1H NMR spectroscopic characterisation of HepG2 cells as a model metabolic system for toxicology studies.

The immortalised human hepatocellular HepG2 cell line is commonly used for toxicology studies as an alternative to animal testing due to its characteristic liver-distinctive functions. However, little is known about the baseline metabolic changes within these cells upon toxin exposure. We have applied high-resolution 1H Nuclear Magnetic Resonance (NMR) spectroscopy to characterise the biochemical composition of HepG2 cells at baseline and post-exposure to hydrogen peroxide (H2O2). Metabolic profiles of live cells, cell extracts, and their spent media supernatants were obtained using 1H high-resolution magic angle spinning (HR-MAS) NMR and 1H NMR spectroscopic techniques. Orthogonal partial least squares discriminant analysis (O-PLS-DA) was used to characterise the metabolites that differed between the baseline and H2O2 treated groups. The results showed that H2O2 caused alterations to 10 metabolites, including acetate, glutamate, lipids, phosphocholine, and creatine in the live cells; 25 metabolites, including acetate, alanine, adenosine diphosphate (ADP), aspartate, citrate, creatine, glucose, glutamine, glutathione, and lactate in the cell extracts, and 22 metabolites, including acetate, alanine, formate, glucose, pyruvate, phenylalanine, threonine, tryptophan, tyrosine, and valine in the cell supernatants. At least 10 biochemical pathways associated with these metabolites were disrupted upon toxin exposure, including those involved in energy, lipid, and amino acid metabolism. Our findings illustrate the ability of NMR-based metabolic profiling of immortalised human cells to detect metabolic effects on central metabolism due to toxin exposure. The established data sets will enable more subtle biochemical changes in the HepG2 model cell system to be identified in future toxicity testing.

Welfare Implications for Tigers in Travelling Circuses.

There are very few studies that have focused on species-specific welfare implications for tigers in a travelling circus. The absence of scientific evidence to inform nationwide legislation means that tigers are still commonly used in travelling circuses across the world. A systematic review of relevant published studies was conducted using the bibliographic databases Web of Science and Scopus, supplemented by a narrative search. In total, 42 relevant studies were identified that assessed the welfare of tigers in captivity, including circuses and zoos. Only eight papers assessed the welfare implications for tigers in circuses directly, evidencing the lack of research in this area. Given that circuses provide a sub-optimal environment compared to zoos, implications for tiger welfare were also inferred from zoo research, within the Five Domains framework. Collectively, these papers infer that the travelling nature of a circus often negatively impacts the welfare domains of nutrition, physical environment, health, and mental state. This is due to limitations in enclosure size, as well as in both environmental and behavioural enrichment. There is also often difficulty in sourcing appropriate food and specialised routine veterinary care. The literature is divided concerning behavioural interactions, specifically whether training can improve welfare by offering mental stimulation. However, circus performances are often associated with negative welfare due to noise disruption from spectators. The collective scientific evidence indicates that tigers are not well suited to circus living, due to the inability of a travelling circus to provide for their species-specific psychological, physiological, and behavioural needs.

Modifications to therapeutic plasma exchange to achieve rapid exchange on cardiopulmonary bypass prior to pediatric cardiac transplant.

