Protein degradation on the global scale.

Protein degradation occurs through proteasomal, endosomal, and lysosomal pathways. Technological advancements have allowed for the determination of protein copy numbers and turnover rates on a global scale, which has provided an overview of trends and rules governing protein degradation. Sharper chemical and gene-editing tools have enabled the specific perturbation of each degradation pathway, whose effects on protein dynamics can now be comprehensively analyzed. We review major studies and innovation in this field and discuss the interdependence between the major pathways of protein degradation.

Microtubule association of TRIM3 revealed by differential extraction proteomics.

The microtubule network is formed from polymerised tubulin subunits and associating proteins, which govern microtubule dynamics and a diverse array of functions. To identify novel microtubule-binding proteins, we have developed an unbiased biochemical assay, which relies on the selective extraction of cytosolic proteins from U2OS cells, while leaving behind the microtubule network. Candidate proteins are linked to microtubules by their sensitivities to the depolymerising drug nocodazole or the microtubule-stabilising drug taxol, which is quantitated by mass spectrometry. Our approach is benchmarked by co-segregation of tubulin and previously established microtubule-binding proteins. We then identify several novel candidate microtubule-binding proteins, from which we have selected the ubiquitin E3 ligase tripartite motif-containing protein 3 (TRIM3) for further characterisation. We map TRIM3 microtubule binding to its C-terminal NHL-repeat region. We show that TRIM3 is required for the accumulation of acetylated tubulin, following treatment with taxol. Furthermore, loss of TRIM3 partially recapitulates the reduction in nocodazole-resistant microtubules characteristic of α-tubulin acetyltransferase 1 (ATAT1) depletion. These results can be explained by a decrease in ATAT1 following depletion of TRIM3 that is independent of transcription.

Membrane compartmentalisation of the ubiquitin system.

We now have a comprehensive inventory of ubiquitin system components. Understanding of any system also needs an appreciation of how components are organised together. Quantitative proteomics has provided us with a census of their relative populations in several model cell types. Here, by examining large scale unbiased data sets, we seek to identify and map those components, which principally reside on the major organelles of the endomembrane system. We present the consensus distribution of > 50 ubiquitin modifying enzymes, E2s, E3s and DUBs, that possess transmembrane domains. This analysis reveals that the ER and endosomal compartments have a diverse cast of resident E3s, whilst the Golgi and mitochondria operate with a more restricted palette. We describe key functions of ubiquitylation that are specific to each compartment and relate this to their signature complement of ubiquitin modifying components.

Characterisation and prion transmission study in mice with genetic reduction of sporadic Creutzfeldt-Jakob disease risk gene Stx6.

Sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is thought to occur when the cellular prion protein (PrPC) spontaneously misfolds and assembles into prion fibrils, culminating in fatal neurodegeneration. In a genome-wide association study of sCJD, we recently identified risk variants in and around the gene STX6, with evidence to suggest a causal increase of STX6 expression in disease-relevant brain regions. STX6 encodes syntaxin-6, a SNARE protein primarily involved in early endosome to trans-Golgi network retrograde transport. Here we developed and characterised a mouse model with genetic depletion of Stx6 and investigated a causal role of Stx6 expression in mouse prion disease through a classical prion transmission study, assessing the impact of homozygous and heterozygous syntaxin-6 knockout on disease incubation periods and prion-related neuropathology. Following inoculation with RML prions, incubation periods in Stx6-/- and Stx6+/- mice differed by 12 days relative to wildtype. Similarly, in Stx6-/- mice, disease incubation periods following inoculation with ME7 prions also differed by 12 days. Histopathological analysis revealed a modest increase in astrogliosis in ME7-inoculated Stx6-/- animals and a variable effect of Stx6 expression on microglia activation, however no differences in neuronal loss, spongiform change or PrP deposition were observed at endpoint. Importantly, Stx6-/- mice are viable and fertile with no gross impairments on a range of neurological, biochemical, histological and skeletal structure tests. Our results provide some support for a pathological role of Stx6 expression in prion disease, which warrants further investigation in the context of prion disease but also other neurodegenerative diseases considering syntaxin-6 appears to have pleiotropic risk effects in progressive supranuclear palsy and Alzheimer's disease.

