An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus.

BACKGROUND: A number of integrase defective lentiviral (IDLV) packaging systems have been developed to produce integration deficient lentiviruses for gene delivery and epichromosomal expression. However, despite their growing demand, a comparative study to systemically evaluate the performance efficiency of different mutants on virus packaging and gene expression has not been done. RESULTS: Site-directed mutagenesis was used to generate five integrasedeficient mutants for non-integrative lentiviral packaging (NILVP). The five mutants were then individually incorporated to make different integrase defective lentivirus plasmid packaging mix, keeping other packaging factors constant. CD511B-1, a lentivectorexpressing GFP from an EF1 promoter, was packaged with each of the five different lentivirus packaging mix to make pseudotypedviral particles. The performance and packaging efficiency of each of the integrase deficient mutants was evaluated based on GFP expression in HT1080 cells, while the wild type lentivirus packaging mix was used as a control. Of the five integrase mutant candidates, one with the highestGFP transgene expression level was chosen for further characterization. The non-integrative nature of this candidate was confirmed by quantitative polymerase chain reaction and characterized using both dividing and non-dividing cells. Finally, a detailed standard protocol for NILVP using this integrase defective mutant was developed. CONCLUSIONS: An efficient lentiviral packaging system for producing on-integrative lentivirus was established. This system is compatible with most existing lentivectors and can be used to transduce both dividing and non-dividing cells.

A TALEN-based strategy for efficient bi-allelic miRNA ablation in human cells.

Significant progress in the functional understanding of microRNAs (miRNAs) has been made in mice, but a need remains to develop efficient tools for bi-allelic knockouts of miRNA in the human genome. Transcription activator-like effector nucleases (TALENs) provide an exciting platform for targeted gene ablation in cultured human cells, but bi-allelic modifications induced by TALENs alone occur at low frequency, making screening for double knockouts a tedious task. Here, we present an approach that is highly efficient in bi-allelic miRNA ablation in the human genome by combining TALENs targeting to the miRNA seed region with a homologous recombination donor vector and a positive selection strategy. A pilot test of this approach demonstrates bi-allelic miR-21 gene disruption at high frequency (∼87%) in cultured HEK293 cells. Analysis of three independent clones showed a total loss of miR-21 expression. Phenotypical analysis revealed increased miR-21 target gene expression, reduced cell proliferation, and alterations of global miRNA expression profiles. Taken together, our study reveals a feasible and efficient approach for bi-allelic miRNA ablation in cultured human cells and demonstrates its usefulness in elucidating miRNA function in human cells.

Differential Treatment and Outcomes for Patients With Heart Attacks in Advantaged and Disadvantaged Communities.

Background Racially and ethnically minoritized groups, people with lower income, and rural communities have worse access to percutaneous coronary intervention (PCI) than their counterparts, but PCI hospitals have preferentially opened in wealthier areas. Our study analyzed disparities in PCI access, treatment, and outcomes for patients with acute myocardial infarction based on the census-derived Area Deprivation Index. Methods and Results We obtained patient-level data on 629 419 patients with acute myocardial infarction in California between January 1, 2006 and December 31, 2020. We linked patient data with population characteristics and geographic coordinates, and categorized communities into 5 groups based on the share of the population in low or high Area Deprivation Index neighborhoods to identify differences in PCI access, treatment, and outcomes based on community status. Risk-adjusted models showed that patients in the most advantaged communities had 20% and 15% greater likelihoods of receiving same-day PCI and PCI during the hospitalization, respectively, compared with patients in the most disadvantaged communities. Patients in the most advantaged communities also had 19% and 16% lower 30-day and 1-year mortality rates, respectively, compared with the most disadvantaged, and a 15% lower 30-day readmission rate. No statistically significant differences in admission to a PCI hospital were observed between communities. Conclusions Patients in disadvantaged communities had lower chances of receiving timely PCI and a greater risk of mortality and readmission compared with those in more advantaged communities. These findings suggest a need for targeted interventions to influence where cardiac services exist and who has access to them.

Patterns in Patient Encounters and Emergency Department Capacity in California, 2011-2021.

