Cutting Edge: Characterization of Low Copy Number Human Angiotensin-Converting Enzyme 2-Transgenic Mice as an Improved Model of SARS-CoV-2 Infection.

A popular mouse model of COVID-19, the K18-hACE2 mouse, expresses the SARS-coronavirus entry receptor, human angiotensin-converting enzyme 2 (hACE2) driven by the keratin-18 promoter. SARS-CoV-2-infected K18-hACE2 mice exhibit neuropathology not representative of human infection. They contain eight transgene (Tg) copies, leading to excess hACE2 expression and rampant viral replication. We generated two new lines of K18-hACE2 mice encoding one and two copies of hACE2 (1-hACE2-Tg and 2-hACE2-Tg, respectively). Relative to the original strain (called 8-hACE2-Tg in this study), 2-hACE2-Tg mice exhibited lower mortality, with less viral replication in the lung and brain. Furthermore, 1-hACE2-Tg mice exhibited no mortality and had no detectable virus in the brain; yet, they exhibited clear viral replication in the lung. All three strains showed SARS-CoV-2-related weight loss commensurate with the mortality rates. 1-hACE2-Tg mice mounted detectable primary and memory T effector cell and Ab responses. We conclude that these strains provide improved models to study hACE2-mediated viral infections.

Serine Threonine Kinase Receptor-Associated Protein Deficiency Impairs Mouse Embryonic Stem Cells Lineage Commitment Through CYP26A1-Mediated Retinoic Acid Homeostasis.

Retinoic acid (RA) signaling is essential for the differentiation of embryonic stem cells (ESCs) and vertebrate development. RA biosynthesis and metabolism are controlled by a series of enzymes, but the molecular regulators of these enzymes remain largely obscure. In this study, we investigated the functional role of the WD-domain protein STRAP (serine threonine kinase receptor-associated protein) in the pluripotency and lineage commitment of murine ESCs. We generated Strap knockout (KO) mouse ESCs and subjected them to spontaneous differentiation. We observed that, despite the unchanged characteristics of ESCs, Strap KO ESCs exhibited defects for lineage differentiation. Signature gene expression analyses revealed that Strap deletion attenuated intracellular RA signaling in embryoid bodies (EBs), and exogenous RA significantly rescued this deficiency. Moreover, loss of Strap selectively induced Cyp26A1 expression in mouse EBs, suggesting a potential role of STRAP in RA signaling. Mechanistically, we identified putative Krüppel-like factor 9 (KLF9) binding motifs to be critical in the enhancement of non-canonical RA-induced transactivation of Cyp26A1. Increased KLF9 expression in the absence of STRAP is partially responsible for Cyp26A1 induction. Interestingly, STRAP knockdown in Xenopus embryos influenced anterior-posterior neural patterning and impaired the body axis and eye development during early Xenopus embryogenesis. Taken together, our study reveals an intrinsic role for STRAP in the regulation of RA signaling and provides new molecular insights for ESC fate determination. Stem Cells 2018;36:1368-1379.

Fusobacterium nucleatum infection of gingival epithelial cells leads to NLRP3 inflammasome-dependent secretion of IL-1ß and the danger signals ASC and HMGB1.

Fusobacterium nucleatum is an invasive anaerobic bacterium that is associated with periodontal disease. Previous studies have focused on virulence factors produced by F. nucleatum, but early recognition of the pathogen by the immune system remains poorly understood. Although an inflammasome in gingival epithelial cells (GECs) can be stimulated by danger-associated molecular patterns (DAMPs) (also known as danger signals) such as ATP, inflammasome activation by this periodontal pathogen has yet to be described in these cells. This study therefore examines the effects of F. nucleatum infection on pro-inflammatory cytokine expression and inflammasome activation in GECs. Our results indicate that infection induces translocation of NF-κB into the nucleus, resulting in cytokine gene expression. In addition, infection activates the NLRP3 inflammasome, which in turn activates caspase-1 and stimulates secretion of mature IL-1ß. Unlike other pathogens studied until now, F. nucleatum activates the inflammasome in GECs in the absence of exogenous DAMPs such as ATP. Finally, infection promotes release of other DAMPs that mediate inflammation, such as high-mobility group box 1 protein and apoptosis-associated speck-like protein, with a similar time-course as caspase-1 activation. Thus, F. nucleatum expresses the pathogen-associated molecular patterns necessary to activate NF-κB and also provides an endogenous DAMP to stimulate the inflammasome and further amplify inflammation through secretion of secondary DAMPs.

