Distributable, metabolic PET reporting of tuberculosis.

Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) - is a mechanism-based reporter of Mycobacteria-selective enzyme activity in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-mediated processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-selective candidate for clinical evaluation. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either custom-made radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.

Increasing Diversity in Academic Medicine Via a Strategic Intermural Housestaff Leadership Development Program.

PROBLEM: By 2055, the United States will no longer have a single race or ethnic majority. As the nation's demographics change, the field of medicine must also change to meet the needs of diverse patients. APPROACH: In 2013, UT Southwestern Medical Center implemented the Housestaff Emerging Academy of Leaders (HEAL) program, which provides leadership development skills and training to underrepresented in medicine physician residents in preparation for academic medicine careers. Program leaders hypothesized that by providing housestaff with structured mentorship, career coaching, and individualized development plans, HEAL would increase interest in pursuing academic careers and prepare residents for faculty positions. HEAL has since expanded to graduate medical education programs nationwide. OUTCOMES: From 2013 to 2018, HEAL included housestaff at UT Southwestern and other Texas medical centers, totaling 392 enrollees. In 2019, the program increased to include housestaff from around the country. The first HEAL USA program had 39 housestaff, which increased to 173 in 2019, including 60 faculty from 31 U.S. academic medical centers. The 2019 HEAL USA preassessment survey (32 trainee responses) revealed that 10 (31%) of the housestaff were "extremely interested" in academic medicine, but only 1 (3%) felt "extremely confident" to pursue an academic medicine career. Postassessment responses to these same items (5 trainee responses) were 3 (60%) and 1 (20%), respectively, with 3 (60%) also feeling "extremely prepared" (1 [20%]) or "very prepared" (2 [40%]) to pursue an academic medicine career. Of 70 evaluable participants who attended at least 2 sessions and have graduated from residency, 47 (67%) have attained academic faculty positions, whereas 23 (33%) have pursued positions at nonacademic centers. NEXT STEPS: The next steps for HEAL USA will be continued expansion to additional medical centers and effective delivery of career development and leadership training to encourage participants to pursue academic medical careers.

Mapping of susceptibility loci for Ebola virus pathogenesis in mice.

Ebola virus (EBOV), a major global health concern, causes severe, often fatal EBOV disease (EVD) in humans. Host genetic variation plays a critical role, yet the identity of host susceptibility loci in mammals remains unknown. Using genetic reference populations, we generate an F2 mapping cohort to identify host susceptibility loci that regulate EVD. While disease-resistant mice display minimal pathogenesis, susceptible mice display severe liver pathology consistent with EVD-like disease and transcriptional signatures associated with inflammatory and liver metabolic processes. A significant quantitative trait locus (QTL) for virus RNA load in blood is identified in chromosome (chr)8, and a severe clinical disease and mortality QTL is mapped to chr7, which includes the Trim5 locus. Using knockout mice, we validate the Trim5 locus as one potential driver of liver failure and mortality after infection. The identification of susceptibility loci provides insight into molecular genetic mechanisms regulating EVD progression and severity, potentially informing therapeutics and vaccination strategies.

Distributable, Metabolic PET Reporting of Tuberculosis.

Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) - can act as a mechanism-based enzyme reporter in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-specific processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-specific, clinical diagnostic candidate. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either bespoke radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.

Ectopically expressed Airn lncRNA deposits Polycomb with a potency that rivals Xist.

We report that when expressed at similar levels from an isogenic locus, the Airn lncRNA induces Polycomb deposition with a potency that rivals Xist . However, when subject to the same degree of promoter activation, Xist is more abundant and more potent than Airn . Our data definitively demonstrate that the Airn lncRNA is functional and suggest that Xist achieved extreme potency in part by evolving mechanisms to promote its own abundance.

A precisely humanized FCRN transgenic mouse for preclinical pharmacokinetics studies.