Importance: The health care system has undergone major changes in the past decade, and emergency department (ED) crowding has worsened over time; however, the most recent patterns in ED capacity and use in California have yet to be studied. Objective: To analyze patterns in ED capacity and utilization in California hospitals from 2011 to 2021. Design, Setting, and Participants: This retrospective cohort study used data from the California Department of Health Care Access and Information and the US Census Bureau to analyze ED facility characteristics from more than 400 general acute care hospitals with more than 320 EDs in California as well as patients who presented to those EDs between January 1, 2011, and December 31, 2021. Main Outcomes and Measures: Linear patterns (measured by percentage change) in total annual ED capacity (volume of hospital beds, EDs, ED treatment stations, and trauma centers) and ED use (ED visits by disposition and acuity) were assessed as primary outcomes. Patterns in ambulance diversion hours and the number of patients who left the ED without being seen were also examined as secondary outcomes. Visit acuity was categorized into 5 levels by increasing severity (minor, low to moderate, moderate, severe without threat, and severe with threat) based on California Department of Health Care Access and Information descriptions corresponding to Current Procedural Terminology codes. Results: In the prepandemic period (2011-2019), the total population of California increased from 37 638 369 to 39 512 223 (5.0%; 95% CI, 4.1%-5.8%), then decreased to 39 237 836 in 2021 (0.7%; 95% CI, -3.9% to 2.5%). Over the entire study period (2011-2021), the total California population increased by 4.2% (95% CI, 3.3%-5.2%). From 2011 to 2019, the annual number of ED visits increased from 12 054 885 to 14 876 653 (23.4%; 95% CI, 20.0%-26.8%) before decreasing to 12 944 692 in 2021 (-13.0%; 95% CI, -33.1% to 7.1%); from 2011 to 2021, total ED visits increased by 7.4% (95% CI, 5.6%-9.1%). From 2011 to 2021, the total number of EDs decreased from 339 to 326 (-3.8%; 95% CI, -4.4% to -3.2%) and the total number of hospital beds decreased from 75 940 to 74 052 (-2.5%; 95% CI, -3.3% to -1.6%), while the number of ED treatment stations in these fewer EDs increased from 7159 to 8667 (21.1%; 95% CI, 19.7%-22.4%). The number of visits rated as severe with threat also increased, from 2 011 637 in 2011 to 3 375 539 in 2021 (67.8%; 95% CI, 59.7%-75.9%), while visits rated as minor decreased from 913 712 to 336 071 (-63.2%; 95% CI, -75.2% to -51.2%) over the same period. Conclusions and Relevance: In this cohort study, multiple measures of ED capacity did not proportionally increase with the increasing demand for services; however, the COVID-19 pandemic appears to have substantially affected some of these patterns. These findings may be helpful to policy makers and health care stakeholders when planning resource allocation of limited health care resources.

Structural Inequities for Historically Underserved Communities in the Adoption of Stroke Certification in the United States.

Importance: Stroke centers are associated with better outcomes. There is substantial literature surrounding disparities in stroke outcomes for underserved populations. However, the existing literature has focused primarily on discrimination at the individual or institutional level, and studies of structural discrimination in stroke care are scant. Objective: To examine differences in hospitals' likelihood of adopting stroke care certification between historically underserved and general communities. Design, Setting, and Participants: This study combined a data set of hospital stroke certification from all general acute nonfederal hospitals in the continental US from January 1, 2009, to December 31, 2019, with national, hospital, and census data to define historically underserved communities by racial and ethnic composition, income distribution, and rurality. For all categories except rurality, communities were categorized by the composition and degree of segregation of each characteristic. Cox proportional hazard models were then estimated to compare the hazard of adopting stroke care certification between historically underserved and general communities, adjusting for population size and hospital bed capacity. Data were analyzed from June 2021 to April 2022. Main Outcomes and Measures: Hospitals' likelihood of adopting stroke care certification. Results: A total of 4984 hospitals were included. From 2009 to 2019, the total number of hospitals with stroke certification grew from 961 to 1763. Hospitals serving Black, racially segregated communities had the highest hazard of adopting stroke care certification (hazard ratio [HR], 1.67; 95% CI, 1.41-1.97) in models not accounting for population size, but their hazard was 26% lower than among those serving non-Black, racially segregated communities (HR, 0.74; 95% CI, 0.62-0.89) in models controlling for population and hospital size. Adoption hazard was lower in low-income communities compared with high-income communities, regardless of their level of economic segregation, and rural hospitals were much less likely to adopt any level of stroke care certification relative to urban hospitals (HR, 0.43; 95% CI, 0.35-0.51). Conclusions and Relevance: In this analysis of stroke certification adoption across acute care hospitals in the US from 2009 to 2019, hospitals in low-income and rural communities had a lower likelihood of receiving stroke certification than hospitals in general communities. Hospitals operating in Black, racially segregated communities had the highest likelihood of adopting stroke care, but because these communities had the largest population, patients in these communities had the lowest likelihood of access to stroke-certified hospitals when the model controlled for population size. These findings provide empirical evidence that the provision of acute neurological services is structurally inequitable across historically underserved communities.

