Inactivation of Escherichia coli, Salmonella enterica, and Listeria monocytogenes using the Contamination Sanitization Inspection and Disinfection (CSI-D) device.

The Contamination Sanitization Inspection and Disinfection (CSI-D) device is a handheld fluorescence-based imaging system designed to disinfect food contact surfaces using ultraviolet-C (UVC) illumination. This study aimed to determine the optimal CSI-D parameters (i.e., UVC exposure time and intensity) for the inactivation of the following foodborne bacteria plated on non-selective media: generic Escherichia coli (indicator organism) and the pathogens enterohemorrhagic E. coli, enterotoxigenic E. coli, Salmonella enterica, and Listeria monocytogenes. Each bacterial strain was spread-plated on non-selective agar and exposed to high-intensity (10 mW/cm2) or low-intensity (5 mW/cm2) UVC for 1-5 s. Control plates were not exposed to UVC. The plates were incubated overnight at 37 °C and then enumerated. Three trials for each bacterial strain were conducted. Statistical analysis was carried out to determine if there were significant differences in bacterial growth between UVC intensities and exposure times. Overall, exposure to low or high intensity for 3-5 s resulted in consistent inhibition of bacterial growth, with reductions of 99.9-100 % for E. coli, 96.8-100 % for S. enterica, and 99.2-100 % for L. monocytogenes. The 1 s exposure time showed inconsistent results, with a 66.0-100 % reduction in growth depending on the intensity and bacterial strain. When the results for all strains within each species were combined, the 3-5 s exposure times showed significantly greater (p < 0.05) growth inhibition than the 1 s exposure time. However, there were no significant differences (p > 0.05) in growth inhibition between the high and low UVC intensities. The results of this study show that, in pure culture conditions, exposure to UVC with the CSI-D device for ≥3 s is required to achieve consistent reduction of E. coli, S. enterica, and L. monocytogenes.

Phenotypic plasticity and genetic diversity shed light on endemism of rare Boechera perstellata and its potential vulnerability to climate warming.

The rapid pace of contemporary environmental change puts many species at risk, especially rare species constrained by limited capacity to adapt or migrate due to low genetic diversity and/or fitness. But the ability to acclimate can provide another way to persist through change. We compared the capacity of rare Boechera perstellata (Braun's rockcress) and widespread B. laevigata to acclimate to change. We investigated the phenotypic plasticity of growth, biomass allocation, and leaf morphology of individuals of B. perstellata and B. laevigata propagated from seed collected from several populations throughout their ranges in a growth chamber experiment to assess their capacity to acclimate. Concurrently, we assessed the genetic diversity of sampled populations using 17 microsatellite loci to assess evolutionary potential. Plasticity was limited in both rare B. perstellata and widespread B. laevigata, but differences in the plasticity of root traits between species suggest that B. perstellata may have less capacity to acclimate to change. In contrast to its widespread congener, B. perstellata exhibited no plasticity in response to temperature and weaker plastic responses to water availability. As expected, B. perstellata also had lower levels of observed heterozygosity than B. laevigata at the species level, but population-level trends in diversity measures were inconsistent due to high heterogeneity among B. laevigata populations. Overall, the ability of phenotypic plasticity to broadly explain the rarity of B. perstellata versus commonness of B. laevigata is limited. However, some contextual aspects of our plasticity findings compared with its relatively low genetic variability may shed light on the narrow range and habitat associations of B. perstellata and suggest its vulnerability to climate warming due to acclimatory and evolutionary constraints.

Pan-caspase inhibition during normothermic machine perfusion of discarded livers mitigates ex situ innate immune responses.

Access to liver transplantation is limited by a significant organ shortage. The recent introduction of machine perfusion technology allows surgeons to monitor and assess ex situ liver function prior to transplantation. However, many donated organs are of inadequate quality for transplant, though opportunities exist to rehabilitate organ function with adjunct therapeutics during normothermic machine perfusion. In this preclinical study, we targeted the apoptosis pathway as a potential method of improving hepatocellular function. Treatment of discarded human livers during normothermic perfusion with an irreversible pan-caspase inhibitor, emricasan, resulted in significant mitigation of innate immune and pro-inflammatory responses at both the transcriptional and protein level. This was evidenced by significantly decreased circulating levels of the pro-inflammatory cytokines, interleukin-6, interleukin-8, and interferon-gamma, compared to control livers. Compared to emricasan-treated livers, untreated livers demonstrated transcriptional changes notable for enrichment in pathways involved in innate immunity, leukocyte migration, and cytokine-mediated signaling. Targeting of unregulated apoptosis may represent a viable therapeutic intervention for immunomodulation during machine perfusion.

