanish patients as well. The patient information provided regarding LMWH injections is important, but there is a need to for patients and their families to be given additional information about CAT itself and future prognosis. CAT also has a profound impact on the patient's family who has similar support needs. It appears that there are several commonalities between UK and Spanish patients, as well as specific local issues. This study justifies expansion of the sampling to other countries.

Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.

Precise gene correction using the CRISPR-Cas9 system in human iPS cells holds great promise for various applications, such as the study of gene functions, disease modeling, and gene therapy. In this review article, we summarize methods for effective editing of genomic sequences of iPS cells based on our experiences correcting dystrophin gene mutations with the CRISPR-Cas9 system. Designing specific sgRNAs as well as having efficient transfection methods and proper detection assays to assess genomic cleavage activities are critical for successful genome editing in iPS cells. In addition, because iPS cells are fragile by nature when dissociated into single cells, a step-by-step confirmation during the cell recovery process is recommended to obtain an adequate number of genome-edited iPS cell clones. We hope that the techniques described here will be useful for researchers from diverse backgrounds who would like to perform genome editing in iPS cells.

An Internet-Based Method for Extracting Nursing Home Resident Sedative Medication Data From Pharmacy Packing Systems: Descriptive Evaluation.

BACKGROUND: Inappropriate use of sedating medication has been reported in nursing homes for several decades. The Reducing Use of Sedatives (RedUSe) project was designed to address this issue through a combination of audit, feedback, staff education, and medication review. The project significantly reduced sedative use in a controlled trial of 25 Tasmanian nursing homes. To expand the project to 150 nursing homes across Australia, an improved and scalable method of data collection was required. This paper describes and evaluates a method for remotely extracting, transforming, and validating electronic resident and medication data from community pharmacies supplying medications to nursing homes. OBJECTIVE: The aim of this study was to develop and evaluate an electronic method for extracting and enriching data on psychotropic medication use in nursing homes, on a national scale. METHODS: An application uploaded resident details and medication data from computerized medication packing systems in the pharmacies supplying participating nursing homes. The server converted medication codes used by the packing systems to Australian Medicines Terminology coding and subsequently to Anatomical Therapeutic Chemical (ATC) codes for grouping. Medications of interest, in this case antipsychotics and benzodiazepines, were automatically identified and quantified during the upload. This data was then validated on the Web by project staff and a "champion nurse" at the participating home. RESULTS: Of participating nursing homes, 94.6% (142/150) had resident and medication records uploaded. Facilitating an upload for one pharmacy took an average of 15 min. A total of 17,722 resident profiles were extracted, representing 95.6% (17,722/18,537) of the homes' residents. For these, 546,535 medication records were extracted, of which, 28,053 were identified as antipsychotics or benzodiazepines. Of these, 8.17% (2291/28,053) were modified during validation and verification stages, and 4.75% (1398/29,451) were added. The champion nurse required a mean of 33 min website interaction to verify data, compared with 60 min for manual data entry. CONCLUSIONS: The results show that the electronic data collection process is accurate: 95.25% (28,053/29,451) of sedative medications being taken by residents were identified and, of those, 91.83% (25,762/28,053) were correct without any manual intervention. The process worked effectively for nearly all homes. Although the pharmacy packing systems contain some invalid patient records, and data is sometimes incorrectly recorded, validation steps can overcome these problems and provide sufficiently accurate data for the purposes of reporting medication use in individual nursing homes.

Antiretroviral adherence and treatment outcomes among adult Ethiopian patients.

Developing appropriate strategies to sustain optimal medication adherence among the increasing number of HIV-positive patients taking antiretroviral therapy (ART) in sub-Saharan Africa is a major challenge. The objective of this study was to determine patient, regimen, disease, patient-provider, and healthcare-related factors associated with adherence with ART over a one-year period, and assess the impact of adherence on treatment outcomes. We performed a prospective, observational study among 246 patients who were initiated on ART in Ethiopia. Of 172 who completed follow-up, 130 (75.6%) had ≥95% adherence. In the multivariate analyses, a higher baseline BMI (OR, 1.2; 95% CI 1.0, 1.4) and use of reminder devices (OR, 9.1; 95% CI 2.0, 41.6) remained positively associated with adherence, while a higher HIV symptom and adverse drug reaction distress score was an independent negative predictor of adherence (OR, 0.90; 95% CI 0.9, 1.0) CD4 count increase was significantly higher in the adherent patients compared to non-adherent patients at 12 months (159 cells/µL [interquartile range (IQR), 72-324 cells/µL] vs. 132 cells/µL [IQR, 43-190 cells/µL]; p = 0.026). Our findings indicate that interventions aimed at improving adherence and thereby treatment outcomes in patients initiated on ART should promote the use of reminder devices, and monitor HIV symptoms and adverse reaction distress and nutritional status.