Trends in Payer Type for Emergency Department Visits in California, 2011-2019.

This cohort study investigates trends in the association between emergency department use and insurance coverage in California between 2011 and 2019.

Is Inpatient Volume Or Emergency Department Crowding A Greater Driver Of Ambulance Diversion?

Inpatient volume has long been believed to be a contributing factor to ambulance diversion, which can lead to delayed treatment and poorer outcomes. We examined the extent to which both daily inpatient and emergency department (ED) volumes at specified hospitals, and diversion levels (that is, the number of hours ambulances were diverted on a given day) at their nearest neighboring hospitals, were associated with diversion levels in the period 2005-12. We found that a 10 percent increase in patient volume was associated with a sevenfold greater increase in diversion hours when the volume increase occurred among inpatients (5 percent) versus ED visitors (0.7 percent). When the next-closest ED experienced mild, moderate, or severe diversion, the study hospital's diversion hours increased by 8 percent, 23 percent, and 44 percent, respectively. These findings suggest that efforts focused on managing inpatient volume and flow might reduce diversion more effectively than interventions focused only on ED dynamics.

Erratum: This Article Corrects: "Trends in Regionalization of Care for ST-Segment Elevation Myocardial Infarction".

[This corrects the article on p. 1010 in vol. 18, PMID: 29085531.].

Impact Of Ambulance Diversion: Black Patients With Acute Myocardial Infarction Had Higher Mortality Than Whites.

This study investigated whether emergency department crowding affects blacks more than their white counterparts and the mechanisms behind which this might occur. Using a nonpublic database of patients in California with acute myocardial infarction between 2001 and 2011 and hospital-level data on ambulance diversion, we found that hospitals treating a high share of black patients with acute myocardial infarction were more likely to experience diversion and that black patients fared worse compared to white patients experiencing the same level of emergency department crowding as measured by ambulance diversion. The ninety-day and one-year mortality rates among blacks exposed to high diversion levels were 2.88 and 3.09 percentage points higher, respectively, relative to whites, representing a relative increase of 19 percent and 14 percent for ninety-day and one-year death, respectively. Interventions that decrease the need for diversion in hospitals serving a high volume of blacks could reduce these disparities.

Viral Cre-LoxP tools aid genome engineering in mammalian cells.

BACKGROUND: Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. RESULTS: We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. CONCLUSIONS: We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

Trends in Regionalization of Care for ST-Segment Elevation Myocardial Infarction.

INTRODUCTION: California has led successful regionalized efforts for several time-critical medical conditions, including ST-segment elevation myocardial infarction (STEMI), but no specific mandated protocols exist to define regionalization of care. We aimed to study the trends in regionalization of care for STEMI patients in the state of California and to examine the differences in patient demographic, hospital, and county trends. METHODS: Using survey responses collected from all California emergency medical services (EMS) agencies, we developed four categories - no, partial, substantial, and complete regionalization - to capture prehospital and inter-hospital components of regionalization in each EMS agency's jurisdiction between 2005-2014. We linked the survey responses to 2006 California non-public hospital discharge data to study the patient distribution at baseline. RESULTS: STEMI regionalization-of-care networks steadily developed across California. Only 14% of counties were regionalized in 2006, accounting for 42% of California's STEMI patient population, but over half of these counties, representing 86% of California's STEMI patient population, reached complete regionalization in 2014. We did not find any dramatic differences in underlying patient characteristics based on regionalization status; however, differences in hospital characteristics were relatively substantial. CONCLUSION: Potential barriers to achieving regionalization included competition, hospital ownership, population density, and financial challenges. Minimal differences in patient characteristics can establish that patient differences unlikely played any role in influencing earlier or later regionalization and can provide a framework for future analyses evaluating the impact of regionalization on patient outcomes.

