Singleplex, multiplex and pooled sample real-time RT-PCR assays for detection of SARS-CoV-2 in an occupational medicine setting.

For workplaces which cannot operate as telework or remotely, there is a critical need for routine occupational SARS-CoV-2 diagnostic testing. Although diagnostic tests including the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (CDC Diagnostic Panel) (EUA200001) were made available early in the pandemic, resource scarcity and high demand for reagents and equipment necessitated priority of symptomatic patients. There is a clearly defined need for flexible testing methodologies and strategies with rapid turnaround of results for (1) symptomatic, (2) asymptomatic with high-risk exposures and (3) asymptomatic populations without preexisting conditions for routine screening to address the needs of an on-site work force. We developed a distinct SARS-CoV-2 diagnostic assay based on the original CDC Diagnostic Panel (EUA200001), yet, with minimum overlap for currently employed reagents to eliminate direct competition for limited resources. As the pandemic progressed with testing loads increasing, we modified the assay to include 5-sample pooling and amplicon target multiplexing. Analytical sensitivity of the pooled and multiplexed assays was rigorously tested with contrived positive samples in realistic patient backgrounds. Assay performance was determined with clinical samples previously assessed with an FDA authorized assay. Throughout the pandemic we successfully tested symptomatic, known contact and travelers within our occupational population with a ~ 24-48-h turnaround time to limit the spread of COVID-19 in the workplace. Our singleplex assay had a detection limit of 31.25 copies per reaction. The three-color multiplexed assay maintained similar sensitivity to the singleplex assay, while tripling the throughput. The pooling assay further increased the throughput to five-fold the singleplex assay, albeit with a subtle loss of sensitivity. We subsequently developed a hybrid 'multiplex-pooled' strategy to testing to address the need for both rapid analysis of samples from personnel at high risk of COVID infection and routine screening. Herein, our SARS-CoV-2 assays specifically address the needs of occupational healthcare for both rapid analysis of personnel at high-risk of infection and routine screening that is essential for controlling COVID-19 disease transmission. In addition to SARS-CoV-2 and COVID-19, this work demonstrates successful flexible assays developments and deployments with implications for emerging highly transmissible diseases and future pandemics.

Dynamic blue light-switchable protein patterns on giant unilamellar vesicles.

The blue light-dependent interaction between the proteins iLID and Nano allows recruiting and patterning proteins on GUV membranes, which thereby capture key features of patterns observed in nature. This photoswitchable protein interaction provides non-invasive, reversible and dynamic control over protein patterns of different sizes with high specificity and spatiotemporal resolution.

Thermophilic fungi in an aridland ecosystem.

We report a comprehensive multi-year study of thermophilic fungi at the Sevilleta National Wildlife Refuge in central New Mexico. Recovery of thermophilic fungi from soils showed seasonal fluctuations, with greater abundance correlating with spring and summer precipitation peaks. In addition to grassland soils, we obtained and characterized isolates from grassland and riparian litter, herbivore dung and biological soil crusts. All strains belonged to either the Eurotiales or Sordariales (Chaetomiaceae). No particular substrate or microhabitat associations were detected. Molecular typing of strains revealed substantial phylogenetic diversity, eight ad hoc phylogroups across the two orders were identified and genetic diversity was present within each phylogroup. Growth tests over a range of temperatures showed substantial variation in maximum growth rates among strains and across phylogroups but consistency within phylogroups. Results demonstrated that 45-50 C represents the optimal temperature for growth of most isolates, with a dramatic decline at 60 C. Most strains grew at 60 C, albeit slowly, whereas none grew at 65 C, providing empirical confirmation that 60 C presents an evolutionary threshold for fungal growth. Our results support the hypothesis that fungal thermophily is an adaptation to transient seasonal and diurnal high temperatures, rather than simply an adaptation to specialized high-temperature environments. We note that the diversity observed among strains and the frequently confused taxonomy within these groups highlight the need for comprehensive biosystematic revision of thermophilic taxa in both orders.

Improvement in survival with peptidyl membrane interactive molecule D4B treatment after burn wound infection.

