Deep scRNA sequencing reveals a broadly applicable Regeneration Classifier and implicates antioxidant response in corticospinal axon regeneration.

Despite substantial progress in understanding the biology of axon regeneration in the CNS, our ability to promote regeneration of the clinically important corticospinal tract (CST) after spinal cord injury remains limited. To understand regenerative heterogeneity, we conducted patch-based single-cell RNA sequencing on rare regenerating CST neurons at high depth following PTEN and SOCS3 deletion. Supervised classification with Garnett gave rise to a Regeneration Classifier, which can be broadly applied to predict the regenerative potential of diverse neuronal types across developmental stages or after injury. Network analyses highlighted the importance of antioxidant response and mitochondrial biogenesis. Conditional gene deletion validated a role for NFE2L2 (or NRF2), a master regulator of antioxidant response, in CST regeneration. Our data demonstrate a universal transcriptomic signature underlying the regenerative potential of vastly different neuronal populations and illustrate that deep sequencing of only hundreds of phenotypically identified neurons has the power to advance regenerative biology.

Probing regenerative heterogeneity of corticospinal neurons with scRNA-Seq.

The corticospinal tract (CST) is clinically important for the recovery of motor functions after spinal cord injury. Despite substantial progress in understanding the biology of axon regeneration in the central nervous system (CNS), our ability to promote CST regeneration remains limited. Even with molecular interventions, only a small proportion of CST axons regenerate1. Here we investigate this heterogeneity in the regenerative ability of corticospinal neurons following PTEN and SOCS3 deletion with patch-based single cell RNA sequencing (scRNA-Seq)2,3, which enables deep sequencing of rare regenerating neurons. Bioinformatic analyses highlighted the importance of antioxidant response and mitochondrial biogenesis along with protein translation. Conditional gene deletion validated a role for NFE2L2 (or NRF2), a master regulator of antioxidant response, in CST regeneration. Applying Garnett4, a supervised classification method, to our dataset gave rise to a Regenerating Classifier (RC), which, when applied to published scRNA-Seq data, generates cell type- and developmental stage-appropriate classifications. While embryonic brain, adult dorsal root ganglion and serotonergic neurons are classified as Regenerators, most neurons from adult brain and spinal cord are classified as Non-regenerators. Adult CNS neurons partially revert to a regenerative state soon after injury, which is accelerated by molecular interventions. Our data indicate the existence of universal transcriptomic signatures underlying the regenerative abilities of vastly different neuronal populations, and further illustrate that deep sequencing of only hundreds of phenotypically identified CST neurons has the power to reveal new insights into their regenerative biology.

Benzodiazepine analysis by an improved LC-MS/MS method illustrates usage patterns in Washington State.

BACKGROUND: The standard approach for benzodiazepine detection often includes immunoassay followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The illicit use of non-prescribed benzodiazepines has been trending up nationally. METHODS: We developed and validated an improved LC-MS/MS assay for benzodiazepine detection in urine. We expanded the testing panel by adding five drugs to the previous panel of ten. We determined the prevalence of individual benzodiazepines in our patient population. Immunoassay results were compared with LC-MS/MS to evaluate assay performance. RESULTS: Clonazepam and alprazolam were the most common benzodiazepines present. Etizolam and flualprazolam were also prevalent in Washington State. Compared with the LC-MS/MS assay, the immunoassay had variable cross-reactivity, which explained false negative and false positive immunoassay results. The inclusion of new drugs in the LC-MS/MS panel significantly reduced the incidence of immunoassay results interpreted as falsely positive. CONCLUSION: New illicit benzodiazepines have emerged regionally and nationally. The inclusion of novel drugs in LC-MS/MS assay was helpful in properly characterizing the epidemiology of benzodiazepine use in our patient population. This information will lead to better assay result interpretations and patient care, and our experiences provide a roadmap for other clinical laboratories looking to expand their testing menu or transition to new instrumentation.

High-throughput method to analyze the cytotoxicity of CAR-T Cells in a 3D tumor spheroid model using image cytometry.