The cost-effectiveness of procalcitonin for guiding antibiotic prescribing in individuals hospitalized with COVID-19: part of the PEACH study.

BACKGROUND: Many hospitals introduced procalcitonin (PCT) testing to help diagnose bacterial coinfection in individuals with COVID-19, and guide antibiotic decision-making during the COVID-19 pandemic in the UK. OBJECTIVES: Evaluating cost-effectiveness of using PCT to guide antibiotic decisions in individuals hospitalized with COVID-19, as part of a wider research programme. METHODS: Retrospective individual-level data on patients hospitalized with COVID-19 were collected from 11 NHS acute hospital Trusts and Health Boards from England and Wales, which varied in their use of baseline PCT testing during the first COVID-19 pandemic wave. A matched analysis (part of a wider analysis reported elsewhere) created groups of patients whose PCT was/was not tested at baseline. A model was created with combined decision tree/Markov phases, parameterized with quality-of-life/unit cost estimates from the literature, and used to estimate costs and quality-adjusted life years (QALYs). Cost-effectiveness was judged at a £20 000/QALY threshold. Uncertainty was characterized using bootstrapping. RESULTS: People who had baseline PCT testing had shorter general ward/ICU stays and spent less time on antibiotics, though with overlap between the groups' 95% CIs. Those with baseline PCT testing accrued more QALYs (8.76 versus 8.62) and lower costs (£9830 versus £10 700). The point estimate was baseline PCT testing being dominant over no baseline testing, though with uncertainty: the probability of cost-effectiveness was 0.579 with a 1 year horizon and 0.872 with a lifetime horizon. CONCLUSIONS: Using PCT to guide antibiotic therapy in individuals hospitalized with COVID-19 is more likely to be cost-effective than not, albeit with uncertainty.

Treatment of Hidradenitis Suppurativa Evaluation Study (THESEUS): a prospective cohort study.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic, painful disease affecting flexures and other skin regions, producing nodules, abscesses and skin tunnels. Laser treatment targeting hair follicles and deroofing of skin tunnels are standard HS interventions in some countries but are rarely offered in the UK. OBJECTIVES: To describe current UK HS management pathways and influencing factors to inform the design of future randomized controlled trials (RCTs). METHODS: THESEUS was a nonrandomized 12-month prospective cohort study set in 10 UK hospitals offering five interventions: oral doxycycline 200 mg daily; oral clindamycin and rifampicin both 300 mg twice daily for 10 weeks, extended for longer in some cases; laser treatment targeting hair follicles; deroofing; and conventional surgery. The primary outcome was the combination of clinician-assessed eligibility and participant hypothetical willingness to receive each intervention. The secondary outcomes were the proportion of participants selecting each intervention as their final treatment option; the proportion who switch treatments; treatment fidelity; and attrition rates. THESEUS was prospectively registered on the ISRCTN registry: ISRCTN69985145. RESULTS: The recruitment target of 150 participants was met after 18 months, in July 2021, with two pauses due to the COVID-19 pandemic. Baseline demographics reflected the HS secondary care population: average age 36 years, 81% female, 20% non-White, 64% current or ex-smokers, 86% body mass index ≥ 25, 68% with moderate disease, 19% with severe disease and 13% with mild disease. Laser was the intervention with the highest proportion (69%) of participants eligible and willing to receive treatment, then deroofing (58%), conventional surgery (54%), clindamycin and rifampicin (44%), and doxycycline (37%). Laser was ranked first choice by the greatest proportion of participants (41%). Attrition rates were 11% and 17% after 3 and 6 months, respectively. Concordance with doxycycline was 52% after 3 months due to lack of efficacy, participant choice and adverse effects. Delays with procedural interventions were common, with only 43% and 26% of participants starting laser and deroofing, respectively, after 3 months. Uptake of conventional surgery was too small to characterize the intervention. Switching treatment was uncommon and there were no serious adverse events. CONCLUSIONS: THESEUS has established laser treatment and deroofing for HS in the UK and demonstrated their popularity with patients and clinicians for future RCTs.

Intrinsic risk factors for alpha-gal syndrome in a case-control study, 2019 to 2020.