Monoclonal antibodies (mAbs) are one of the fastest-growing classes of drugs and have been approved to treat several diseases, including cancers and autoimmune disorders. Preclinical pharmacokinetics studies are performed to determine the therapeutically meaningful dosages and efficacy of candidate drugs. These studies are typically performed in non-human primates; however, using primates is costly and raises ethical considerations. As a result, rodent models that better mimic human-like pharmacokinetics have been generated and remain an area of active investigation. Pharmacokinetic characteristics of a candidate drug, such as half-life, are partly controlled by antibody binding to the human neonatal receptor hFCRN. Due to the abnormally high binding of human antibodies to mouse FCRN, traditional laboratory rodents do not accurately model the pharmacokinetics of human mAbs. In response, humanized rodents expressing hFCRN have been generated. However, these models generally use large inserts randomly integrated into the mouse genome. Here, we report the production and characterization of a CRISPR/Cas9-mediated hFCRN transgenic mouse termed SYNB-hFCRN. Using CRISPR/Cas9-assisted gene targeting, we prepared a strain with a simultaneous knockout of mFcrn and insertion of a hFCRN mini-gene under the control of the endogenous mouse promoter. These mice are healthy and express hFCRN in the appropriate tissues and immune cell subtypes. Pharmacokinetic evaluation of human IgG and adalimumab (Humira®) demonstrate hFCRN-mediated protection. These newly generated SYNB-hFCRN mice provide another valuable animal model for use in preclinical pharmacokinetics studies during early drug development.

Viral protease cleavage of MAVS in genetically modified mice with hepatitis A virus infection.

BACKGROUND & AIMS: Consistent with its relatively narrow host species range, hepatitis A virus (HAV) cannot infect C57BL/6 mice. However, in Mavs-/- mice with genetic deficiency of the innate immune signaling adaptor MAVS, HAV replicates robustly in the absence of disease. The HAV 3ABC protease cleaves MAVS in human cells, thereby disrupting virus-induced IFN responses, but it cannot cleave murine MAVS (mMAVS) due to sequence differences at the site of scission. Here, we sought to elucidate the role of 3ABC MAVS cleavage in determining HAV pathogenesis and host species range. METHODS: Using CRISPR/Cas9 gene editing, we established two independent lineages of C57BL/6 mice with knock-in mutations altering two amino acids in mMAVS ('mMAVS-VS'), rendering it susceptible to 3ABC cleavage without loss of signaling function. We challenged homozygous Mavsvs/vs mice with HAV, and compared infection outcomes with C57BL/6 and genetically deficient Mavs-/- mice. RESULTS: The humanized murine mMAVS-VS protein was cleaved as efficiently as human MAVS when co-expressed with 3ABC in Huh-7 cells. In embyronic fibroblasts from Mavsvs/vs mice, mMAVS-VS was cleaved by ectopically expressed 3ABC, significantly disrupting Sendai virus-induced IFN responses. However, in contrast to Mavs-/- mice with genetic MAVS deficiency, HAV failed to establish infection in Mavsvs/vs mice, even with additional genetic knockout of Trif or Irf1. Nonetheless, when crossed with permissive Ifnar1-/- mice lacking type I IFN receptors, Mavsvs/vsIfnar1-/- mice demonstrated enhanced viral replication coupled with significant reductions in serum alanine aminotransferase, hepatocellular apoptosis, and intrahepatic inflammatory cell infiltrates compared with Ifnar1-/- mice. CONCLUSIONS: MAVS cleavage by 3ABC boosts viral replication and disrupts disease pathogenesis, but it is not by itself sufficient to break the host-species barrier to HAV infection in mice. IMPACT AND IMPLICATIONS: The limited host range of human hepatitis viruses could be explained by species-specific viral strategies that disrupt innate immune responses. Both hepatitis A virus (HAV) and hepatitis C virus express viral proteases that cleave the innate immune adaptor protein MAVS, in human but not mouse cells. However, the impact of this immune evasion strategy has never been assessed in vivo. Here we show that HAV 3ABC protease cleavage of MAVS enhances viral replication and lessens liver inflammation in mice lacking interferon receptors, but that it is insufficient by itself to overcome the cross-species barrier to infection in mice. These results enhance our understanding of how hepatitis viruses interact with the host and their impact on innate immune responses.

Endocardium-to-coronary artery differentiation during heart development and regeneration involves sequential roles of Bmp2 and Cxcl12/Cxcr4.