Solid tumors account for approximately 90% of all adult human cancers. As such, the development of novel cellular therapies has become of increasing importance to target solid tumor malignancies, such as prostate, lung, breast, bladder, colon, and liver cancers. One such cellular therapy relies on the use of chimeric antigen receptor T cells (CAR-T cells). CAR-T cells are engineered to target specific antigens on tumor cells. To date, there are six FDA-approved CAR-T cell therapies that have been utilized for hematologic B cell malignancies. Immune cell trafficking and immunosuppressive factors within the tumor microenvironment increase the relative difficulty in developing a robust CAR-T cell therapy against solid tumors. Therefore, it is critical to develop novel methodologies for high-throughput phenotypic and functional assays using 3D tumor spheroid models to assess CAR-T cell products against solid tumors. In this manuscript, we discuss the use of CAR-T cells targeted towards PSMA, an antigen that is found on prostate cancer tumor cells, the second most common cause of cancer deaths among men worldwide. We demonstrate the use of high-throughput, plate-based image cytometry to characterize CAR-T cell-mediated cytotoxic potency against 3D prostate tumor spheroids. We were able to kinetically evaluate the efficacy and therapeutic value of PSMA CAR-T cells by analyzing the cytotoxicity against prostate tumor spheroids. In addition, the CAR-T cells were fluorescently labeled to visually identify the location of the T cells as cytotoxicity occurs, which may provide more meaningful information for assessing the functionality of the CAR-T cells. The proposed image cytometry method can overcome limitations placed on traditional methodologies to effectively assess cell-mediated 3D tumor spheroid cytotoxicity and efficiently generate time- and dose-dependent results.

From education to empowerment: Redesigning the role of students in college health promotion.

College health promotion departments frequently employ peer health educators to disseminate relevant education and conduct outreach to their student body. While there are certainly benefits to these programs, this approach is outdated and does little to empower students or engage them in the process of health promotion. In this viewpoint article, I describe the Community Health Organizer model, which expands students' role in peer engagement and advocacy beyond the traditional peer educator or peer counseling programs. Finally, I provide recommendations for college health practitioners interested in implementing a similar model on their campuses.

CDC-Funded HIV Testing and Linkage to HIV Medical Care Among American Indian and Alaska Native People in the United States, 2014-2020.

OBJECTIVE: An estimated 1 in 5 American Indian and Alaska Native (AI/AN) adults living with HIV are unaware of their status. We investigated HIV testing among AI/AN people receiving a Centers for Disease Control and Prevention (CDC)-funded test from 2014 through 2020. METHODS: We analyzed data on CDC-funded HIV tests reported by health departments and community-based organizations in the United States. We described the number of CDC-funded HIV tests, the percentage of people with newly and previously diagnosed HIV, and linkage to HIV medical care within 90 days of diagnosis. RESULTS: CDC-funded health departments and community-based organizations provided 99 227 HIV tests to AI/AN people during 2014-2020. Seven hundred thirty-five (0.7%) AI/AN people were diagnosed with HIV; 361 (0.4%) were newly diagnosed, 319 (0.3%) had a previous HIV diagnosis, and 55 (0.1%) had a previously unknown HIV status. Positivity for new diagnoses was highest among the following population groups tested in non-health care settings: men who had sex with men (MSM; n = 72, 1.2%), MSM who inject drugs (n = 12, 1.8%), and transgender people (n = 12, 1.5%). The percentage of linkage to HIV medical care was 80.6% for newly diagnosed people and 78.2% for previously diagnosed people. CONCLUSIONS: MSM AI/AN, including those who inject drugs, and transgender AI/AN may benefit from prioritized HIV testing. All AI/AN people with HIV, whether newly or previously diagnosed, should rapidly link to HIV medical care and receive support throughout the continuum of care. Our findings can inform which AI/AN population subgroups may benefit from enhanced HIV testing efforts and interventions.

Low SARS-CoV-2 Seroprevalence and No Active Infections among Dogs and Cats in Animal Shelters with Laboratory-Confirmed COVID-19 Human Cases among Employees.