Eco-evolutionary causes and consequences of rarity in plants: a meta-analysis.

Species differ dramatically in their prevalence in the natural world, with many species characterized as rare due to restricted geographic distribution, low local abundance and/or habitat specialization. We investigated the ecoevolutionary causes and consequences of rarity with phylogenetically controlled metaanalyses of population genetic diversity, fitness and functional traits in rare and common congeneric plant species. Our syntheses included 252 rare species and 267 common congeners reported in 153 peer-reviewed articles published from 1978 to 2020 and one manuscript in press. Rare species have reduced population genetic diversity, depressed fitness and smaller reproductive structures than common congeners. Rare species also could suffer from inbreeding depression and reduced fertilization efficiency. By limiting their capacity to adapt and migrate, these characteristics could influence contemporary patterns of rarity and increase the susceptibility of rare species to rapid environmental change. We recommend that future studies present more nuanced data on the extent of rarity in focal species, expose rare and common species to ecologically relevant treatments, including reciprocal transplants, and conduct quantitative genetic and population genomic analyses across a greater array of systems. This research could elucidate the processes that contribute to rarity and generate robust predictions of extinction risks under global change.

Point-of-Care Ultrasound on Management of Cellulitis Versus Local Angioedema in the Pediatric Emergency Department.

OBJECTIVES: To evaluate whether ultrasound can differentiate between cellulitis and angioedema from insect bites in pediatric patients. METHODS: A prospective, pre-post study in an urban pediatric emergency department of patients younger than 21 years with soft tissue swelling from insect bites without abscesses were enrolled. Treating physician's pretest opinions regarding the diagnosis and need for antibiotics were determined. Ultrasound of the affected areas was performed, and effects on management were recorded. Further imaging, medications, and disposition were at the discretion of the enrolling physician. Phone call follow-ups were made within a week of presentation. RESULTS: Among 103 patients enrolled with soft tissue swelling secondary to insect bites, ultrasound changed the management in 27 (26%) patients (95% confidence interval [CI], 18-35%). Of the patients who were indeterminate or believed to require antibiotics, ultrasound changed management in 6 (23%) of 26 patients (95% CI, 6%-40%). In those patients who were believed not to require antibiotics, ultrasound changed management in 12 (16%) 77 patients (95% CI, 7%-24%). Patients with diagnosis of local angioedema achieved symptom resolution 1.4 days sooner than patients diagnosed with cellulitis (mean, -1.389; 95% CI, -2.087 to -0.690; P < 0.001). No patient who was initially diagnosed as local angioedema received antibiotics upon patient follow-up. CONCLUSIONS: Point-of-care ultrasound changed physician management in 1 of 4 patients in the pediatric emergency department with soft tissue swelling secondary to insect bites. Ultrasound may guide the management in these patients and lead to improved antibiotic stewardship in conjunction with history and physical examination.

Activation of autophagy during normothermic machine perfusion of discarded livers is associated with improved hepatocellular function.

Liver transplantation is hampered by a severe shortage of donor organs. Normothermic machine perfusion (NMP) of donor livers allows dynamic preservation in addition to viability assessment before transplantation. Little is known about the injury and repair mechanisms induced during NMP. To investigate these mechanisms, we examined gene and protein expression changes in a cohort of discarded human livers, stratified by hepatocellular function, during NMP. Six human livers acquired through donation after circulatory death (DCD) underwent 12 h of NMP. Of the six livers, three met predefined criteria for adequate hepatocellular function. We applied transcriptomic profiling and protein analysis to evaluate temporal changes in gene expression during NMP between functional and nonfunctional livers. Principal component analysis segregated the two groups and distinguished the various perfusion time points. Transcriptomic analysis of biopsies from functional livers indicated robust activation of innate immunity after 3 h of NMP followed by enrichment of prorepair and prosurvival mechanisms. Nonfunctional livers demonstrated delayed and persistent enrichment of markers of innate immunity. Functional livers demonstrated effective induction of autophagy, a cellular repair and homeostasis pathway, in contrast to nonfunctional livers. In conclusion, NMP of discarded DCD human livers results in innate immune-mediated injury, while also activating autophagy, a presumed mechanism for support of cellular repair. More pronounced activation of autophagy was seen in livers that demonstrated adequate hepatocellular function.NEW & NOTEWORTHY We demonstrate that ischemia-reperfusion injury occurs in all livers during NMP, though there are notable differences in gene expression between functional and nonfunctional livers. We further demonstrate that activation of the liver's repair and homeostasis mechanisms through autophagy plays a vital role in the graft's response to injury and may impact liver function. These findings indicate that liver autophagy might be a key therapeutic target for rehabilitating the function of severely injured or untransplantable livers.