Glasgow Coma Scale Scoring is Often Inaccurate.

INTRODUCTION: The Glasgow Coma Scale (GCS) is widely applied in the emergency setting; it is used to guide trauma triage and for the application of essential interventions such as endotracheal intubation. However, inter-rater reliability of GCS scoring has been shown to be low for inexperienced users, especially for the motor component. Concerns regarding the accuracy and validity of GCS scoring between various types of emergency care providers have been expressed. Hypothesis/Problem The objective of this study was to determine the degree of accuracy of GCS scoring between various emergency care providers within a modern Emergency Medical Services (EMS) system. METHODS: This was a prospective observational study of the accuracy of GCS scoring using a convenience sample of various types of emergency medical providers using standardized video vignettes. Ten video vignettes using adults were prepared and scored by two board-certified neurologists. Inter-rater reliability was excellent (Cohen's κ = 1). Subjects viewed the video and then scored each scenario. The scoring of subjects was compared to expert scoring of the two board-certified neurologists. RESULTS: A total of 217 emergency providers watched 10 video vignettes and provided 2,084 observations of GCS scoring. Overall total GCS scoring accuracy was 33.1% (95% CI, 30.2-36.0). The highest accuracy was observed on the verbal component of the GCS (69.2%; 95% CI, 67.8-70.4). The eye-opening component was the second most accurate (61.2%; 95% CI, 59.5-62.9). The least accurate component was the motor component (59.8%; 95% CI, 58.1-61.5). A small number of subjects (9.2%) assigned GCS scores that do not exist in the GCS scoring system. CONCLUSIONS: Glasgow Coma Scale scoring should not be considered accurate. A more simplified scoring system should be developed and validated.

Risk of pure red cell aplasia in patients with hepatitis C receiving antiviral therapy and an erythropoiesis-stimulating agent.

Antibody-mediated pure red cell aplasia (PRCA) has been primarily observed in patients with chronic kidney disease treated with an erythropoiesis-stimulating agent (ESA); only a few anecdotal cases have been reported in other patient populations. We searched the Amgen Global Safety Adverse Event Database and identified 14 patients with hepatitis C who developed severe anemia, anti-erythropoietin antibodies, and bone marrow biopsy-proven PRCA, while receiving interferon therapy (with or without ribavirin) and an ESA. During the follow-up period and after ESA treatment stopped, 11 patients no longer required transfusions and 3 did. Analysis of antibody isotypes showed that, contrary to reports of patients with chronic kidney disease, immunoglobulin G1 was the predominant isotype rather than immunoglobulin G4 (immunoglobulin G4 was detected in only 1 of 6 patients). Epitope mapping showed the anti-erythropoietin antibodies bound domains required for receptor binding. Therefore, the potential benefits of ESA therapy must be weighed against the risk for PRCA in patients with hepatitis C who are receiving treatment with interferon and ribavirin.

EMS provider compliance with infection control recommendations is suboptimal.

INTRODUCTION: Standard precautions are disease transmission prevention strategies recommended by both the World Health Organization (WHO) and by the Centers for Disease Control and Prevention (CDC). Emergency medical services (EMS) personnel are expected to utilize standard precautions. METHODS: This was a prospective observational study of the use of standard precautions by EMS providers arriving at a large urban emergency department (ED). Research assistants (RAs) observed EMS crews throughout their arrival and delivery of patients and recorded data related to the use of gloves, hand hygiene, and equipment disinfection. RESULTS: A total of 423 EMS deliveries were observed, allowing for observation of 899 EMS providers. Only 512 (56.9%) EMS providers arrived wearing gloves. Hand washing was observed in 250 (27.8%) of providers. Reusable equipment disinfection was noted in only 31.6% of opportunities. The most commonly disinfected item was the stretcher (55%). CONCLUSION: EMS provider compliance with standard precautions and equipment disinfection recommendations is suboptimal. Strategies must be developed to improve EMS provider compliance with internationally recognized infection control guidelines. Key words: Emergency medical services, hand washing, hygiene, disinfection, disease prevention.

Mitochondrial genetic background modulates bioenergetics and susceptibility to acute cardiac volume overload.