BACKGROUND: Cardiac transplants increasingly occur following placement of ventricular assist devices (VADs). A strong association exists between human leukocyte antigen (HLA) sensitization and VAD placement; however, desensitization protocols that utilize therapeutic plasma exchange (TPE) are fraught with technical challenges and are at increased risk of adverse events. In response to increased VAD utilization in our pre-transplant population, we developed a new institutional standard for TPE in the operating room. METHODS: Through a multidisciplinary effort, we developed an institutional protocol for intraoperative TPE immediately prior to cardiac transplantation after cannulation onto cardiopulmonary bypass (CPB). All procedures used the standard TPE protocol on the Terumo Optia (Terumo BCT, Lakewood, CO, USA), but incorporated multiple modifications to limit patients' bypass times, and to coordinate with the surgical teams. These modifications included deliberate misidentification of replacement fluid and maximization of the citrate infusion rate. RESULTS: These adjustments allowed the machine to run at maximal inlet speeds, minimizing duration of TPE. To date, 11 patients have been treated with this protocol. All survived their cardiac transplantation operation. Hypocalcemia and hypotension were noted; however, none of these adverse events appeared to have clinical impact. Technical complications included unexpected fibrin deposition in the TPE circuit and air in the inlet line due to surgical manipulation of the CPB cannula. No thromboembolic complications occurred in any patient. CONCLUSION: We feel that this procedure can be rapidly and safely performed in HLA sensitized pediatric patients on CPB to limit the risk of antibody mediated rejection of their heart transplant.

Collateral Impacts of the COVID-19 Pandemic: The New York City Experience.

OBJECTIVE: To adapt an existing surveillance system to monitor the collateral impacts of the COVID-19 pandemic on health outcomes in New York City across 6 domains: access to care, chronic disease, sexual/reproductive health, food/economic insecurity, mental/behavioral health, and environmental health. DESIGN: Epidemiologic assessment. Public health surveillance system. SETTING: New York City. PARTICIPANTS: New York City residents. MAIN OUTCOME MEASURES: We monitored approximately 30 indicators, compiling data from 2006 to 2022. Sources of data include clinic visits, surveillance surveys, vital statistics, emergency department visits, lead and diabetes registries, Medicaid claims, and public benefit enrollment. RESULTS: We observed disruptions across most indicators including more than 50% decrease in emergency department usage early in the pandemic, which rebounded to prepandemic levels by late 2021, changes in reporting levels of probable anxiety and depression, and worsening birth outcomes for mothers who identified as Asian/Pacific Islander or Black. Data are processed in SAS and analyzed using the R Surveillance package to detect possible inflections. Data are updated monthly to an internal Tableau Dashboard and shared with agency leadership. CONCLUSIONS: As the COVID-19 pandemic continues into its third year, public health priorities are returning to addressing non-COVID-19-related diseases and conditions, their collateral impacts, and postpandemic recovery needs. Substantial work is needed to return even to a suboptimal baseline across multiple health topic areas. Our surveillance framework offers a valuable starting place to effectively allocate resources, develop interventions, and issue public communications.

Atopic Polygenic Risk Score Is Associated with Paradoxical Eczema Developing in Patients with Psoriasis Treated with Biologics.

Biologic therapies for psoriasis can cause paradoxical eczema. The role of genetic factors in its pathogenesis is unknown. To identify risk variants, we conducted a GWAS of 3,212 patients with psoriasis, of whom 88 developed paradoxical eczema. Two lead SNPs reached genome-wide significance (P ≤ 5 × 10-8) for association with paradoxical eczema: rs192705221 (near UNC5B, P = 9.52 × 10-10) and rs72925168 (within SLC1A2, P = 1.66 × 10-9). Genome-wide significant SNPs from published GWAS were used to generate polygenic risk scores (PRSs) for atopic eczema, general atopic disease, or a combination, which were tested for association with paradoxical eczema. Improvement over a clinical risk model was assessed by the area under the curve. All three atopy polygenic risk scores were associated with paradoxical eczema (P < 0.05); polygenic risk score for a combination of atopic eczema and general atopic disease had the strongest association (OR = 1.83, 95% CI = 1.17-2.84, P = 0.0078). Including atopic polygenic risk scores in the multivariable model, which included age, sex, atopic background, and psoriatic arthritis history, increased the area under the curve from 0.671 to 0.681-0.686. Atopic genetic burden is associated with paradoxical eczema occurring in biologic-treated patients with psoriasis, indicating shared underlying mechanisms. Incorporating genetic risk may improve treatment outcome prediction models for psoriasis.