Seed Cryopreservation and Germination of Rhus glabra and the Critically Endangered Species Rhus michauxii.

Rhus michauxii is a perennial rhizomatous shrub native to the southeastern United States that is found mainly in sunny, dry, open rocky or sandy woodlands. Moreover, it is found on ridges or river bluffs in the inner coastal plane and lower piedmont of Virginia, Georgia, and the Carolinas. Habitat conversion to agriculture, suppression of fires, and low reproduction have caused R. michauxii to become rare and it is now federally listed as threatened. Methods are needed to multiply and conserve R. michauxii. Protocols were developed for seed cryopreservation, in vitro germination, and micropropagation for R. glabra and R. michauxii. Seed scarification in concentrated sulfuric acid for 6 h and germination on ½ MS medium resulted in germination up to 96% for control and cryopreserved seeds of R. glabra and 70 and 40% for control and cryopreserved seeds of R. michauxii. Shortly after germination in vitro, young seedlings were established in a greenhouse potting mix providing new plants from the endemic Georgia R. michauxii populations. Several of the findings meet goals within the R. michauxii recovery plan by providing methods for sexual and asexual multiplication and long-term seed storage under cryogenic conditions. The protocols developed will assist in the safeguarding and conservation of dwindling natural R. michauxii populations.

A subset of CB002 xanthine analogs bypass p53-signaling to restore a p53 transcriptome and target an S-phase cell cycle checkpoint in tumors with mutated-p53.

Mutations in TP53 occur commonly in the majority of human tumors and confer aggressive tumor phenotypes, including metastasis and therapy resistance. CB002 and structural-analogs restore p53 signaling in tumors with mutant-p53 but we find that unlike other xanthines such as caffeine, pentoxifylline, and theophylline, they do not deregulate the G2 checkpoint. Novel CB002-analogs induce pro-apoptotic Noxa protein in an ATF3/4-dependent manner, whereas caffeine, pentoxifylline, and theophylline do not. By contrast to caffeine, CB002-analogs target an S-phase checkpoint associated with increased p-RPA/RPA2, p-ATR, decreased Cyclin A, p-histone H3 expression, and downregulation of essential proteins in DNA-synthesis and DNA-repair. CB002-analog #4 enhances cell death, and decreases Ki-67 in patient-derived tumor-organoids without toxicity to normal human cells. Preliminary in vivo studies demonstrate anti-tumor efficacy in mice. Thus, a novel class of anti-cancer drugs shows the activation of p53 pathway signaling in tumors with mutated p53, and targets an S-phase checkpoint.

Mitochondrial genotype alters the impact of rapamycin on the transcriptional response to nutrients in Drosophila.

BACKGROUND: In addition to their well characterized role in cellular energy production, new evidence has revealed the involvement of mitochondria in diverse signaling pathways that regulate a broad array of cellular functions. The mitochondrial genome (mtDNA) encodes essential components of the oxidative phosphorylation (OXPHOS) pathway whose expression must be coordinated with the components transcribed from the nuclear genome. Mitochondrial dysfunction is associated with disorders including cancer and neurodegenerative diseases, yet the role of the complex interactions between the mitochondrial and nuclear genomes are poorly understood. RESULTS: Using a Drosophila model in which alternative mtDNAs are present on a common nuclear background, we studied the effects of this altered mitonuclear communication on the transcriptomic response to altered nutrient status. Adult flies with the 'native' and 'disrupted' genotypes were re-fed following brief starvation, with or without exposure to rapamycin, the cognate inhibitor of the nutrient-sensing target of rapamycin (TOR). RNAseq showed that alternative mtDNA genotypes affect the temporal transcriptional response to nutrients in a rapamycin-dependent manner. Pathways most greatly affected were OXPHOS, protein metabolism and fatty acid metabolism. A distinct set of testis-specific genes was also differentially regulated in the experiment. CONCLUSIONS: Many of the differentially expressed genes between alternative mitonuclear genotypes have no direct interaction with mtDNA gene products, suggesting that the mtDNA genotype contributes to retrograde signaling from mitochondria to the nucleus. The interaction of mitochondrial genotype (mtDNA) with rapamycin treatment identifies new links between mitochondria and the nutrient-sensing mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway.