Selectable and inheritable gene silencing through RNA interference in the unicellular alga Chlamydomonas reinhardtii.

Reverse genetic approaches have become invaluable tools to tap into the wealth of information provided by sequenced genomes. In 2007, sequencing of the Chlamydomonas reinhardtii genome was completed, and with this an increased demand for the development of reverse genetic strategies for gene analysis. In a variety of organisms, including Chlamydomonas, inverted repeat transgenes have been used to produce strains silenced for a specific gene due to the production of double stranded RNA (dsRNA). Here, we describe a tandem inverted repeat system designed to overcome some of the typical challenges that arise when transgenes are used to trigger gene silencing including the lack of a screenable phenotype, unpredictable levels of silencing, silencing of the transgene itself and thus loss of target gene silencing, and finally silencing of unintended genes (off-target genes). The described strategy allows selection of target gene silencing by inducing co-silencing of the target gene and a gene, MAA7, silencing of which produces a selectable RNAi-induced phenotype. This selection, therefore, precludes extensive molecular screening for transgenic strains exhibiting target gene silencing, and also ensures heritable silencing through many generations.

Functional specialization of Chlamydomonas reinhardtii cytosolic thioredoxin h1 in the response to alkylation-induced DNA damage.

DNA damage occurs as a by-product of intrinsic cellular processes, like DNA replication, or as a consequence of exposure to genotoxic agents. Organisms have evolved multiple mechanisms to avoid, tolerate, or repair DNA lesions. To gain insight into these processes, we have isolated mutants hypersensitive to DNA-damaging agents in the green alga Chlamydomonas reinhardtii. One mutant, Ble-1, showed decreased survival when it was treated with methyl methanesulfonate (MMS), bleomycin, or hydrogen peroxide (H2O2) but behaved like the wild type when it was exposed to UVC irradiation. Ble-1 carries an extensive chromosomal deletion that includes the gene encoding cytosolic thioredoxin h1 (Trxh1). Transformation of Ble-1 with a wild-type copy of Trxh1 fully corrected the MMS hypersensitivity and partly restored the tolerance to bleomycin. Trxh1 also complemented a defect in the repair of MMS-induced DNA strand breaks and alkali-labile sites. In addition, a Trxh1-beta-glucuronidase fusion protein translocated to the nucleus in response to treatment with MMS. However, somewhat surprisingly, Trxh1 failed to correct the Ble-1 hypersensitivity to H2O2. Moreover, Trxh1 suppression by RNA interference in a wild-type strain resulted in enhanced sensitivity to MMS and DNA repair defects but no increased cytotoxicity to H2O2. Thioredoxins have been implicated in oxidative-stress responses in many organisms. Yet our results indicate a specific role of Chlamydomonas Trxh1 in the repair of MMS-induced DNA damage, whereas it is dispensable for the response to H2O2. These observations also suggest functional specialization among cytosolic thioredoxins since another Chlamydomonas isoform (Trxh2) does not compensate for the lack of Trxh1.

Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas.

RNA interference (RNAi), the double-stranded RNA (dsRNA) triggered post-transcriptional gene silencing, is becoming a powerful tool for reverse genetics studies. Stable RNAi, induced by the expression of inverted repeat (IR) transgenes, has been achieved in protozoa, algae, fungi, plants, and metazoans. However, the level of gene silencing is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene. This is a hindrance in functional genomics studies, where it is desirable to suppress target genes reliably to analyze unknown phenotypes. Consequently, we explored strategies for direct selection of effective transgenic RNAi lines in Chlamydomonas reinhardtii. We initially attempted to suppress expression of the Rubisco small subunit multigene family by placing an IR, homologous to the conserved coding sequence, in the 3'UTR of a transgene conferring resistance to bleomycin. However, this approach was fairly inefficient at inducing RNAi as many strains displayed defective transgene integration, resulting in partial or complete deletion of the IR, or low levels of dsRNA expression, presumably due to transcriptional silencing of the integrated IR transgenes. To overcome these problems we designed a system consisting of tandem IR transgenes that consistently triggered co-silencing of a gene with a selectable RNAi-induced phenotype (encoding tryptophan synthase beta subunit) and another gene of interest (encoding either Ku80, an RNA-binding protein, or a thioredoxin isoform). We anticipate that this approach will be useful for generating stable hypomorphic epi-mutants in high-throughput phenotypic screens.