OBJECTIVE: To examine the effects of peptidyl membrane interactive molecule D4B in a murine model of lethal burn wound infection. EXPERIMENTAL DESIGN: Four experiments were performed: (1) growth inhibition assays of Pseudomonas aeruginosa treated with D4B, 0 to 100 micromol/L; (2) in vitro coculture of bone marrow cells with D4B, 0 to 100 micromol/L; (3) D4B treatment survival studies after burn injury only or burn wound infection in mice; and (4) peripheral white blood cell count, burn wound tissue bacterial culture, and burn wound morphological analysis at days 1, 2, and 3 after injury. SETTING: University medical center laboratory. SUBJECTS: Groups of B6D2F1 male mice (20 each) were studied. INTERVENTIONS: Full-thickness scald burn, 15% of total body surface area, with P aeruginosa topical infection, and subeschar injections of D4B at 200 microg or 0.25 mL of placebo per mouse at 2 and 24 hours after injury. MAIN OUTCOME MEASURES: Animal survival after thermal burn wound bacterial infection, circulating leukocyte numbers, in vitro clonal cell culture of granulocyte-macrophage progenitor cells, and wound histopathological analysis. RESULTS: The survival rate in the D4B-treated group was nearly 2-fold greater than that in controls (P<.01) during 14 days of study. Bacterial quantitative wound cultures disclosed significant reductions in bacterial numbers at days 1, 2, and 3 in D4B-treated animals as compared with controls (P<.05 to <.01). D4B induced a dose-dependent inhibition of bacterial cell growth when added to in vitro P aeruginosa cultures (P<.01). Granulocyte-macrophage progenitor cell growth in culture was not altered by D4B treatment. D4B-treated animals displayed no signs of toxic effects or impairment in wound healing. CONCLUSIONS: The peptidyl membrane interactive molecule D4B had the ability to improve survival after gram-negative burn wound sepsis via direct antimicrobial effects. Peptidyl membrane interactive molecules may offer the potential of alternative treatments to standard topical agents or in patients with drug-resistant microbes.

Prostaglandin E2 alterations during sepsis are partially mediated by endotoxin-induced inhibition of prostaglandin 15-hydroxydehydrogenase.

Prostaglandin E2 (PGE2) is significantly elevated in the plasma of septic or injured patients and is thought to be a component of the resultant immune suppression associated with augmented rates of infection and mortality. Many studies have examined the effect of burn injury and sepsis on PGE2 synthesis. However, the effect of sepsis or burn injury on the expression of prostaglandin 15-hydroxydehydrogenase (PGDH), the key enzyme responsible for PGE2 degradation, has not been explored. The aim of this study was to examine the effect of endotoxin treatment and/or burn injury on the expression of PGDH. Male BDF1 mice were assigned to four groups (n = 4/group): sham, lipopolysaccharide (LPS) (2.5 mg/kg, Escherichia coli LPS, i.p.), burn (15% body surface area scald injury), and burn + LPS (15% body surface area + 2.5 mg/kg LPS, i.p.). Lung tissue was harvested at specific time points after treatment and subsequently was processed for total RNA and protein. Northern and Western blot analyses were used to examine differences in PGDH protein and mRNA expression. Total RNA was probed with the riboprobe for murine PGDH, and the 100,000 g protein fraction was immunoblotted using an rabbit antimurine PGDH antibody. PGDH was expressed in lung at t = 0 in both the saline and LPS-treated animals. A decrease in mRNA expression was initially observed at 2 hours after LPS treatment. The decrease was also significant (p < 0.05) at 3 hours after LPS and maximal decrease in mRNA and protein expression was observed at 6 hours. At 24 hours after LPS administration, the PGDH mRNA and protein expression was still significantly depressed to 49% of control expression. PGDH expression was similar and not statistically different in both burn and burn + LPS treatment at t = 0. At 2 hours after LPS, PGDH mRNA expression in the burn + LPS treatment group had significantly decreased to 47% in comparison with the burn alone group. Maximal decrease in PGDH mRNA and protein expression in lung from burn + LPS was observed at 6 hours after LPS treatment. This change represents a 73% decrease in mRNA in comparison with the time-matched burn control. At 24 hours after LPS administration, PGDH mRNA but not protein expression in the lung from burn + LPS treated mice was still significantly decreased. In summary, LPS treatment alters PGDH mRNA expression at the transcriptional and protein levels. Consequently, sepsis-induced increases in PGE2 levels may not be only due to increased PGE2 synthesis but also due to decreased PGDH expression and, hence, PGE2 degradation.