Supraglottic airway devices for pediatric airway management in the emergency department.

Endotracheal intubation can be difficult in the emergent situation, and it is important to have an appropriate backup strategy. Supraglottic airway devices have provided an alternative method for pediatric airway management that is relatively easy to learn, with a high success rate. This issue reviews the use of supraglottic airway devices in pediatric patients including common devices, indications and techniques for placement, and complications associated with their use. The use of supraglottic airway devices in the patient with a difficult airway is also discussed.

PuHox52-mediated hierarchical multilayered gene regulatory network promotes adventitious root formation in Populus ussuriensis.

Adventitious root (AR) formation is critically important in vegetative propagation through cuttings in some plants, especially woody species. However, the underlying molecular mechanisms remain elusive. Here, we report the identification of a poplar homeobox gene, PuHox52, which was induced rapidly (within 15 min) at the basal ends of stems upon cutting and played a key regulatory role in adventitious rooting. We demonstrated that overexpression of PuHox52 significantly increased the number of ARs while suppression of PuHox52 had the opposite effect. A multilayered hierarchical gene regulatory network (ML-hGRN) mediated by PuHox52 was reverse-engineered and demonstrated to govern AR formation. PuHox52 regulated AR formation through upregulation of nine hub regulators, including a jasmonate signaling pathway gene, PuMYC2, and an auxin signaling pathway gene, PuAGL12. We also identified coherent type 4 feed-forward loops within this ML-hGRN; PuHox52 repressed PuHDA9, which encodes a histone deacetylase, and led to an increase in acetylation and presumably expression of three hub regulators, PuWRKY51, PuLBD21 and PuIAA7. Our results indicate that the ML-hGRN mediated by PuHox52 governs AR formation at the basal ends of stem cuttings from poplar trees.

Features of COVID-19 post-infectious cytokine release syndrome in children presenting to the emergency department.

The 2019 coronavirus disease (COVID-19) has not appeared to affect children as severely as adults. However, approximately 1 month after the COVID-19 peak in New York City in April 2020, cases of children with prolonged fevers abruptly developing inflammatory shock-like states have been reported in Western Europe and the United States. This case series describes four previously healthy children with COVID-19 infection confirmed by serologic antibody testing, but negative by nasopharyngeal RT-PCR swab, presenting to the Pediatric Emergency Department (PED) with prolonged fever (5 or more days) and abrupt onset of hemodynamic instability with elevated serologic inflammatory markers and cytokine levels (IL-6, IL-8 and TNF-α). Emergency physicians must maintain a high clinical suspicion for this COVID-19 associated post-infectious cytokine release syndrome, with features that overlap with Kawasaki Disease (KD) and Toxic Shock Syndrome (TSS) in children with recent or current COVID-19 infection, as patients can decompensate quickly.

Emergency department management of pediatric septic arthritis and osteomyelitis

Septic arthritis and osteomyelitis in pediatric patients represent true emergencies, and can quickly threaten life and limb. A high index of suspicion should be maintained, as these conditions often present with a subacute course of illness and vague signs and symptoms. Septic arthritis and osteomyelitis can occur concurrently, so suspicion for one should also prompt investigation for the other. The diagnostic evaluation should include blood work as well as samples from the infected joint or bone for culture. Management with antibiotics is a standard approach, but the duration of antibiotic therapy is controversial. This issue reviews the current literature and provides an evidence-based approach for the evaluation and management of pediatric patients with septic arthritis and osteomyelitis.

Distribution and Community Assembly of Trees Along an Andean Elevational Gradient.

Highlighting patterns of distribution and assembly of plants involves the use of community phylogenetic analyses and complementary traditional taxonomic metrics. However, these patterns are often unknown or in dispute, particularly along elevational gradients, with studies finding different patterns based on elevation. We investigated how patterns of tree diversity and structure change along an elevation gradient using taxonomic and phylogenetic diversity metrics. We sampled 595 individuals (36 families; 53 genera; 88 species) across 15 plots along an elevational gradient (2440-3330 m) in Ecuador. Seventy species were sequenced for the rbcL and matK gene regions to generate a phylogeny. Species richness, Shannon-Weaver diversity, Simpson's Dominance, Simpson's Evenness, phylogenetic diversity (PD), mean pairwise distance (MPD), and mean nearest taxon distance (MNTD) were evaluated for each plot. Values were correlated with elevation and standardized effect sizes (SES) of MPD and MNTD were generated, including and excluding tree fern species, for comparisons across elevation. Taxonomic and phylogenetic metrics found that species diversity decreases with elevation. We also found that overall the community has a non-random phylogenetic structure, dependent on the presence of tree ferns, with stronger phylogenetic clustering at high elevations. Combined, this evidence supports the ideas that tree ferns have converged with angiosperms to occupy the same habitat and that an increased filtering of clades has led to more closely related angiosperm species at higher elevations.