Does kindergarten instruction matter for sustaining the prekindergarten (PreK) boost? Evidence from individual- and classroom-level survey and observational data.

The sustaining environments hypothesis theorizes that the lasting effects of PreK programs are contingent on the quality of the subsequent learning environment in early elementary school. The current study tests this theory by leveraging data from students (N = 462) who did and did not enroll in the Boston Public Schools (BPS) prekindergarten (PreK) program as well as features of their kindergarten instruction measured at the child- and classroom-levels using surveys and observations. Taken together, findings revealed limited evidence for the sustaining environments hypothesis. The bulk of the results were null, indicating that in general, associations between enrollment in BPS PreK and language, literacy, and math skills through the spring of kindergarten did not vary by kindergarten instructional experiences. When examining distinct types of instructional experiences, there were some inklings that child-level observational measures of kindergarten learning experiences-particularly those capturing constrained versus unconstrained instruction-were more predictive of PreK persistence than observed global classroom quality measures or survey-based measures of advanced instruction. However, these associations were not always specific to outcomes matching the content delivered during this instruction (math vs. literacy), consistent with the possibility of either cross-domain effects or that instructional variables are proxies for more general instructional practices. Findings for future research and theory are discussed. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Creating a More Perfect Union: Modeling Intestinal Bacteria-Epithelial Interactions Using Organoids.

Intestinal organoids have become indispensable tools for many gastrointestinal researchers, advancing their studies of nontransformed intestinal epithelial cells, and their roles in an array of diseases, including inflammatory bowel disease and colon cancer. In many cases. these diseases, as well as many enteric infections, reflect pathogenic interactions between bacteria and the gut epithelium. The complexity of studying this microbe-epithelial interface in vivo has led to significant focus on modeling this cross-talk using organoid models. Considering how quickly the organoid field is advancing, it can be difficult to keep up to date with the latest techniques, as well as their respective strengths and weaknesses. This review addresses the advantages of using organoids derived from adult stem cells and the recently identified differences that biopsy location and patient age can have on organoids and their interactions with microbes. Several approaches to introducing bacteria in a relevant (apical) manner (ie, microinjecting 3-dimensional spheroids, polarity-reversed organoids, and 2-dimensional monolayers) also are addressed, as are the key readouts that can be obtained using these models. Lastly, the potential for new approaches, such as air-liquid interface, to facilitate studying bacterial interactions with important but understudied epithelial subsets such as goblet cells and their products, is evaluated.

Evolutionary diversification of Pseudomonas aeruginosa in an artificial sputum model.

BACKGROUND: During chronic lung infections of cystic fibrosis patients Pseudomonas aeruginosa populations undergo extensive evolutionary diversification. However, the selective drivers of this evolutionary process are poorly understood. To test the effects of temperate phages on diversification in P. aeruginosa biofilms we experimentally evolved populations of P. aeruginosa for approximately 240 generations in artificial sputum medium with or without a community of three temperate phages. RESULTS: Analysis of end-point populations using a suite of phenotypic tests revealed extensive phenotypic diversification within populations, but no significant differences between the populations evolved with or without phages. The most common phenotypic variant observed was loss of all three types of motility (swimming, swarming and twitching) and resistance to all three phages. Despite the absence of selective pressure, some members of the population evolved antibiotic resistance. The frequency of antibiotic resistant isolates varied according to population and the antibiotic tested. However, resistance to ceftazidime and tazobactam-piperacillin was observed more frequently than resistance to other antibiotics, and was associated with higher prevelence of isolates exhibiting a hypermutable phenotype and increased beta-lactamase production. CONCLUSIONS: We observed considerable within-population phenotypic diversity in P. aeruginosa populations evolving in the artificial sputum medium biofilm model. Replicate populations evolved both in the presence and absence of phages converged upon similar sets of phenotypes. The evolved phenotypes, including antimicrobial resistance, were similar to those observed amongst clinical isolates from cystic fibrosis infections.

