Immortalization and transformation of primary cells mediated by engineered ecDNAs.

Focal gene amplifications are among the most common cancer-associated mutations, but their evolution and contribution to tumorigenesis have proven challenging to recapitulate in primary cells and model organisms. Here we describe a general approach to engineer large (>1 Mbp) focal amplifications mediated by extrachromosomal circular DNAs (ecDNAs, also known as "double minutes") in a spatiotemporally controlled manner in cancer cell lines and in primary cells derived from genetically engineered mice. With this strategy, ecDNA formation can be coupled with expression of fluorescent reporters or other selectable markers to enable the identification and tracking of ecDNA-containing cells. We demonstrate the feasibility of this approach by engineering MDM2-containing ecDNAs in near-diploid human cells, showing that GFP expression can be used to track ecDNA dynamics under physiological conditions or in the presence of specific selective pressures. We also apply this approach to generate mice harboring inducible Myc - and Mdm2 -containing ecDNAs analogous to those spontaneously occurring in human cancers. We show that the engineered ecDNAs rapidly accumulate in primary cells derived from these animals, promoting proliferation, immortalization, and transformation.

Surgical Methods in Postmetamorphic Xenopus laevis: Optic Nerve Crush Injury Model.

Many human optic neuropathies lead to crippling conditions resulting in partial or complete loss of vision. While the retina is made up of several different cell types, retinal ganglion cells (RGCs) are the only cell type connecting the eye to the brain. Optic nerve crush injuries, wherein RGC axons are damaged without severing the optic nerve sheath, can serve as a model for traumatic optical neuropathies as well as some progressive neuropathies such as glaucoma. In this chapter, we describe two different surgical methods for establishing an optic nerve crush (ONC) injury in the postmetamorphic frog, Xenopus laevis. Why use the frog as an animal model? Mammals lose the ability to regenerate damaged CNS neurons, but amphibians and fish retain the ability to regenerate new RGC bodies and regrow RGC axons following an injury. In addition to presenting two different surgical ONC injury methods, we highlight their advantages and disadvantages and discuss the distinctive characteristics of Xenopus laevis as an animal model for studying CNS regeneration.

Effect of Corticosteroid-Sparing Treatment With Mycophenolate Mofetil vs Methotrexate on Inflammation in Patients With Uveitis: A Randomized Clinical Trial.

Importance: Methotrexate and mycophenolate mofetil are commonly used immunomodulatory therapies for achieving corticosteroid-sparing control of noninfectious uveitis, but there is uncertainty about which drug is more effective. Objective: To compare the effect of methotrexate and mycophenolate for achieving corticosteroid-sparing control of noninfectious intermediate uveitis, posterior uveitis, and panuveitis. Design, Setting, and Participants: The First-line Antimetabolites as Steroid-sparing Treatment (FAST) uveitis trial screened 265 adults with noninfectious uveitis requiring corticosteroid-sparing immunosuppressive therapy from 9 referral eye centers in India, the United States, Australia, Saudi Arabia, and Mexico between August 22, 2013, and August 16, 2017. Follow-up ended on August 20, 2018. Interventions: Patients were randomized to receive oral methotrexate, 25 mg weekly (n = 107), or oral mycophenolate mofetil, 3 g daily (n = 109). Main Outcomes and Measures: The primary outcome was treatment success at 6 months, which was defined as having control of inflammation in both eyes, no more than 7.5 mg prednisone daily and less than or equal to 2 drops of prednisolone acetate 1%, and no treatment failure due to safety or intolerability. Patients underwent follow-up to 12 months while receiving the same treatment or switched to the other antimetabolite, depending on their 6-month outcome. Results: Among 216 patients who were randomized (median age, 38 years; 135 (62.5%) women), 194 (89.8%) completed follow-up through 6 months. Treatment success occurred in 64 (66.7%) patients in the methotrexate group vs 56 (57.1%) in the mycophenolate group (difference, 9.5% [95% CI, -5.3% to 21.8%]; odds ratio [OR], 1.50 [95% CI, 0.81 to 2.81]; P = .20). Among patients with posterior uveitis or panuveitis, treatment success was achieved in 58 (74.4%) in the methotrexate group vs 42 (55.3%) in the mycophenolate group (difference, 19.1% [95% CI, 3.6% to 30.6%]; OR, 2.35 [95% CI, 1.16 to 4.90]; P = .02); whereas among patients with intermediate uveitis treatment success occurred in 6 (33.3%) in the methotrexate group vs 14 (63.6%) in the mycophenolate group (difference, -30.3% [95% CI, -51.6% to 1.1%]; OR, 0.29 [95% CI, 0.08 to 1.05]; P = .07; P for interaction = .004). Elevated liver enzymes were the most common nonserious laboratory adverse event, occurring in 14 patients (13.0%) in the methotrexate group and 8 patients (7.4%) in the mycophenolate group. Conclusions and Relevance: Among adults with noninfectious uveitis, the use of mycophenolate mofetil compared with methotrexate as first-line corticosteroid-sparing treatment did not result in superior control of inflammation. Further research is needed to determine if either drug is more effective based on the anatomical subtype of uveitis. Trial Registration: ClinicalTrials.gov Identifier: NCT01829295.