Characterization of functional relationships of R-loops with gene transcription and epigenetic modifications in rice.

We conducted genome-wide identification of R-loops followed by integrative analyses of R-loops with relation to gene expression and epigenetic signatures in the rice genome. We found that the correlation between gene expression levels and profiled R-loop peak levels was dependent on the positions of R-loops within gene structures (hereafter named "genic position"). Both antisense only (ASO)-R-loops and sense/antisense (S/AS)-R-loops sharply peaked around transcription start sites (TSSs), and these peak levels corresponded positively with transcript levels of overlapping genes. In contrast, sense only (SO)-R-loops were generally spread over the coding regions, and their peak levels corresponded inversely to transcript levels of overlapping genes. In addition, integrative analyses of R-loop data with existing RNA-seq, chromatin immunoprecipitation sequencing (ChIP-seq), DNase I hypersensitive sites sequencing (DNase-seq), and whole-genome bisulfite sequencing (WGBS or BS-seq) data revealed interrelationships and intricate connections among R-loops, gene expression, and epigenetic signatures. Experimental validation provided evidence that the demethylation of both DNA and histone marks can influence R-loop peak levels on a genome-wide scale. This is the first study in plants that reveals novel functional aspects of R-loops, their interrelations with epigenetic methylation, and roles in transcriptional regulation.

SLC1A5 glutamine transporter is a target of MYC and mediates reduced mTORC1 signaling and increased fatty acid oxidation in long-lived Myc hypomorphic mice.

Mice that express reduced levels of the c-Myc gene (Myc+/- heterozygotes) are long-lived. Myc hypomorphic mice display reduced rates of protein translation and decreased activity of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Given the prominent effect of mTOR on aging, lower mTORC1 activity could contribute to the exceptional longevity and enhanced healthspan of Myc+/- animals. However, given the downstream position of MYC in these signaling cascades, the mechanism through which mTORC1 activity is downregulated in Myc+/- mice is not understood. We report that the high-affinity glutamine transporter SLC1A5, which is critical for activation of mTORC1 activity by amino acids, is a transcriptional target of MYC. Myc+/- cells display decreased Slc1a5 gene expression that leads to lower glutamine uptake and consequently reduced mTORC1 activity. Decreased mTORC1 activity in turn mediates an elevation of fatty acid oxidation (FAO) by indirectly upregulating the expression of carnitine palmitoyltransferase 1a (Cpt1a) that mediates the rate-limiting step of ß-oxidation. Increased FAO has been noted in a number of long-lived mouse models. Taken together, our results show that transcriptional feedback loops regulated by MYC modulate upstream signaling pathways such as mTOR and impact FAO on an organismal level.

Transcriptional changes during hepatic ischemia-reperfusion in the rat.

There are few effective targeted strategies to reduce hepatic ischemia-reperfusion (IR) injury, a contributor to poor outcomes in liver transplantation recipients. It has been proposed that IR injury is driven by the generation of reactive oxygen species (ROS). However, recent studies implicate other mediators of the injury response, including mitochondrial metabolic dysfunction. We examined changes in global gene expression after transient hepatic ischemia and at several early reperfusion times to identify potential targets that could be used to protect against IR injury. Male Wistar rats were subjected to 30 minutes of 70% partial warm ischemia followed by 0, 0.5, 2, or 6 hours of reperfusion. RNA was extracted from the reperfused and non-ischemic lobes at each time point for microarray analysis. Identification of differentially expressed genes and pathway analysis were used to characterize IR-induced changes in the hepatic transcriptome. Changes in the reperfused lobes were specific to the various reperfusion times. We made the unexpected observation that many of these changes were also present in tissue from the paired non-ischemic lobes. However, the earliest reperfusion time, 30 minutes, showed a marked increase in the expression of a set of immediate-early genes (c-Fos, c-Jun, Atf3, Egr1) that was exclusive to the reperfused lobe. We interpreted these results as indicating that this early response represented a tissue autonomous response to reperfusion. In contrast, the changes that occurred in both the reperfused and non-ischemic lobes were interpreted as indicating a non-autonomous response resulting from hemodynamic changes and/or circulating factors. These tissue autonomous and non-autonomous responses may serve as targets to ameliorate IR injury.