Host factor SAMHD1 restricts DNA viruses in non-dividing myeloid cells.

SAMHD1 is a newly identified anti-HIV host factor that has a dNTP triphosphohydrolase activity and depletes intracellular dNTP pools in non-dividing myeloid cells. Since DNA viruses utilize cellular dNTPs, we investigated whether SAMHD1 limits the replication of DNA viruses in non-dividing myeloid target cells. Indeed, two double stranded DNA viruses, vaccinia and herpes simplex virus type 1, are subject to SAMHD1 restriction in non-dividing target cells in a dNTP dependent manner. Using a thymidine kinase deficient strain of vaccinia virus, we demonstrate a greater restriction of viral replication in non-dividing cells expressing SAMHD1. Therefore, this study suggests that SAMHD1 is a potential innate anti-viral player that suppresses the replication of a wide range of DNA viruses, as well as retroviruses, which infect non-dividing myeloid cells.

Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases.

Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9), hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9.

SARS-CoV-2-induced disruption of a vascular bed in a microphysiological system caused by type-I interferon from bronchial organoids.

Blood vessels show various COVID-19-related conditions including thrombosis and cytokine propagation. Existing in vitro blood vessel models cannot represent the consequent changes in the vascular structure or determine the initial infection site, making it difficult to evaluate how epithelial and endothelial tissues are damaged. Here, we developed a microphysiological system (MPS) that co-culture the bronchial organoids and the vascular bed to analyze infection site and interactions. In this system, virus-infected organoids caused damage in vascular structure. However, vasculature was not damaged or infected when the virus was directly introduced to vascular bed. The knockout of interferon-related genes and inhibition of the JAK/STAT pathway reduced the vascular damage, indicating the protective effect of interferon response suppression. The results demonstrate selective infection of bronchial epithelial cells and vascular damage by cytokines and also indicate the applicability of MPS to investigate how the infection influences vascular structure and functions.

Review of the management of childhood asthma in Tasmania.

AIM: To review the supply of medications to children with asthma and parent-reported management of childhood asthma in Tasmania and highlight evidence-practice gaps for future interventions. METHODS: Participating pharmacies ran a software application that extracted data from dispensing records and helped to identify children with asthma. Parents of identified children were mailed a survey evaluating components of asthma management. Dispensing and survey data were analysed. RESULTS: A total of 939 children from 23 pharmacies were identified by the software and deemed eligible for inclusion. Surveys were received from 353 (37.6%) parents. In the past year, short-acting beta-2 agonists were supplied to 56.1% of the cohort, preventers to 76.5% (inhaled corticosteroids 52.3%; leukotriene receptor antagonists 31.3%; inhaled cromones 0.6%), long-acting beta-2 agonists (LABAs) to 25.7% and oral corticosteroids to 21.5%. Approximately half of the children receiving inhaled corticosteroids were concurrently receiving a LABA. Among children with indicators of inadequately controlled asthma, up to 73.7% of their parents reported that their asthma was adequately controlled, up to 38.2% did not possess an Asthma Action Plan, up to 36.8% were not regularly using a spacer and up to 22.8% had not received a preventer. CONCLUSION: These results indicate gaps in childhood asthma management, in particular, undersupply of preventers in high-risk patient groups, high supply of LABAs and insufficient spacer and asthma action plan usage. These areas should be targeted for interventions to improve childhood asthma management.

Preparation of NanoMEDIC Extracellular Vesicles to Deliver CRISPR-Cas9 Ribonucleoproteins for Genomic Exon Skipping.

The CRISPR-Cas9 system has quickly become the standard tool for genome editing. To deliver this system to target cells, adeno-associated virus (AAV) vectors are commonly used. In fact, AAV vectors have been utilized to deliver the CRISPR-Cas9 system to induce genomic exon skipping and restore the dystrophin protein in various Duchenne muscular dystrophy model animals. Despite the high transduction efficiency, AAV vector-mediated delivery has several limitations, such as the packaging size, prolonged overexpression of Cas9, immunogenicity against the AAV capsid, and the risk of integrating a part of the AAV genomic sequence into the host cell. To overcome these issues, we have recently engineered a transient delivery system utilizing VSV-G pseudotyped extracellular vesicles (EVs) termed NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo). NanoMEDIC utilizes an HIV-derived Gag protein to package Cas9 protein and gRNA into EVs. The Cas9 and Gag proteins are fused to a heterodimerizer and conditionally dimerized by the addition of an inducible chemical ligand to recruit Cas9 protein into EVs. sgRNA is packaged into EVs through an HIV-derived RNA packaging signal and is subsequently released by two self-cleaving ribozymes. Utilizing these features, NanoMEDIC can achieve highly efficient packaging of the Cas9 protein and gRNA for genome editing into a variety of target cells and in vivo. Here, we describe a step-by-step protocol, including the gRNA-expressing vector construction and large-scale NanoMEDIC production, for in vivo genome editing.