Dysfunctional bioenergetics has emerged as a key feature in many chronic pathologies such as diabetes and cardiovascular disease. This has led to the mitochondrial paradigm in which it has been proposed that mtDNA sequence variation contributes to disease susceptibility. In the present study we show a novel animal model of mtDNA polymorphisms, the MNX (mitochondrial-nuclear exchange) mouse, in which the mtDNA from the C3H/HeN mouse has been inserted on to the C57/BL6 nuclear background and vice versa to test this concept. Our data show a major contribution of the C57/BL6 mtDNA to the susceptibility to the pathological stress of cardiac volume overload which is independent of the nuclear background. Mitochondria harbouring the C57/BL6J mtDNA generate more ROS (reactive oxygen species) and have a higher mitochondrial membrane potential relative to those with C3H/HeN mtDNA, independent of nuclear background. We propose this is the primary mechanism associated with increased bioenergetic dysfunction in response to volume overload. In summary, these studies support the 'mitochondrial paradigm' for the development of disease susceptibility, and show that the mtDNA modulates cellular bioenergetics, mitochondrial ROS generation and susceptibility to cardiac stress.

Low-fractional oxygen concentration continuous positive airway pressure is effective in the prehospital setting.

OBJECTIVE: The objective of this study was to determine the effects of low-fractional concentration of inspired oxygen (FiO(2)) continuous positive airway pressure (CPAP) in prehospital noninvasive ventilation (NIV). With increasing concerns about the detrimental effects of hyperoxia, we sought to determine whether CPAP using a low FiO(2) (28%-30%) was effective in the prehospital setting. METHODS: The study was a six-month prospective, nonblinded observational study conducted in a large, busy urban emergency medical services (EMS) system (Las Vegas, NV). RESULTS: A total of 340 patients participated in the study. Most patients presented with symptoms consistent with a diagnosis of congestive heart failure/acute pulmonary edema (47.4%), followed by chronic obstructive pulmonary disease (COPD) (40.9%), asthma (22.7%), and pneumonia (15.9%). Improvements were seen in respiratory rate (p = 0.00) and oxygen saturation (p = 0.00). The overall CPAP discontinuation rate was 16.5%. The most common reason for CPAP discontinuation was anxiety/claustrophobia. The total number of patients requiring prehospital intubation was 5.6%. Subjective paramedic assessment of patient status at hospital delivery found that 71.5% of patients' conditions were improved, 15.1% remained unchanged, and 13.4% were worse. CONCLUSIONS: CPAP using a low FiO(2) (28%-30%) was highly effective in the treatment of commonly encountered prehospital respiratory emergencies.

Can emergency medical services personnel effectively place and use the Supraglottic Airway Laryngopharyngeal Tube (SALT) airway?

BACKGROUND: Various alternative airway devices have been developed in the last several years. Among these is the Supraglottic Airway Laryngopharyngeal Tube (SALT), which was designed to function as a basic mechanical airway and as an endotracheal tube (ET) introducer for blind endotracheal intubation (ETI). OBJECTIVE: To determine the rate of successful placement of the SALT and the success rate of subsequent blind ET insertion by a cohort of emergency medical services (EMS) providers of varying levels of EMS certification. METHODS: This study was a two-phase, two-group nonblinded, prospective time trial using a convenience cohort of prehospital providers to determine the success rate for SALT placement (i.e., the basic life support [BLS] phase) and ET placement using the SALT (i.e., the advanced life support [ALS] phase) in an unembalmed human cadaver model. The part 1 cohort (group 1) comprised predominantly basic and intermediate emergency medical technician (EMT)-level providers, whereas the part 2 cohort (group 2) comprised exclusively paramedic-level providers. RESULTS: In group 1, 51 (98%) of the subjects were able to successfully place the SALT and ventilate the cadaver (BLS phase), with 48 (92.3%) subjects successfully placing it on the first attempt. In group 2, 21 (96%) of the subjects were able to successfully place the SALT, with 19 (86%) placing the SALT on the first attempt. Successful blind placement of an ET through the SALT (ALS phase) by group 1 was 48.1% (95% confidence interval [CI]: 34-62), with 37% (95% CI: 24-51) placing the ET on the first attempt. In group 2, 20 subjects (91% [95% CI: 71-99]) were able to successfully place an ET through the SALT, with 13 (59% [95% CI: 36-79]) doing so on the first attempt. CONCLUSIONS: Emergency medical services providers of varying levels can successfully and rapidly place the SALT and ventilate a cadaver specimen. The success rate for blind placement of an ET through the SALT was suboptimal.

Hypothenar hammer syndrome: an update with algorithms for diagnosis and treatment.