BACKGROUND: Alpha-gal syndrome (AGS) is an allergy to galactose-α-1,3-galactose (alpha-gal), a carbohydrate found in most mammals. Evidence indicates that AGS develops after a tick bite, and in the United States, AGS is most associated with bites from Amblyomma americanum (lone star tick); however, not all persons bitten by ticks develop clinical AGS. OBJECTIVE: To investigate intrinsic risk factors associated with the development of AGS. METHODS: We performed a case-control study among adults presenting for diagnosis or management of AGS at an allergy clinic in North Carolina during 2019 to 2020 and compared them with controls enrolled from 2 nearby internal medicine clinics. A questionnaire gathered epidemiologic and tick exposure data, and blood was obtained for alpha-gal-specific IgE and other testing. RESULTS: The 82 enrolled case patients and 191 controls did not differ significantly by age or sex. Case patients were more likely than controls to have A or O blood types (non B-antigen), have experienced childhood allergies, and have a family history of AGS and other food allergies. Case patients were also more likely to report experiencing long healing times for insect bites or stings and a family history of allergy to stinging or biting insects. CONCLUSION: This study suggested that intrinsic factors contribute to risk of developing AGS. Some traits are genetic, but common behaviors among households and family units likely also contribute. Identification of these risk factors can inform personal risk, aid health care providers in understanding susceptible populations, and contribute to ongoing understanding of AGS epidemiology.

ISGylation-independent protection of cell growth by USP18 following interferon stimulation.

Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signalling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation, a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non-catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity.

Fasting for 20 h does not affect exercise-induced increases in circulating BDNF in humans.

Intermittent fasting and exercise provide neuroprotection from age-related cognitive decline. A link between these two seemingly distinct stressors is their capability to steer the brain away from exclusively glucose metabolism. This cerebral substrate switch has been implicated in upregulating brain-derived neurotrophic factor (BDNF), a protein involved in neuroplasticity, learning and memory, and may underlie some of these neuroprotective effects. We examined the isolated and interactive effects of (1) 20-h fasting, (2) 90-min light exercise, and (3) high-intensity exercise on peripheral venous BDNF in 12 human volunteers. A follow-up study isolated the influence of cerebrovascular shear stress on circulating BDNF. Fasting for 20 h decreased glucose and increased ketones (P ≤ 0.0157) but had no effect on BDNF (P ≥ 0.4637). Light cycling at 25% of peak oxygen uptake ( V ̇ O 2 peak ${\dot V_{{{\rm{O}}_{\rm{2}}}{\rm{peak}}}}$ ) increased serum BDNF by 6 ± 8% (independent of being fed or fasted) and was mediated by a 7 ± 6% increase in platelets (P < 0.0001). Plasma BDNF was increased from 336 pg l-1 [46,626] to 390 pg l-1 [127,653] by 90-min of light cycling (P = 0.0128). Six 40-s intervals at 100% of V ̇ O 2 peak ${\dot V_{{{\rm{O}}_{\rm{2}}}{\rm{peak}}}}$ increased plasma and serum BDNF, as well as the BDNF-per-platelet ratio 4- to 5-fold more than light exercise did (P ≤ 0.0044). Plasma BDNF was correlated with circulating lactate during the high-intensity intervals (r = 0.47, P = 0.0057), but not during light exercise (P = 0.7407). Changes in cerebral shear stress - whether occurring naturally during exercise or induced experimentally with inspired CO2 - did not correspond with changes in BDNF (P ≥ 0.2730). BDNF responses to low-intensity exercise are mediated by increased circulating platelets, and increasing either exercise duration or particularly intensity is required to liberate free BDNF. KEY POINTS: Intermittent fasting and exercise both have potent neuroprotective effects and an acute upregulation of brain-derived neurotrophic factor (BDNF) appears to be a common mechanistic link. Switching the brain's fuel source from glucose to either ketone bodies or lactate, i.e. a cerebral substrate switch, has been shown to promote BDNF production in the rodent brain. Fasting for 20 h caused a 9-fold increase in ketone body delivery to the brain but had no effect on any metric of BDNF in peripheral circulation at rest. Prolonged (90 min) light cycling exercise increased plasma- and serum-derived BDNF irrespective of being fed or fasted and seemed to be independent of changes in cerebral shear stress. Six minutes of high-intensity cycling intervals increased every metric of circulating BDNF by 4 to 5 times more than prolonged low-intensity cycling; the increase in plasma-derived BDNF was correlated with a 6-fold increase in circulating lactate irrespective of feeding or fasting. Compared to 1 day of fasting with or without prolonged light exercise, high-intensity exercise is a much more efficient means to increase BDNF in circulation.