Endocardial cells lining the heart lumen are coronary vessel progenitors during embryogenesis. Re-igniting this developmental process in adults could regenerate blood vessels lost during cardiac injury, but this requires additional knowledge of molecular mechanisms. Here, we use mouse genetics and scRNA-seq to identify regulators of endocardial angiogenesis and precisely assess the role of CXCL12/CXCR4 signaling. Time-specific lineage tracing demonstrated that endocardial cells differentiated into coronary endothelial cells primarily at mid-gestation. A new mouse line reporting CXCR4 activity-along with cell-specific gene deletions-demonstrated it was specifically required for artery morphogenesis rather than angiogenesis. Integrating scRNA-seq data of endocardial-derived coronary vessels from mid- and late-gestation identified a Bmp2-expressing transitioning population specific to mid-gestation. Bmp2 stimulated endocardial angiogenesis in vitro and in injured neonatal mouse hearts. Our data demonstrate how understanding the molecular mechanisms underlying endocardial angiogenesis can identify new potential therapeutic targets promoting revascularization of the injured heart.

Genetic deletion of platelet PAR4 results in reduced thrombosis and impaired hemostatic plug stability.

BACKGROUND: Protease-activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes, and lung epithelial cells. It is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and reduced thrombosis. OBJECTIVE: We aimed to generate a mouse line with a megakaryocyte/platelet-specific deletion of PAR4 (PAR4fl/fl ;PF4Cre+ ) and use the mouse line to investigate the role of platelet PAR4 in hemostasis and thrombosis in mice. METHODS: Platelets from PAR4fl/fl ;PF4Cre+ were characterized in vitro. Arterial and venous thrombosis was analyzed. Hemostatic plug formation was analyzed using a saphenous vein laser injury model in mice with global or megakaryocyte/platelet-specific deletion of PAR4 or wild-type mice treated with thrombin or glycoprotein VI (GPVI) inhibitors. RESULTS: PAR4fl/fl ;PF4Cre+ platelets were unresponsive to thrombin or specific PAR4 stimulation but not to other agonists. PAR4-/- and PAR4fl/fl ;PF4Cre+ mice both exhibited a similar reduction in arterial thrombosis compared to their respective controls. More importantly, we show for the first time that platelet PAR4 is critical for venous thrombosis in mice. In addition, PAR4-/- mice and PAR4fl/fl ;PF4Cre+ mice exhibited a similar impairment in hemostatic plug stability in a saphenous vein laser injury model. Inhibition of thrombin in wild-type mice gave a similar phenotype. Combined PAR4 deficiency on platelets with GPVI inhibition did not impair hemostatic plug formation but further reduced plug stability. CONCLUSION: We generated a novel PAR4fl/fl ;PF4Cre+ mouse line. We used this mouse line to show that PAR4 signaling in platelets is critical for arterial and venous thrombosis and hemostatic plug stability.

Generation of a Matrix Gla (Mgp) floxed mouse, followed by conditional knockout, uncovers a new Mgp function in the eye.

The ability to ablate a gene in a given tissue by generating a conditional knockout (cKO) is crucial for determining its function in the targeted tissue. Such tissue-specific ablation is even more critical when the gene's conventional knockout (KO) is lethal, which precludes studying the consequences of its deletion in other tissues. Therefore, here we describe a successful strategy that generated a Matrix Gla floxed mouse (Mgp.floxed) by the CRISPR/Cas9 system, that subsequently allowed the generation of cKOs by local viral delivery of the Cre-recombinase enzyme. MGP is a well-established inhibitor of calcification gene, highly expressed in arteries' smooth muscle cells and chondrocytes. MGP is also one of the most abundant genes in the trabecular meshwork, the eye tissue responsible for maintenance of intraocular pressure (IOP) and development of Glaucoma. Our strategy entailed one-step injection of two gRNAs, Cas9 protein and a long-single-stranded-circular DNA donor vector (lsscDNA, 6.7 kb) containing two loxP sites in cis and 900-700 bp 5'/3' homology arms. Ocular intracameral injection of Mgp.floxed mice with a Cre-adenovirus, led to an Mgp.TMcKO mouse which developed elevated IOP. Our study discovered a new role for the Mgp gene as a keeper of physiological IOP in the eye.

Emergent behavior in an adversarial synchronization and swarming model.