Hypothenar hammer syndrome is an uncommon cause of symptomatic ischemia of the hand secondary to the formation of aneurysm or thrombosis of the ulnar artery as a result of repetitive trauma to the hypothenar region. The injury to the palmar ulnar artery occurs in subjects who use the hypothenar part of the hand as a hammer; the hook of the hamate strikes the superficial palmar branch of the ulnar artery in the Guyon space. Such injuries of the ulnar artery may lead to severe vascular insufficiency in the hand with thrombosis and distal embolization of the digital arteries. Imaging of vascular lesions of the hand has greatly improved in recent years with the use of Doppler, multislice computed tomographic angiography, magnetic resonance imaging, magnetic resonance angiography, and digital subtraction angiography. The aim of this article is to describe a case report using current-day imaging and to present a review of literature. Algorithms for the diagnosis and treatment of hypothenar hammer syndrome will be proposed.

Role of Sertoli cell number and function on regulation of spermatogenesis.

Testicular function is under the control of expression and repression of several genes and gene products, and many of these works through Sertoli cells. The capability of Sertoli cells to regulate spermatogenesis is dependent on Sertoli cell functions and Sertoli cell number. Sertoli cell number has long been thought to be stable in adults with no proliferation of Sertoli cells once adult numbers have been reached. However, adult horses do not have stable Sertoli cell numbers, and new studies indicate that adult Sertoli cells can be made to re-enter mitotic phase under certain experimental conditions. This review discusses roles of Sertoli cells in regulation of spermatogenesis and methods for estimating the number of Sertoli cells, in a testis, that overcome the problems (assumptions) associated with the indented, pear-shaped of Sertoli cell nuclei which make it difficult to estimate the volume of individual nuclei. Using several approaches to overcome the problems associated with any one method, the horse is identified as a species in which Sertoli cell number is not fixed, but it fluctuates with season. In addition to Sertoli cell numbers, the functions of Sertoli cells that are very important in signaling and controlling spermatogenesis are discussed. Recent studies have shown that "post-mitotic terminally differentiated Sertoli cells" from adult animals could, under certain conditions, re-enter the cell division cycle. Can seasonal influences be a natural set of conditions to induce the Sertoli cells of the horse testis to seasonally re-enter the cell division cycle and explain the seasonal differences in Sertoli cell number as summarized in this review? Alternatively, can seasonal differences in Sertoli cell number reflect, in the horse to a greater extent, but in adults of most species, the presence of some mitotic-capable Sertoli cells in adults? In any case, both Sertoli cell number and function are important in regulation of spermatogenesis.

Oral infection of mice with Fusobacterium nucleatum results in macrophage recruitment to the dental pulp and bone resorption.

BACKGROUND: Fusobacterium nucleatum is a Gram-negative anaerobic bacterium associated with periodontal disease. Some oral bacteria, like Porphyromonas gingivalis, evade the host immune response by inhibiting inflammation. On the other hand, F. nucleatum triggers inflammasome activation and release of danger-associated molecular patterns (DAMPs) in infected gingival epithelial cells. METHODS: In this study, we characterized the pro-inflammatory response to F. nucleatum oral infection in BALB/c mice. Western blots and ELISA were used to measure cytokine and DAMP (HMGB1) levels in the oral cavity after infection. Histology and flow cytometry were used to observe recruitment of immune cells to infected tissue and pathology. RESULTS: Our results show increased expression and production of pro-inflammatory cytokines during infection. Furthermore, we observe that F. nucleatum infection leads to recruitment of macrophages in different tissues of the oral cavity. Infection also contributes to osteoclast recruitment, which could be involved in the observed bone resorption. CONCLUSIONS: Overall, our findings suggest that F. nucleatum infection rapidly induces inflammation, release of DAMPs, and macrophage infiltration in gingival tissues and suggest that osteoclasts may drive bone resorption at early stages of the inflammatory process.

Regulation of airway tight junctions by proinflammatory cytokines.

Epithelial tight junctions (TJs) provide an important route for passive electrolyte transport across airway epithelium and provide a barrier to the migration of toxic materials from the lumen to the interstitium. The possibility that TJ function may be perturbed by airway inflammation originated from studies reporting (1) increased levels of the proinflammatory cytokines interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and IL-1beta in airway epithelia and secretions from cystic fibrosis (CF) patients and (2) abnormal TJ strands of CF airways as revealed by freeze-fracture electron microscopy. We measured the effects of cytokine exposure of CF and non-CF well-differentiated primary human airway epithelial cells on TJ properties, including transepithelial resistance, paracellular permeability to hydrophilic solutes, and the TJ proteins occludin, claudin-1, claudin-4, junctional adhesion molecule, and ZO-1. We found that whereas IL-1beta treatment led to alterations in TJ ion selectivity, combined treatment of TNF-alpha and IFN-gamma induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. CF bronchi in vivo exhibited the same pattern of expression of TJ-associated proteins as cultures exposed in vitro to prolonged exposure to TNF-alpha and IFN-gamma. These data indicate that the TJ of airway epithelia exposed to chronic inflammation may exhibit parallel changes in the barrier function to both solutes and ions.