Human-to-animal and animal-to-animal transmission of SARS-CoV-2 has been documented; however, investigations into SARS-CoV-2 transmission in congregate animal settings are lacking. We investigated four animal shelters in the United States that had identified animals with exposure to shelter employees with laboratory-confirmed COVID-19. Of the 96 cats and dogs with specimens collected, only one dog had detectable SARS-CoV-2 neutralizing antibodies; no animal specimens had detectable viral RNA. These data indicate a low probability of human-to-animal transmission events in cats and dogs in shelter settings with early implementation of infection prevention interventions.

A qualitative examination of the impacts of financial stress on college students' well-being: Insights from a large, private institution.

OBJECTIVE: This qualitative research aims to provide deeper insight into college students' experiences by examining the impact of financial stress on their well-being. METHODS: Four focus groups were conducted at a large, private, urban university in the United States over the course of 1 month, each lasting approximately 1 h. Facilitators used a structured moderator guide to maintain consistency. Four focus groups were conducted and a total of 30 students participated. Students were primarily Asian (66.7%) and White (30.0%), and a majority were female (86.7%). Student participants were 43.3% undergraduate and 56.6% graduate. Transcripts were analyzed in Atlas.ti 8 software using line-by-line open coding guided by the principles of qualitative content analysis. An inductive approach was utilized to code the data. Emergent categories and concepts were then organized hierarchically into themes and subthemes. RESULTS: Two overarching themes emerged from the focus group analysis. In these students' perspectives, financial stress impedes their ability to succeed academically. Another major theme is the impact of finances on students' social lives. Students experiencing financial stress find it challenging to navigate relationships with wealthier peers, often leading to feelings of isolation and embarrassment. CONCLUSION: Given the reported negative impact on students' well-being, further research is needed to determine methods for mitigating financial stress.

CDC's Community-Based Organization Behavioral Outcomes Project: Perspectives for Researchers, Implementers and Funders.

Behavioral interventions have been a crucial tool for the prevention of HIV transmission since early in the epidemic. The Centers for Disease Control and Prevention (CDC) has provided funding for evidence-based behavioral interventions (EBIs) at health departments and community-based organizations (CBOs) since 2004. From 2006 to 2015, CDC funded 25 CBOs to evaluate one or more of seven EBIs designed to prevent HIV through the Community-based Organization Behavioral Outcomes Project (CBOP) as implemented outside of a research setting. For each EBI, CBOP showed that most HIV risk behaviors improved after the intervention, and improvements were similar to those observed in research studies. Our findings show that behavioral interventions can be successfully implemented in real-world settings. Although the focus of HIV prevention has largely shifted toward biomedical interventions in recent years, successful implementation often depends on behavioral components. Lessons from CBOP can inform future efforts to develop and implement behavioral interventions for HIV and other areas of public health.

Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency.

One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A.

Identification of Key Coagulation Activity Determining Elements in Canine Factor VIII.

It is well known that canine factor VIII (cFVIII) has a higher specific activity than does human FVIII (hFVIII), and it has been previously demonstrated that cFVIII light chain is able to enhance hFVIII activity. The goal of this study was to first determine which amino acids in cFVIII light chain were responsible for enhancing hFVIII activity, and second to use these amino acids to develop a hFVIII variant with enhanced functional activity. We systemically screened segments of cFVIII light chain by testing an array of human-canine light chain hybrids and found that canine amino acids 1857-2147 were key to this enhancement. Each canine amino acid within this span was screened individually using a negative selection method, which led to the identification of 12 aa (JF12) in the FVIII light chain that could enhance activity. Substitution of the corresponding 12 aa into hFVIII (hFVIIIJF12BDD) elevated the specific activity profile in vitro. Furthermore, hFVIIIJF12BDD expressed an in vivo-displayed increased coagulation activity compared to wild-type, while maintaining normal secretion efficiency. In conclusion, we identified the amino acids in cFVIII that are the key determinants for higher specific activity and may be the basis for future development of therapeutic treatments for hemophilia A.

Parental characteristics and perspectives pertaining to neonatal visits to the emergency department: a multicentre survey.