On the Relative Role of Different Age Groups During Epidemics Associated With Respiratory Syncytial Virus.

Background: While circulation of respiratory syncytial virus (RSV) results in high rates of hospitalization, particularly among young children and elderly individuals, little is known about the role of different age groups in propagating annual RSV epidemics. Methods: We evaluate the roles played by individuals in different age groups during RSV epidemics in the United States between 2001 and 2012, using the previously defined relative risk (RR) statistic estimated from the hospitalization data from the Healthcare Cost and Utilization Project. Transmission modeling was used to examine the robustness of our inference method. Results: Children aged 3-4 years and 5-6 years each had the highest RR estimate for 5 of 11 seasons included in this study, with RSV hospitalization rates in infants being generally higher during seasons when children aged 5-6 years had the highest RR estimate. Children aged 2 years had the highest RR estimate during one season. RR estimates in infants and individuals aged ≥11 years were mostly lower than in children aged 1-10 years. Highest RR values aligned with groups for which vaccination had the largest impact on epidemic dynamics in most model simulations. Conclusions: Our estimates suggest the prominent relative roles of children aged ≤10 years (particularly among those aged 3-6 years) in propagating RSV epidemics. These results, combined with further modeling work, should help inform RSV vaccination policies.

Exogenous arachidonic acid mediates permeability of human brain microvessel endothelial cells through prostaglandin E2 activation of EP3 and EP4 receptors.

The blood-brain barrier, formed by microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. Arachidonic acid (ARA; 5,8,11,14-cis-eicosatetraenoic acid) is a conditionally essential polyunsaturated fatty acid [20:4(n-6)] and is a major constituent of brain lipids. The current study examined the transport processes for ARA in confluent monolayers of human brain microvascular endothelial cells (HBMEC). Addition of radioactive ARA to the apical compartment of HBMEC cultured on Transwell(®) inserts resulted in rapid incorporation of radioactivity into the basolateral medium. Knock down of fatty acid transport proteins did not alter ARA passage into the basolateral medium as a result of the rapid generation of prostaglandin E2 (PGE2 ), an eicosanoid known to facilitate opening of the blood-brain barrier. Permeability following ARA or PGE2 exposure was confirmed by an increased movement of fluorescein-labeled dextran from apical to basolateral medium. ARA-mediated permeability was attenuated by specific cyclooxygenase-2 inhibitors. EP3 and EP4 receptor antagonists attenuated the ARA-mediated permeability of HBMEC. The results indicate that ARA increases permeability of HBMEC monolayers likely via increased production of PGE2 which acts upon EP3 and EP4 receptors to mediate permeability. These observations may explain the rapid influx of ARA into the brain previously observed upon plasma infusion with ARA. The blood-brain barrier, formed by microvessel endothelial cells, is a restrictive barrier between the brain parenchyma and the circulating blood. Radiolabeled arachidonic acid (ARA) movement across, and monolayer permeability in the presence of ARA, was examined in confluent monolayers of primary human brain microvessel endothelial cells (HBMECs) cultured on Transwell(®) plates. Incubation of HBMECs with ARA resulted in a rapid increase in HBMEC monolayer permeability. The mechanism was mediated, in part, through increased prostaglandin E2 production from ARA which acted upon EP3 and EP4 receptors to increase HBMEC monolayer permeability.

The importance of measuring ionized calcium in characterizing calcium status and diagnosing primary hyperparathyroidism.