Burn wound infection-induced myeloid suppression: the role of prostaglandin E2, elevated adenylate cyclase, and cyclic adenosine monophosphate.

Suppressed granulocyte and macrophage growth after burn infection or endotoxicosis appears to be mediated by macrophage-derived products. In this study, we found that after burn, burn plus infection, or endotoxicosis, peritoneal-elicited macrophages or bone marrow cells released increased amounts of prostaglandin E2 (PGE2) and inhibited growth of granulocyte-macrophage progenitor cells (GM-CFC). PGE2, when added in culture, inhibited in vitro GM-CFC growth in a dose-dependent manner. Pretreatment of bone marrow cells with either dibutyryl cyclic adenosine monophosphate or Forskolin in vitro mimicked the PGE2 inhibition, further aggravated the inhibition induced by burn, burn plus infection, or endotoxicosis, and was not blocked by co-culture with indomethacin. Pretreatment of bone marrow cells with SQ22536, an adenylate cyclase inhibitor, significantly restored the suppressed GM-CFC growth found after burn, burn plus infection, or endotoxicosis. Alterations in myeloid production after burn infection appear to be related in part to the level of intracellular cyclic adenosine monophosphate for the GM-CFC and are responsive to PGE2.

Effects of chronic ethanol consumption and aging on 5-HT2A receptors and 5-HT reuptake sites.

The serotonergic system in brain is adversely affected by both aging and chronic ethanol consumption. The present study examined the combined effects of aging and chronic ethanol consumption on two components of the serotonergic system. Serotonin (5-HT) reuptake sites and 5-HT2A receptors were quantitated in brain areas of 5-, 14-, and 24-month-old male Fischer 344 rats that were pair-fed a control or 6.6% (v/v) ethanol-containing liquid diet on a chronic basis. The regions examined include those containing the cell bodies and projections of serotonergic neurons. These experiments demonstrated the sensitivity of the serotonergic system of male Fischer 344 rats to both aging and chronic ethanol consumption. In control rats, aging was associated with a decline in the concentration of 5-HT2A receptors in the nucleus accumbens and four cortical regions: frontal, parietal, piriform, and cingulate cortex. 5-HT2A receptors were also reduced in the frontal, parietal, and cingulate cortex of aged ethanol-fed rats. In contrast, 5-HT reuptake sites were increased in older rats in the frontal cortex, nucleus accumbens, amygdala, and CA3 region of the hippocampus. If comparable changes in 5-HT2A receptors and 5-HT reuptake sites occur in elderly humans, they may contribute to ethanol consumption, and lead to cognitive and other age-related problems. These changes may also alter the effectiveness of serotonergic drugs used in the treatment of alcoholism and mental disorders. The effects of chronic ethanol consumption were more limited. The only significant ethanol effect was an increase of 5-HT2A receptors in the nucleus accumbens of 5-month-old ethanol-fed rats.

Effects of chronic alcohol consumption and aging on dopamine D1 receptors in Fischer 344 rats.

The present study examined the hypothesis that chronic alcoholism augments the age-related loss of dopamine D1 receptors. This hypothesis was investigated because previous studies reported that both aging and chronic alcoholism produce significant changes in dopaminergic systems, and because chronic alcoholism potentiates some age-related CNS losses. In addition, this study investigated the effects of aging on D1 receptors in animals 1 and 7 days after withdrawal from chronic ethanol. Quantitative autoradiography was used to measure [3H]SCH 23390 binding to D1 receptors in brain areas associated with both the nigrostriatal and mesocorticolimbic dopamine systems. Receptors were assessed in 5-, 14-, and 24-month-old male Fischer 344 rats that were pair-fed a control or 6.6% (v/v) ethanol-containing liquid diet for 6 weeks. The results of these studies demonstrated that aging is associated with a significant decline in D1 receptors in the rostral and caudal striatum, and substantia nigra of both control and ethanol-fed rats. These receptor changes in the nigrostriatal system may be associated with motor abnormalities. In addition, there was an age-related decline in D1 receptors in two brain areas of the mesocorticolimbic system: the nucleus accumbens and frontal cortex. The latter findings may be important because of the involvement of this system with the rewarding properties of ethanol and other drugs of abuse. There were no age-related differences in the response of D1 receptors to ethanol withdrawal in the caudal and rostral striatum, substantia nigra, and nucleus accumbens.(ABSTRACT TRUNCATED AT 250 WORDS)