BACKGROUND: Parents take neonates to the emergency department for many reasons, often nonurgent, pressuring an already burdened system. We aimed to characterize these visits and families to identify potential strategies to decrease neonatal emergency department visits. METHODS: We developed and implemented a survey that explored characteristics of neonates and parents/guardians evaluated in the emergency department, perspectives of parents and use of health care services. Parents presenting with a neonate to the emergency department in 5 large academic hospitals in Ontario were surveyed between December 2013 and June 2015. We used descriptive statistics to report survey data and explored correlations between factors. RESULTS: A total of 1533 surveys were completed. The most common reasons for presenting were jaundice (441 [28.8%]) and feeding issues (251 [16.4%]). The majority of respondents (73.9% [1104/1494]) had received advice before going to the emergency department. In most cases (86.4% [954/1104]), this was from a health care provider, who frequently advised going to the emergency department. Although most parents (86.8% [1280/1475]) reported high confidence in caring for a sick or injured child, 42.3% (643/1519) were unsure of the severity, and most (90.4% [578/639]) of these parents felt that the infant required assessment immediately or the same day. Of parents who felt the condition was not serious, 83.2% (198/238) thought that same-day evaluation was required. Nearly half of respondents (44.4% [621/1400]) said they would have gone to their health care provider with a same-day appointment, and 28.1% (344/1225) would have gone to their care provider with a next-day appointment. INTERPRETATION: Parents' reported confidence in caring for sick or injured infants does not match the perceived urgency of neonatal conditions, which likely contributes to emergency department overuse. Any system to decrease nonurgent emergency department use by neonates would need to be immediately responsive, providing same-day help.

HIV Testing, Linkage to HIV Medical Care, and Interviews for Partner Services Among Women - 61 Health Department Jurisdictions, United States, Puerto Rico, and the U.S. Virgin Islands, 2015.

Diagnoses of human immunodeficiency virus (HIV) infection among women declined 17% during 2011-2015, and a total of 7,498 women received a diagnosis of HIV infection in 2015 (1). Although black or African American (black) women accounted for only 12% of the U.S. female population, 60% of women with newly diagnosed HIV infection were black (1,2). By the end of 2014, an estimated 255,900 women were living with HIV infection (3), including approximately 12% who did not know they were infected; in addition, approximately 45% of women who had received a diagnosis had not achieved viral suppression (3). HIV testing is an important public health strategy for identifying women with HIV infection and linking them to HIV medical care. Analysis of CDC-funded program data submitted by 61 health departments in 2015 indicated that among 4,749 women tested who received a diagnosis of HIV infection, 2,951 (62%) had received a diagnosis in the past (previous diagnosis), and 1,798 (38%) were receiving a diagnosis for the first time (new diagnosis). Of those who had received a previous diagnosis, 87% were not in HIV medical care at the time of the current test. Testing and identifying women who are living with HIV infection but who are not in care (regardless of when they received their first diagnosis) and rapidly linking them to care so they can receive antiretroviral therapy and become virally suppressed are essential for reducing HIV infection among all women.

Vaccinia virus as a subhelper for AAV replication and packaging.

Adeno-associated virus (AAV) has been widely used as a gene therapy vector to treat a variety of disorders. While these vectors are increasingly popular and successful in the clinic, there is still much to learn about the viruses. Understanding the biology of these viruses is essential in engineering better vectors and generating vectors more efficiently for large-scale use. AAV requires a helper for production and replication making this aspect of the viral life cycle crucial. Vaccinia virus (VV) has been widely cited as a helper virus for AAV. However, to date, there are no detailed analyses of its helper function. Here, the helper role of VV was studied in detail. In contrast to common belief, we demonstrated that VV was not a sufficient helper virus for AAV replication. Vaccinia failed to produce rAAV and activate AAV promoters. While this virus could not support rAAV production, Vaccinia could initiate AAV replication and packaging when AAV promoter activation is not necessary. This activity is due to the ability of Vaccinia-driven Rep78 to transcribe in the cytoplasm and subsequently translate in the nucleus and undergo typical functions in the AAV life cycle. As such, VV is subhelper for AAV compared to complete helper functions of adenovirus.

Efficient production of dual recombinant adeno-associated viral vectors for factor VIII delivery.