CONTEXT: Serum total calcium (tCa) is routinely measured for diagnosing calcium disorders but may not reflect levels of biologically active ionized calcium (iCa) in disease or detect all cases of primary hyperparathyroidism. OBJECTIVE: We investigated the utility of measuring iCa and tCa for diagnosing primary hyperparathyroidism. DESIGN: This was an observational, retrospective, cross-sectional study. PATIENTS: We studied a biochemistry cohort of consecutive ambulatory outpatients with suspected bone or calcium metabolism disorders referred for calcium metabolism biochemistry panels and a surgical cohort of consecutive tertiary hospital patients whose parathyroid specimens were submitted to a single center, and consecutive parathyroidectomy patients of a single surgeon with specimens submitted to a different center. RESULTS: In 5490 biochemistry cohort patients, discordance between iCa and tCa in classifying calcium status occurred in 12.6% of cases overall but was worse in hypercalcemic (whether defined by tCa and/or iCa) cases (49%) and hypocalcemic cases (92%). Reliance on tCa alone would miss 45% with ionized hypercalcemia. In 315 biochemistry cohort cases with PTH-dependent hypercalcemia, 130 (41%) had isolated ionized hypercalcemia at diagnosis. In 143 patients with histologically proven parathyroid disease, 24% had isolated ionized hypercalcemia at diagnosis. These patients were younger (P = 0.022) with milder ionized hypercalcemia and better renal function (both P ≤ 0.001) than patients presenting with concurrently elevated iCa and tCa. CONCLUSION: In abnormal calcium states, tCa frequently disagrees with iCa in classifying calcium status. Histologically proven parathyroid disease can present with isolated ionized hypercalcemia. Measurement of iCa is required to accurately assess calcium status and improve diagnostic accuracy.

Carboetomidate: a pyrrole analog of etomidate designed not to suppress adrenocortical function.

BACKGROUND: Etomidate is a sedative hypnotic that is often used in critically ill patients because it provides superior hemodynamic stability. However, it also binds with high affinity to 11beta-hydroxylase, potently suppressing the synthesis of steroids by the adrenal gland that are necessary for survival. The authors report the results of studies to define the pharmacology of (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate), a pyrrole analog of etomidate specifically designed not to bind with high affinity to 11beta-hydroxylase. METHODS: The hypnotic potency of carboetomidate was defined in tadpoles and rats using loss of righting reflex assays. Its ability to enhance wild-type alpha1beta2gamma2l and etomidate-insensitive mutant alpha1beta2M286Wgamma2l human gamma-aminobutyric acid type A receptor activities was assessed using electrophysiologic techniques. Its potency for inhibiting in vitro cortisol synthesis was defined using a human adrenocortical cell assay. Its effects on in vivo hemodynamic and adrenocortical function were defined in rats. RESULTS: Carboetomidate was a potent hypnotic in tadpoles and rats. It increased currents mediated by wild-type but not etomidate-insensitive mutant gamma-aminobutyric acid type A receptors. Carboetomidate was a three orders of magnitude less-potent inhibitor of in vitro cortisol synthesis by adrenocortical cells than was etomidate. In rats, carboetomidate caused minimal hemodynamic changes and did not suppress adrenocortical function at hypnotic doses. CONCLUSIONS: Carboetomidate is an etomidate analog that retains many beneficial properties of etomidate, but it is dramatically less potent as an inhibitor of adrenocortical steroid synthesis. Carboetomidate is a promising new sedative hypnotic for potential use in critically ill patients in whom adrenocortical suppression is undesirable.

Methoxycarbonyl-etomidate: a novel rapidly metabolized and ultra-short-acting etomidate analogue that does not produce prolonged adrenocortical suppression.

BACKGROUND: Etomidate is a rapidly acting sedative-hypnotic that provides hemodynamic stability. It causes prolonged suppression of adrenocortical steroid synthesis; therefore, its clinical utility and safety are limited. The authors describe the results of studies to define the pharmacology of (R)-3-methoxy-3-oxopropyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate (MOC-etomidate), the first etomidate analogue designed to be susceptible to ultra-rapid metabolism. METHODS: The gamma-aminobutyric acid type A receptor activities of MOC-etomidate and etomidate were compared by using electrophysiological techniques in human alpha1beta2gamma2l receptors. MOC-etomidate's hypnotic concentration was determined in tadpoles by using a loss of righting reflex assay. Its in vitro metabolic half-life was measured in human liver S9 fraction, and the resulting metabolite was provisionally identified by using high-performance liquid chromatography/mass spectrometry techniques. The hypnotic and hemodynamic actions of MOC-etomidate, etomidate, and propofol were defined in rats. The abilities of MOC-etomidate and etomidate to inhibit corticosterone production were assessed in rats. RESULTS: MOC-etomidate potently enhanced gamma-aminobutyric acid type A receptor function and produced loss of righting reflex in tadpoles. Metabolism in human liver S9 fraction was first-order, with an in vitro half-life of 4.4 min versus more than 40 min for etomidate. MOC-etomidate's only detectable metabolite was a carboxylic acid. In rats, MOC-etomidate produced rapid loss of righting reflex that was extremely brief and caused minimal hemodynamic changes. Unlike etomidate, MOC-etomidate produced no adrenocortical suppression 30 min after administration. CONCLUSIONS: MOC-etomidate is an etomidate analogue that retains etomidate's important favorable pharmacological properties. However, it is rapidly metabolized, ultra-short-acting, and does not produce prolonged adrenocortical suppression after bolus administration.