Temperate phages both mediate and drive adaptive evolution in pathogen biofilms.

Temperate phages drive genomic diversification in bacterial pathogens. Phage-derived sequences are more common in pathogenic than nonpathogenic taxa and are associated with changes in pathogen virulence. High abundance and mobilization of temperate phages within hosts suggests that temperate phages could promote within-host evolution of bacterial pathogens. However, their role in pathogen evolution has not been experimentally tested. We experimentally evolved replicate populations of Pseudomonas aeruginosa with or without a community of three temperate phages active in cystic fibrosis (CF) lung infections, including the transposable phage, ɸ4, which is closely related to phage D3112. Populations grew as free-floating biofilms in artificial sputum medium, mimicking sputum of CF lungs where P. aeruginosa is an important pathogen and undergoes evolutionary adaptation and diversification during chronic infection. Although bacterial populations adapted to the biofilm environment in both treatments, population genomic analysis revealed that phages altered both the trajectory and mode of evolution. Populations evolving with phages exhibited a greater degree of parallel evolution and faster selective sweeps than populations without phages. Phage ɸ4 integrated randomly into the bacterial chromosome, but integrations into motility-associated genes and regulators of quorum sensing systems essential for virulence were selected in parallel, strongly suggesting that these insertional inactivation mutations were adaptive. Temperate phages, and in particular transposable phages, are therefore likely to facilitate adaptive evolution of bacterial pathogens within hosts.

The integration of BRCA testing into oncology clinics.

PURPOSE: The PARP inhibitor, Olaparib, is approved for women with BRCA-mutated ovarian cancer. Therefore there is an urgent need to test patients and obtain results in time to influence treatment. Models of BRCA testing, such as the mainstreaming oncogenetic pathway, involving oncology health professionals are being used. The authors report on the establishment of the extended role of the clinical nurse specialist in consenting women for BRCA testing in routine gynaecology-oncology clinics using the mainstreaming model. METHODS: Nurses undertook generic consent training and specific counselling training for BRCA testing in the form of a series of online videos, written materials and checklists before obtaining approval to consent patients for germline BRCA1 and BRCA2 mutations. RESULTS: Between July 2013 and December 2015, 108 women with ovarian cancer were counselled and consented by nurses in the medical oncology clinics at a single centre (The Royal Marsden, UK). This represented 36% of all ovarian cancer patients offered BRCA testing in the oncology clinics at the centre. Feedback from patients and nurses was encouraging with no significant issues raised in the counselling and consenting process. CONCLUSION: The mainstreaming model allows for greater access to BRCA testing for ovarian cancer patients, many of whom may benefit from personalised therapy (PARP inhibitors). This is the first report of oncology nurses in the BRCA testing pathway. Specialist oncology nurses trained in BRCA testing have an important role within a multidisciplinary team counselling and consenting patients to undergo BRCA testing.

Temperate phages enhance pathogen fitness in chronic lung infection.

The Liverpool Epidemic Strain (LES) is a polylysogenic, transmissible strain of Pseudomonas aeruginosa, capable of superinfecting existing P. aeruginosa respiratory infections in individuals with cystic fibrosis (CF). The LES phages are highly active in the CF lung and may have a role in the competitiveness of the LES in vivo. In this study, we tested this by competing isogenic PAO1 strains that differed only by the presence or absence of LES prophages in a rat model of chronic lung infection. Lysogens invaded phage-susceptible populations, both in head-to-head competition and when invading from rare, in the spatially structured, heterogeneous lung environment. Appreciable densities of free phages in lung tissue confirmed active phage lysis in vivo. Moreover, we observed lysogenic conversion of the phage-susceptible competitor. These results suggest that temperate phages may have an important role in the competitiveness of the LES in chronic lung infection by acting as anti-competitor weapons.

The role of temperate bacteriophages in bacterial infection.