We consider a red-versus-blue coupled synchronization and spatial swarming (i.e., swarmalator) model that incorporates attraction and repulsion terms and an adversarial game of phases. The model exhibits behaviors such as spontaneous emergence of tactical manoeuvres of envelopment (e.g., flanking, pincer, and envelopment) that are often proposed in military theory or observed in nature. We classify these states based on a large set of features such as spatial densities, synchronization between clusters, and measures of cluster distances. These features are used to study the influence of coupling parameters on the expected presence of these states and the-sometimes sharp-transitions between them.

Elements at the 5' end of Xist harbor SPEN-independent transcriptional antiterminator activity.

The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5' end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of downstream polyadenylation sequences. Readthrough required Repeat A and the ∼750 nucleotides downstream, did not require SPEN, and was attenuated by splicing. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a 5.5-kb Xist transgene robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5' end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.

Mentoring Black Men in Medicine.

In recent years, there have been multiple publications about the dearth of Black men in medicine. Appreciating the fact that underrepresented minority physicians disproportionately care for America's underserved communities, the lack of diversity in health care is particularly disturbing. Of imminent concern is the critical shortage of Black men doctors. In this Perspective, the authors contend that while mentoring is often considered among the most important strategies to increase the number of Black men in medicine, unique challenges in this demographic can diminish its effectiveness. Among these challenges are below average primary school educational experiences and a general mistrust of society on the part of Black men, as well as difficulties overcoming stereotypes and social biases that others hold against them. Furthermore, acknowledging that mentorship is paramount in achieving success in the medical field, the authors provide a framework to assist mentors in recognizing and addressing situations and obstacles that may disrupt the mentoring relationship and hinder its potential to best serve Black men pursuing advancement in medicine. This framework is represented by the acronym RACE: Reluctance to discuss race, Access to mentors, Cultural mistrust and racial concordance, and Empathy.

Baseline and innate immune response characterization of a Zfp30 knockout mouse strain.

Airway neutrophilia is correlated with disease severity in a number of chronic and acute pulmonary diseases, and dysregulation of neutrophil chemotaxis can lead to host tissue damage. The gene Zfp30 was previously identified as a candidate regulator of neutrophil recruitment to the lungs and secretion of CXCL1, a potent neutrophil chemokine, in a genome-wide mapping study using the Collaborative Cross. ZFP30 is a putative transcriptional repressor with a KRAB domain capable of inducing heterochromatin formation. Using a CRISPR-mediated knockout mouse model, we investigated the role that Zfp30 plays in recruitment of neutrophils to the lung using models of allergic airway disease and acute lung injury. We found that the Zfp30 null allele did not affect CXCL1 secretion or neutrophil recruitment to the lungs in response to various innate immune stimuli. Intriguingly, despite the lack of neutrophil phenotype, we found there was a significant reduction in the proportion of live Zfp30 homozygous female mutant mice produced from heterozygous matings. This deviation from the expected Mendelian ratios implicates Zfp30 in fertility or embryonic development. Overall, our results indicate that Zfp30 is an essential gene but does not influence neutrophilic inflammation in this particular knockout model.

Randomized Controlled Trial of Compression Interventions for Managing Hand Burn Edema, as Measured by Bioimpedance Spectroscopy.

Compression, a common treatment of choice for the management of edema, is one intervention that is applied with little objective understanding of the optimal parameters of application or efficacy in acute burn wounds. The aim of this study was to determine the effectiveness of different methods of compression for the management of hand edema following burn injury. The primary hypothesis tested was that in acute hand burn injury, the application of cohesive bandage will reduce edema faster than a generic compression glove. It is a randomized controlled study of 100 patients presenting with hand burn injury. Compression was randomized to one of the three methods of application: 1) spiral application of Coban to fingers, figure of eight to hand and wrist; 2) pinch application of Coban to fingers, spiral application to hand and wrist; or 3) a generic compression glove (control condition). Bioimpedance spectroscopy was used to measure hand volumes. Hand and wrist range of movement, pain scores, and QuickDASH were recorded. One hundred patients (68 males) demonstrated significant reductions in hand volumes, using all compression methods. Both methods of applying Coban resulted in significantly greater reductions in edema compared to the generic compression glove. Notwithstanding compression method, all range of movement measures improved, with significant improvement in thumb opposition (P = .046), hand span (P = .020), and wrist flexion (P = .020). QuickDASH decreased between sessions (P < .001). Different methods of applying Coban are superior to generic compression gloves for managing acute hand burn edema.