Preliminary evaluation of an after-school resistance training program for improving physical fitness in middle school-age boys.

Most after-school physical activity programs for youth focus on aerobic games and activities. The purpose of this study was to evaluate the efficacy of an after-school resistance training program on improving the physical fitness of middle school-age boys. 22 boys (M = 13.9 yr., SD = .4 yr.) participated in a periodized, multiple-set, 9-wk. (2x/week) resistance training program. All subjects were pre- and post-tested on their 10-repetition maximum squat, 10-repetition maximum bench press, vertical jump, medicine ball toss, flexibility, and also percentage of body fat and the progressive aerobic cardiovascular endurance run (PACER). Statistical analysis indicated that subjects significantly improved performance on the squat (19%), bench press (15%), flexibility (10%), vertical jump (5%), medicine ball toss (12%), and the PACER (36%). Although this design minus a control group limits interpretation, this after-school resistance-training program can improve muscular fitness and cardiovascular fitness in boys and should be replicated with appropriate experimental controls.

Improved Oviduct Transfer Surgery for Genetically Modified Rat Production.

Rat embryo transfer surgeries are becoming more common with targeted nucleases increasing the demand for rat models. This protocol details pre-surgical preparation, improved surgical techniques for placing embryos into the oviduct, and post-surgical care of rats to parturition. Direct application of mouse oviduct transfer protocols results in limited success in the rat. By combining techniques from several widely used protocols in the field, increased yield of live pups born to healthy dams is possible. This protocol is distinct from previously published protocols by the use of a modified anesthesia protocol (Smith et al., 2004), use of analgesia, the addition of peritoneal sutures (Filipiak and Saunders, 2006), incision location and number of transfers per animal (Krinke et al., 2000).

A multicomponent quality improvement intervention to improve blood pressure and reduce racial disparities in rural primary care practices.

The Southeastern United States has the highest prevalence of hypertension and African Americans have disproportionately worse blood pressure control. The authors sought to evaluate the effect of a multicomponent practice-based quality improvement intervention on lowering mean systolic blood pressure (SBP) at 12 and 24 months compared with baseline among 525 patients, and to assess for a differential effect of the intervention by race (African Americans vs white). At 12 months, both African Americans (-5.0 mm Hg) and whites (-7.8 mm Hg) had a significant decrease in mean SBP compared with baseline, with no significant between-group difference. Similarly, at 24 months, mean SBP decreased in both African Americans (-6.0 mm Hg) and whites (-7.2 mm Hg), with no significant difference between groups. Notably, no significant racial disparity in mean SBP at baseline was shown. The intervention was effective in lowering mean SBP in both African Americans and whites but there was no differential effect of the intervention by race.

Manganese acts centrally to activate reproductive hormone secretion and pubertal development in male rats.

Manganese (Mn) is an important element for normal growth and reproduction. Because Mn accumulates in the hypothalamus and is capable of stimulating puberty-related hormones in female rats, we assessed whether this metal could cause similar effects in male rats. We have demonstrated that MnCl2, when administered acutely into the third ventricle of the brain, acts dose dependently to stimulate luteinizing hormone (LH) release. Furthermore, there was a dose dependent stimulation in the secretion of LH-releasing hormone (LHRH) from the medial basal hypothalamus in vitro, and administration of an LHRH receptor antagonist in vivo blocks Mn-induced LH release. To assess potential chronic effects of the metal, male pups were supplemented with 10 or 25 mg MnCl2 per kg by gastric gavage from day 15 until days 48 or 55, at which times developmental signs of spermatogenesis were assessed. Results demonstrate that while significant effects were not observed with the 10 mg/kg dose, the animals receiving the 25 mg/kg dose showed increased LH (p<0.05), FSH (p<0.01) and testosterone (p<0.01) levels at 55 days of age. Furthermore, there was a concomitant increase in both daily sperm production (p<0.05) and efficiency of spermatogenesis (p<0.05), demonstrating a Mn-induced acceleration in spermatogenesis. Our results suggest Mn is a stimulator of prepubertal LHRH/LH secretion and may facilitate the normal onset of male puberty. These data also suggest that the metal may contribute to male precocious pubertal development should an individual be exposed to low but elevated levels of Mn too early in life.