Ten-Year Trends in Psychotropic Prescribing and Polypharmacy in Australian General Practice Patients with and without Dementia.

OBJECTIVE: Little research has evaluated trends in psychotropic prescribing and polypharmacy in primary care patients, especially those with dementia. We sought to examine this in Australia from 2011 to 2020 using the primary care dataset, MedicineInsight. METHODS: Ten consecutive serial cross-sectional analyses were performed to evaluate the proportion of patients aged 65 years or more, with a recorded diagnosis of dementia, who were prescribed psychotropic medications within the first six months of each year from 2011 to 2020. This proportion was compared with propensity score-matched control patients without dementia. RESULTS: Before matching, 24,701 patients (59.2% females) with, and 72,105 patients (59.2% females) without, a recorded diagnosis of dementia were included. In 2011, 42% (95% confidence interval [CI] 40.5-43.5%) of patients in the dementia group had at least one recorded prescription of a psychotropic medication, which declined to 34.2% (95% CI 33.3-35.1%; p for trend < 0.001) by 2020. However, it remained unchanged for matched controls (36% [95% CI 34.6-37.5%] in 2011 and 36.7% [95% CI 35.7-37.6%] in 2020). The greatest decline in the dementia groups by medication class was for antipsychotics (from 15.9% [95% CI 14.8-17.0%] to 8.8% [95% CI 8.2-9.4%]; p for trend < 0.001). During this period, the prevalence of psychotropic polypharmacy (use of two or more individual psychotropics) also decreased from 21.7% (95% CI 20.5-22.9%) to 18.1% (95% CI 17.4-18.9%) in the dementia groups, and slightly increased from 15.2% (95% CI 14.1-16.3%) to 16.6% (95% CI 15.9-17.3%) in the matched controls. CONCLUSIONS: The decline in psychotropic prescribing, particularly antipsychotics, in Australian primary care patients with dementia is encouraging. However, psychotropic polypharmacy still occurred in almost one in five patients with dementia at the end of the study period. Programs focused on encouraging further reductions in the use of multiple psychotropic drugs in patients with dementia are recommended, particularly in rural and remote regions.

Elucidation of the liver pathophysiology of COVID-19 patients using liver-on-a-chips.

SARS-CoV-2 induces severe organ damage not only in the lung but also in the liver, heart, kidney, and intestine. It is known that COVID-19 severity correlates with liver dysfunction, but few studies have investigated the liver pathophysiology in COVID-19 patients. Here, we elucidated liver pathophysiology in COVID-19 patients using organs-on-a-chip technology and clinical analyses. First, we developed liver-on-a-chip (LoC) which recapitulating hepatic functions around the intrahepatic bile duct and blood vessel. We found that hepatic dysfunctions, but not hepatobiliary diseases, were strongly induced by SARS-CoV-2 infection. Next, we evaluated the therapeutic effects of COVID-19 drugs to inhibit viral replication and recover hepatic dysfunctions, and found that the combination of anti-viral and immunosuppressive drugs (Remdesivir and Baricitinib) is effective to treat hepatic dysfunctions caused by SARS-CoV-2 infection. Finally, we analyzed the sera obtained from COVID-19 patients, and revealed that COVID-19 patients, who were positive for serum viral RNA, are likely to become severe and develop hepatic dysfunctions, as compared with COVID-19 patients who were negative for serum viral RNA. We succeeded in modeling the liver pathophysiology of COVID-19 patients using LoC technology and clinical samples.

APOBEC1-mediated editing and attenuation of herpes simplex virus 1 DNA indicate that neurons have an antiviral role during herpes simplex encephalitis.

APOBEC1 (A1) is a cytidine deaminase involved in the regulation of lipids in the small intestine. Herpes simplex virus 1 (HSV-1) is a ubiquitous pathogen that is capable of infecting neurons in the brain, causing encephalitis. Here, we show that A1 is induced during encephalitis in neurons of rats infected with HSV-1. In cells stably expressing A1, HSV-1 infection resulted in significantly reduced virus replication compared to that in control cells. Infectivity could be restored to levels comparable to those observed for control cells if A1 expression was silenced by specific A1 short hairpin RNAs (shRNA). Moreover, cytidine deaminase activity appeared to be essential for this inhibition and led to an impaired accumulation of viral mRNA transcripts and DNA copy numbers. The sequencing of viral gene UL54 DNA, extracted from infected A1-expressing cells, revealed G-to-A and C-to-T transitions, indicating that A1 associates with HSV-1 DNA. Taken together, our results demonstrate a model in which A1 induction during encephalitis in neurons may aid in thwarting HSV-1 infection.