Recombinant adeno-associated viral (rAAV) vectors have gained attention for human gene therapy because of their high safety and clinical efficacy profile. For factor VIII gene delivery, splitting the coding region between two AAV vectors remains a viable strategy to avoid the packaging capacity limitation (∼5.0 kb). However, it is time-consuming and labor-intensive to produce two rAAV vectors in separate batches. Here we demonstrated successful production of dual rAAV vectors for hemophilia A gene therapy in a single preparation. When the AAV vector plasmids carrying the human factor VIII heavy chain (hHC) and the light chain (hLC) expression cassettes were cotransfected into 293 cells along with the AAV rep&cap and mini-adenovirus helper plasmids, both rAAV-hHC and rAAV-hLC were produced at the desired ratio and in high titer. Interestingly, the rAAV-hHC vectors always yielded higher titers than rAAV-hLC vectors as a result of more efficient replication of rAAV-hHC genomes. The resulting vectors were effective in transducing the tissue culture cells in vitro. When these vectors were administered to hemophilia A mice, factor VIII was detected in the mouse plasma by both the activated partial thromboplastin time assay and enzyme-linked immunosorbent assay. The functional activity as well as the antigen levels of secreted factor VIII were similar to those of vectors produced by the traditional method. The dual-vector production method has been successfully extended to both AAV2 and AAV8 serotypes. In conclusion, cotransfection of vector plasmids presents an efficient method for producing dual or multiple AAV vectors at significantly reduced cost and labor.

An evaluation of the RapidHIT(®) system for reliably genotyping reference samples.

Short tandem repeat (STR) typing is used routinely for associating or excluding individuals with biological evidence left at a crime scene. Improvements have been made to reduce the turnaround time and labor involved with profile generation, but there is still some lag time between sample collection and interpretation of results. The RapidHIT(®) (IntegenX; Pleasanton, CA, USA) system is an automated instrument that is configured to perform DNA extraction, bead-based DNA normalization, amplification, electrophoresis of PCR amplicons, and data analysis of five reference swabs simultaneously. The RapidHIT system provided reliable STR profiles from reference buccal swabs in approximately 90min with nominal "hands-on" sample loading time with no evidence of contamination between samples. The overall success rate of typing buccal swabs was comparable to standard typing systems. In the event of a failed run due to instrument failure, the swab can be removed from the cartridge and reanalyzed in the RapidHIT system or with standard STR genotyping workflows.

A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy.

Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.

Babies are still dying of SIDS.

OVERVIEW: The following account of a devastating, preventable death helped change licensing regulations and brought about a safer sleep environment for all infants in licensed child-care facilities in Arkansas. Coauthor Andrea Moore responded to her own personal and family tragedy with a determination to save lives by reducing risk and promoting public awareness of sudden infant death syndrome (SIDS). She teamed up with coauthor Rebecca Matthews, a community health nurse, and Rebecca's husband, pediatrician David Matthews, president of the Arkansas chapter of the American Academy of Pediatrics, to initiate a four-year public health intervention. This article provides information on SIDS, suggests ways that nurses can help educate caregivers and contribute to the prevention of SIDS, and details the progress of the team's educational initiative.

Native molecular state of adeno-associated viral vectors revealed by single-molecule sequencing.

The single-stranded genome of adeno-associated viral (AAV) vectors is one of the key factors leading to slow-rising but long-term transgene expression kinetics. Previous molecular studies have established what is now considered a textbook molecular model of AAV genomes with two copies of inverted tandem repeats at either end. In this study, we profiled hundreds of thousands of individual molecules of AAV vector DNA directly isolated from capsids, using single-molecule sequencing (SMS), which avoids any intermediary steps such as plasmid cloning. The sequence profile at 3' ends of both the regular and oversized vector did show the presence of an inverted terminal repeat (ITR), which provided direct confirmation that AAV vector packaging initiates from its 3' end. Furthermore, the vector 5'-terminus profile showed inconsistent termination for oversized vectors. Such incomplete vectors would not be expected to undergo canonical synthesis of the second strand of their genomic DNA and thus could function only via annealing of complementary strands of DNA. Furthermore, low levels of contaminating plasmid DNA were also detected. SMS may become a valuable tool during the development phase of vectors that are candidates for clinical use and for facilitating/accelerating studies on vector biology.