Bacteriophages are viruses that infect bacteria. There are an estimated 10(31) phage on the planet, making them the most abundant form of life. We are rapidly approaching the centenary of their identification, and yet still have only a limited understanding of their role in the ecology and evolution of bacterial populations. Temperate prophage carriage is often associated with increased bacterial virulence. The rise in use of technologies, such as genome sequencing and transcriptomics, has highlighted more subtle ways in which prophages contribute to pathogenicity. This review discusses the current knowledge of the multifaceted effects that phage can exert on their hosts and how this may contribute to bacterial adaptation during infection.

Safety of topical corticosteroids in pregnancy.

BACKGROUND: Topical corticosteroids are the most frequently prescribed dermatological treatment and are often used by pregnant women with skin conditions. However, little is known about their safety in pregnancy. OBJECTIVES: To assess the effects of topical corticosteroids on pregnancy outcomes in pregnant women. SEARCH METHODS: This is an update of a review previously published in 2009. We updated our searches of the following databases to July 2015: the Cochrane Skin Group Specialised Register, the Cochrane Pregnancy and Childbirth Group Specialised Register, the Cochrane Central Register of Controlled Trials (CENTRAL) (2015, Issue 6), MEDLINE, EMBASE, and LILACS. We also searched five trials registers and checked the reference lists of included studies, published reviews, articles that had cited the included studies, and one author's literature collection, for further references to relevant RCTs. SELECTION CRITERIA: Randomised controlled trials and cohort studies of topical corticosteroids in pregnant women, as well as case-control studies comparing maternal exposure to topical corticosteroids between cases and controls when studies reported pre-specified outcomes. The primary outcomes included mode of delivery, major congenital abnormality, birth weight, and preterm delivery (delivery before 37 completed weeks gestation); the secondary outcomes included foetal death, minor congenital abnormality, and low Apgar score (less than seven at 5 min). DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. Two authors independently applied selection criteria, extracted data, and assessed the quality of the included studies. A third author was available for resolving differences of opinion. A further author independently extracted data from included studies that were conducted by authors of this systematic review. MAIN RESULTS: We included 7 new observational studies in this update, bringing the total number to 14, including 5 cohort and 9 case-control studies, with 1,601,515 study subjects.Most studies found no causal associations between maternal exposure to topical corticosteroids of any potency and pregnancy outcomes when compared with no exposure. These outcomes included: mode of delivery (risk ratio (RR) 1.04, 95% confidence interval (CI) 0.95 to 1.15, 1 cohort study, n = 9904, low quality evidence); congenital abnormalities, including orofacial cleft or cleft palate and hypospadias (where the urethral opening is on the underside of the penis) (RR 0.82, 95% CI 0.34 to 1.96, 2 cohort studies, n = 9512, low quality evidence; and odds ratio (OR) 1.07, 95% CI 0.71 to 1.60, 1 case-control study, n = 56,557); low birth weight (RR 1.08, 95% CI 0.86 to 1.36; n = 59,419, 4 cohort studies; very low quality evidence); preterm delivery (RR 0.93, 95% CI 0.81 to 1.08, 4 cohort studies, n = 59,419, low quality evidence); foetal death (RR 1.02, 95% CI 0.60 to 1.73, 4 cohort studies, n = 63,885, very low quality evidence); and low Apgar score (RR 0.84, 95% CI 0.54 to 1.31, 1 cohort study, n = 9220, low quality evidence).We conducted stratified analyses of mild or moderate potency, and potent or very potent topical corticosteroids, but we found no causal associations between maternal exposure to topical corticosteroid of any potency and congenital abnormality, orofacial clefts, preterm delivery, or low Apgar score. For low birth weight, although the meta-analysis based on study-level data was not significant for either mild to moderate corticosteroids (pooled RR 0.90, 95% CI 0.74 to 1.09, 3 cohort studies, n > 55,713) or potent to very potent corticosteroids (pooled RR 1.58, 95% CI 0.96 to 2.58, 4 cohort studies, n > 47,651), there were significant differences between the two subgroups (P = 0.04). The results from three of the individual studies in the meta-analysis indicated an increased risk of low birth weight in women who received potent to very potent topical corticosteroids. Maternal use of mild to moderate potency topical steroids was associated with a decreased risk of foetal death (pooled RR 0.70, 95% CI 0.64 to 0.77, 2 studies, n = 48,749; low quality evidence), but we did not observe this effect when potent to very potent topical corticosteroids were given during pregnancy (pooled RR 1.14, 95% CI 0.69 to 1.88, 3 studies, n = 37,086, low quality evidence).We used the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) Working Group approach to rate the overall quality of the evidence. Data from observational studies started at low quality. We further downgraded the evidence because of imprecision in low birth weight and inconsistency in foetal death. Lower quality evidence resulted in lower confidence in the estimate of effect for those outcomes. AUTHORS' CONCLUSIONS: This update adds more evidence showing no causal associations between maternal exposure to topical corticosteroids of all potencies and pregnancy outcomes including mode of delivery, congenital abnormalities, preterm delivery, foetal death, and low Apgar score, which is consistent with the previous version of this review. This update provides stratified analyses based on steroid potency; we found no association between maternal use of topical corticosteroids of any potency and an increase in adverse pregnancy outcomes, including mode of delivery, congenital abnormality, preterm delivery, foetal death, and low Apgar score. Similar to the previous version of the review, this update identified a probable association between low birth weight and maternal use of potent to very potent topical corticosteroids, especially when the cumulative dosage of topical corticosteroids throughout the pregnancy is very large, which warrants further investigation. The finding of a possible protective effect of mild to moderate topical corticosteroids on foetal death could also be examined.