Identification of amino acids in the human tetherin transmembrane domain responsible for HIV-1 Vpu interaction and susceptibility.

Tetherin, also known as BST-2/CD317/HM1.24, is an antiviral cellular protein that inhibits the release of HIV-1 particles from infected cells. HIV-1 viral protein U (Vpu) is a specific antagonist of human tetherin that might contribute to the high virulence of HIV-1. In this study, we show that three amino acid residues (I34, L37, and L41) in the transmembrane (TM) domain of human tetherin are critical for the interaction with Vpu by using a live cell-based assay. We also found that the conservation of an additional amino acid at position 45 and two residues downstream of position 22, which are absent from monkey tetherins, are required for the antagonism by Vpu. Moreover, computer-assisted structural modeling and mutagenesis studies suggest that an alignment of these four amino acid residues (I34, L37, L41, and T45) on the same helical face in the TM domain is crucial for the Vpu-mediated antagonism of human tetherin. These results contribute to the molecular understanding of human tetherin-specific antagonism by HIV-1 Vpu.

A 12-month follow-up study of "RedUSe": a trial aimed at reducing antipsychotic and benzodiazepine use in nursing homes.

BACKGROUND: To assess the long-term impact of the "Reducing Use of Sedatives" (RedUSe) trial on antipsychotic and benzodiazepine prevalence and dosage. METHODS: RedUSe was a six-month controlled trial conducted in 25 Tasmanian nursing homes in 2008-9 which led to significant reductions in benzodiazepine and antipsychotic use and a doubling of dose reductions of these agents. In a follow-up study, data on psychotropic use was collected from all nursing homes a year after the final RedUSe measure. Mean daily doses for each home were calculated by converting antipsychotic and benzodiazepine doses to chlorpromazine and diazepam equivalents, respectively. To determine the long-term impact of the project, 6-month and initial baseline data were compared to the 18-month follow-up data. RESULTS: 1578 residents were audited for the follow-up measure. In the 18 months since the RedUSe project was instigated, benzodiazepine prevalence fell by 25% in intervention nursing homes. Similarly, the mean daily diazepam equivalence in these homes had fallen by 24%. In contrast, after a significant reduction during the RedUSe trial, antipsychotic prevalence returned to baseline levels in intervention nursing homes, with mean chlorpromazine equivalence remaining relatively constant with time. There was a delayed reduction in benzodiazepine and antipsychotic use in the control homes. CONCLUSIONS: Both benzodiazepine usage and mean daily diazepam equivalence continued to decline in intervention nursing homes in the year following the RedUSe trial. However, the effect of the RedUSe intervention on antipsychotic prevalence and dosage was not sustained.

In vitro and in planta compatibility of insecticides and the endophytic entomopathogen, Lecanicillium lecanii.

In an attempt to clarify the potential role of endophytic fungi in integrated pest management, the compatibility of an endophytic isolate of Lecanicillium lecanii (Zimmermann) Gams & Zare (Hyphomycetes) with nine insecticides used against Aphis gossypii Glover (Homoptera : Aphididae) was examined both in vitro over 14 days and in planta. In the laboratory, most insecticides partially or completely inhibited the germination of conidia and growth of hyphae in nutrient-rich conditions. Endosulfan completely inhibited the germination of conidia and hyphal growth. In contrast, all insecticides were compatible with L. lecanii in planta, and the fungus was readily recovered from inoculated, colonized leaves. These data support the hypothesis that endophytic L. lecanii will be unaffected by insecticides and could be integrated in the management of pests in cotton.

Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells.

Selection-free, scarless genome editing in human pluripotent stem cells (PSCs) by utilizing ribonucleoprotein (RNP) of CRISPR-Cas9 is a useful tool for a variety of applications. However, the process can be hampered by time-consuming subcloning steps and inefficient delivery of the RNP complex and ssDNA template. Here, we describe the optimized protocol to introduce a single nucleotide change or a loxP site insertion in feeder-free, xeno-free iPSCs by utilizing MaxCyte and 4D-Nucleofector electroporators. For complete details on the use and execution of this protocol, please refer to Kagita et al. (2021) and Xu et al. (2019).