Re-Aiming Equity Evaluation in Clinical Decision Support: A Scoping Review of Equity Assessments in Surgical Decision Support Systems.

OBJECTIVE: We critically evaluated the surgical literature to explore the prevalence and describe how equity assessments occur when using clinical decision support systems. BACKGROUND: Clinical decision support (CDS) systems are increasingly used to facilitate surgical care delivery. Despite formal recommendations to do so, equity evaluations are not routinely performed on CDS systems and underrepresented populations are at risk of harm and further health disparities. We explored surgical literature to determine frequency and rigor of CDS equity assessments and offer recommendations to improve CDS equity by appending existing frameworks. METHODS: We performed a scoping review up to Augus 25, 2021 using PubMed and Google Scholar for the following search terms: clinical decision support, implementation, RE-AIM, Proctor, Proctor's framework, equity, trauma, surgery, surgical. We identified 1415 citations and 229 abstracts met criteria for review. A total of 84 underwent full review after 145 were excluded if they did not assess outcomes of an electronic CDS tool or have a surgical use case. RESULTS: Only 6% (5/84) of surgical CDS systems reported equity analyses, suggesting that current methods for optimizing equity in surgical CDS are inadequate. We propose revising the RE-AIM framework to include an Equity element (RE 2 -AIM) specifying that CDS foundational analyses and algorithms are performed or trained on balanced datasets with sociodemographic characteristics that accurately represent the CDS target population and are assessed by sensitivity analyses focused on vulnerable subpopulations. CONCLUSION: Current surgical CDS literature reports little with respect to equity. Revising the RE-AIM framework to include an Equity element (RE 2 -AIM) promotes the development and implementation of CDS systems that, at minimum, do not worsen healthcare disparities and possibly improve their generalizability.

Estimating the early impact of the US COVID-19 vaccination programme on COVID-19 cases, emergency department visits, hospital admissions, and deaths among adults aged 65 years and older: an ecological analysis of national surveillance data.

BACKGROUND: In the USA, COVID-19 vaccines became available in mid-December, 2020, with adults aged 65 years and older among the first groups prioritised for vaccination. We estimated the national-level impact of the initial phases of the US COVID-19 vaccination programme on COVID-19 cases, emergency department visits, hospital admissions, and deaths among adults aged 65 years and older. METHODS: We analysed population-based data reported to US federal agencies on COVID-19 cases, emergency department visits, hospital admissions, and deaths among adults aged 50 years and older during the period Nov 1, 2020, to April 10, 2021. We calculated the relative change in incidence among older age groups compared with a younger reference group for pre-vaccination and post-vaccination periods, defined by the week when vaccination coverage in a given age group first exceeded coverage in the reference age group by at least 1%; time lags for immune response and time to outcome were incorporated. We assessed whether the ratio of these relative changes differed when comparing the pre-vaccination and post-vaccination periods. FINDINGS: The ratio of relative changes comparing the change in the COVID-19 case incidence ratio over the post-vaccine versus pre-vaccine periods showed relative decreases of 53% (95% CI 50 to 55) and 62% (59 to 64) among adults aged 65 to 74 years and 75 years and older, respectively, compared with those aged 50 to 64 years. We found similar results for emergency department visits with relative decreases of 61% (52 to 68) for adults aged 65 to 74 years and 77% (71 to 78) for those aged 75 years and older compared with adults aged 50 to 64 years. Hospital admissions declined by 39% (29 to 48) among those aged 60 to 69 years, 60% (54 to 66) among those aged 70 to 79 years, and 68% (62 to 73), among those aged 80 years and older, compared with adults aged 50 to 59 years. COVID-19 deaths also declined (by 41%, 95% CI -14 to 69 among adults aged 65-74 years and by 30%, -47 to 66 among those aged ≥75 years, compared with adults aged 50 to 64 years), but the magnitude of the impact of vaccination roll-out on deaths was unclear. INTERPRETATION: The initial roll-out of the US COVID-19 vaccination programme was associated with reductions in COVID-19 cases, emergency department visits, and hospital admissions among older adults. FUNDING: None.