Reporter mice for isolating and auditing cell type-specific extracellular vesicles in vivo.

Extracellular vesicles (EVs) are abundant, lipid-enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type-specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR-Cas9-mediated genome editing to generate mice bearing a CD63-emGFPloxP/stop/loxP knock-in cassette that enables the specific labeling of circulating CD63+ vesicles from any cell type when crossed with lineage-specific Cre recombinase driver mice. As proof-of-principle, we have crossed these mice with Cdh5-CreERT2 mice to generate CD63emGFP+ vasculature. Using these mice, we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females. In adult mice, quiescent vasculature and angiogenic vasculature (in tumors) is also marked with emGFP. Moreover, whole plasma-purified EVs contain a subpopulation of emGFP+ vesicles that are derived from the endothelium, co-express additional EV (e.g., CD9 and CD81) and endothelial cell (e.g., CD105) markers, and they harbor specific miRNAs (e.g., miR-126, miR-30c, and miR-125b). This new mouse strain should be a useful genetic tool for generating cell type-specific, CD63+ EVs that freely circulate in serum and can subsequently be isolated and characterized using standard methodologies.

Bioimpedance Spectroscopy Is a Valid and Reliable Measure of Edema Following Hand Burn Injury (Part 1-Method Validation).

The assessment of swelling following burn injury is complicated by the presence of wounds and dressings and due to patients experiencing significant pain and impaired movement. There remains a lack of sensitive objective measures for edema in patients presenting with hand burn injury. Bioimpedance spectroscopy (BIS) is a measure of body composition that has been demonstrated by our group to be reliable for measuring whole body and limb edema during resuscitation and to be sensitive to edema changes within healing wounds. The aim of this study was to determine the reliability and validity of BIS as a measure of edema following hand burn injury specifically. One hundred patients presenting with burn injury including a portion of a hand were recruited to this trial. Repeated measures of the hand were recorded using a novel application of BIS and in parallel with water displacement volumetry (WDV). The results were analyzed using mixed-effects regressions. Paired repeated measures were obtained for 195 hands, using four electrode configurations. BIS demonstrated high reliability in measuring hand BIS-Intraclass Correlation Coefficient 0.995 to 0.999 (95% CI 0.992-1.000) and sensitivity-Minimum Detectable Difference 0.74 to 3.86 Ω (0.09-0.48 Ω/cm). A strong correlation was shown with WDV, Pearson's r = -0.831 to -0.798 (P < .001). BIS is a sensitive and reliable measure of edema following acute hand burn injury.

Alternate Electrode Positions for the Measurement of Hand Volumes Using Bioimpedance Spectroscopy.

Background: Bioimpedance spectroscopy (BIS) is a tool that can be used to measure body composition in a variety of populations. Previous studies have investigated novel applications to utilize BIS to measure localized body composition, including in the hand. According to BIS guidelines, there should be no skin wounds at the site of electrodes, and that electrode positions may be modified in specific circumstances, as our group has validated previously in burn wound populations. Methods and Results: To determine in noninjured participants, whether BIS measurements recorded using alternate electrode positions on the palm of the hand and forearm, or a combination of electrodes on the dorsum and volar surface of the hand and forearm, were comparable with electrode positions on the dorsum of the hand and forearm. The study demonstrated that drive and sense electrodes on the palm of the hand and volar forearm, and a combination of electrodes on the palm of the hand and dorsum of the forearm, resulted in comparable measures of impedance of extracellular water (difference from reference position: 1.26%-4.75%, p = 0.411-0.558) and total water (difference from reference: 2.15%-2.40%, p = 0.258-0.781). Electrodes on the dorsum of the hand and volar forearm resulted in significantly different measures for the same BIS variables (percentage difference range 4.66%-6.15%, p < 0.001-0.003). Conclusion: Electrode positions on the palm of the hand and volar forearm, or on the palm of the hand and dorsum of the forearm, are interchangeable as clinical measures of hand lymphedema and total water impedance.