Lytic activity by temperate phages of Pseudomonas aeruginosa in long-term cystic fibrosis chronic lung infections.

Pseudomonas aeruginosa is the most common bacterial pathogen infecting the lungs of cystic fibrosis (CF) patients. The transmissible Liverpool epidemic strain (LES) harbours multiple inducible prophages (LESϕ2; LESϕ3; LESϕ4; LESϕ5; and LESϕ6), some of which are known to confer a competitive advantage in an in vivo rat model of chronic lung infection. We used quantitative PCR (Q-PCR) to measure the density and dynamics of all five LES phages in the sputa of 10 LES-infected CF patients over a period of 2 years. In all patients, the densities of free-LES phages were positively correlated with the densities of P. aeruginosa, and total free-phage densities consistently exceeded bacterial host densities 10-100-fold. Further, we observed a negative correlation between the phage-to-bacterium ratio and bacterial density, suggesting a role for lysis by temperate phages in regulation of the bacterial population densities. In 9/10 patients, LESϕ2 and LESϕ4 were the most abundant free phages, which reflects the differential in vitro induction properties of the phages. These data indicate that temperate phages of P. aeruginosa retain lytic activity after prolonged periods of chronic infection in the CF lung, and suggest that temperate phage lysis may contribute to regulation of P. aeruginosa density in vivo.

Encephalopathy in children: an approach to assessment and management.

Childhood encephalopathy is an uncommon but significant paediatric presentation and is associated with significant mortality and long-term morbidity in survivors. By definition it is a somewhat non-specific presentation with a wide differential diagnosis and long list of possible investigations. Choice of investigation, including neuroimaging modality, can be a daunting prospect for the clinician faced with the encephalopathic child and it is important to select appropriately for a high diagnostic yield. Initial management centres on good emergency care irrespective of cause. More specific therapies however vary enormously, and appropriate treatment is important and influences outcome. Evidence exists for management of many of the individual conditions causing encephalopathy. This review aims to outline a clinical approach to selecting investigations to identify a specific cause and provides an overview of the treatment for the commoner causes of encephalopathy that a general paediatrician may reasonably expect to be faced with.