Clinical and laboratory features of patients diagnosed with alpha-gal syndrome-2010-2019.

BACKGROUND: Alpha-gal syndrome (AGS) is an IgE-mediated allergy to galactose-alpha-1,3-galactose. Clinical presentation ranges from hives to anaphylaxis; episodes typically occur 2-6 h after exposure to alpha-gal-containing products. In the United States, lone star tick bites are associated with the development of AGS. To characterize features of AGS, we evaluated a cohort of patients presenting for care at the University of North Carolina, focusing on symptoms, severity, and identifying features unique to specific alpha-gal-containing product exposures. METHODS: We performed a chart review and descriptive analysis of 100 randomly selected patients with AGS during 2010-2019. RESULTS: Median age at onset was 53 years, 56% were female, 95% reported White race, 86% reported a history of tick bite, and 75% met the criteria for anaphylaxis based on the involvement of ≥2 organ systems. Those reporting dairy reactions were significantly less likely to report isolated mucocutaneous symptoms (3% vs. 24%; ratio [95% CI]: 0.1 [0.1, 0.3]) than those who tolerated dairy, and were more likely to report gastrointestinal symptoms (79% vs. 59%; ratio [95% CI]: 1.3 [0.7, 2.6]), although this difference was not statistically significant. Dairy-tolerant patients demonstrated higher alpha-gal sIgE titers (as a percentage of total IgE) than dairy-reactive patients (GM 4.1 [95% CI: 2.7, 6.1] vs. GM 2.5 [95% CI: 1.3, 4.8], respectively; ratio -1.6 [95% CI: -1.0, 3.9]). CONCLUSION: While tick exposure is common in the southern United States, nearly all AGS patients reported a tick bite. Gastrointestinal symptoms were prominent among those reporting reactions to dairy. Anaphylaxis was common, underscoring the severity and need to raise awareness of AGS among patients and providers.

Tick bite as a risk factor for alpha-gal-specific immunoglobulin E antibodies and development of alpha-gal syndrome.

BACKGROUND: The disaccharide galactose-α-1,3-galactose (alpha-gal) is expressed in mammals other than humans, apes, and old-world monkeys. In humans, elevated immunoglobulin E (IgE) antibodies specific for alpha-gal can result in allergic hypersensitivity known as alpha-gal syndrome (AGS). Case reports and series suggest that tick bites can induce alpha-gal-specific IgE (sIgE) antibodies. OBJECTIVE: To evaluate tick exposure as a risk factor for AGS and elevated alpha-gal sIgE level. METHODS: We conducted a case-control study comparing patients with AGS from a North Carolina allergy clinic with controls who were patients at a nearby internal medicine clinic. Cases and controls were administered a questionnaire to obtain information about demographics, home environment, outdoor activities, and recollection of tick bite. Serum samples taken at the time of enrollment were tested for total IgE, alpha-gal sIgE, and antibodies to other tick-borne pathogens. RESULTS: The patients with AGS were more likely to recall finding a tick on themselves (odds ratio [OR], 11.20; 95% confidence interval [CI], 4.97-25.15), live near wooded forest (OR, 2.27; 95% CI, 0.92-5.55), and spend 17 or more hours per week outdoors in wooded areas (OR, 5.58; 95% CI, 2.56-12.19). The patients with AGS were also more likely to report 4 or more tick bites (OR, 33.05; 95% CI, 9.92-155.12) and reactions at the site of tick bites (OR, 7.93; 95% CI, 3.74-16.80). Furthermore, elevated alpha-gal sIgE level was observed in 33% of the controls and was associated with tick exposure in the controls (OR, 4.25; 95% CI, 2.21-8.18). CONCLUSION: The results define tick bite as a risk factor for AGS and elevated alpha-gal sIgE level.

Overlooked Symptoms in Autoimmune Hepatitis Negatively Impact Many Facets of Life.

BACKGROUND: Significant reduction in quality of life among patients with autoimmune hepatitis (AIH) patients has been observed in several studies. While acute symptoms associated with AIH have been well described, little is known about the overall impact of living with AIH on patients' quality of life. The aim of this qualitative descriptive study was to describe the impact of AIH and associated symptoms on quality of life from the perspectives of patients living with AIH. METHODS: Patients from Autoimmune Hepatitis Association support groups were recruited to participate in one of five online focus groups conducted between August and September 2020. After enrollment, patients were asked to complete a brief demographic and disease history questionnaire. A single moderator conducted interviews with each group guided by seven questions focused on the impact of AIH on the participants' quality of life. Each session was recorded, transcribed, and verified. Content analysis was used to summarize the participants' responses. RESULTS: The participants' discussed three overarching topics: (a) symptoms of AIH and medication side effects, (b) the impact the disease and symptoms/side effects on five domains of quality of life (work life, relationships with friends and family, social life, leisure activities, and diet and exercise) and (c) interactions with healthcare providers and recommendations for future research. CONCLUSIONS: Living with AIH can have profound effects on patients' quality of life in several domains. Healthcare providers and the AIH research community should focus on developing further strategies that can improve the quality of life in persons suffering from AIH.

COVID-19 vaccination in pregnancy: ambiguity in decision-making.

Throughout the COVID-19 pandemic, pregnant women/people were identified as an at-risk group of severe COVID-19 disease. Consequently, vaccine uptake among this group became a public health priority. However, the relationship between pregnancy and vaccination decision-making is complex, and the heightened uncertainty and anxiety produced through the pandemic further exacerbated this immunization decision. This study explores COVID-19 vaccination decision-making during pregnancy in Aotearoa New Zealand by using an online story completion survey tool. Ninety-five responses were received and analysed using thematic analysis where ambiguity was a core facet within and across stories. Three ambiguities were identified, including who makes the decision (agential), what the risks are (risk) and how immunity to this threat can be best achieved (immunity). We discuss the implications of this ambiguity and how the strong desire to protect the baby persisted across accounts. The recognition of the rather persistent ambiguity in vaccination decision-making helps conceptualize influencing factors taken into account in a more nuanced manner for further research, public health campaigns and health professionals. Future public health campaigns can consider redistributing responsibility for vaccination decision-making in pregnancy, traverse an either/or perspective of 'natural' and 'artificial' immunity-boosting and consider how risk is perceived through anecdotes and viral immediacy.

Continuing bonds following stillbirth: protective and risk factors associated with parental bereavement adaptation.

OBJECTIVE: To investigate any association between expressions of parents' continuing bond with their stillborn baby and bereavement adaptation. BACKGROUND: Continuing bonds theory suggests that bereaved parents adapt to the loss of their child by sharing and transforming mental representations of the child, allowing them to be integrated into parents' everyday lives. Little is known about the mental health benefits of expressing continuing bonds following stillbirth. This study examined any association between aspects of parents' relationship with their stillborn baby, social support for the relationship, and bereavement adaptation. METHODS: Cross-sectional questionnaire study. Parents of stillborn babies (N=170) completed an online questionnaire examining engagement in continuing bonds expressions; characteristics of parents' relationship with their stillborn baby and their experience of sharing it; social support, and meaning-making. Measures of mental health were included to quantify bereavement adaptation. RESULTS: Regression analyses showed that time since death, meaning-making, engaging with nature, and legacy building are positively linked to bereavement adaptation. Risk factors included inadequate social support for the relationship, a greater desire to share it more freely, an increased sense of integration with baby, and societal pressure to move on. CONCLUSION: Key aspects of parents' ongoing relationship with their stillborn baby and the social context are related to bereavement adaptation.

Predominance of Severe Plasma Leakage in Pediatric Patients With Severe Dengue in Puerto Rico.

BACKGROUND: We evaluated clinical and laboratory findings among patients with nonsevere or severe dengue in Puerto Rico to examine whether clinical manifestations vary by age. METHODS: During 2012-2014, we enrolled patients who arrived at the emergency department with fever or history of fever within 7 days of presentation. Serum samples were tested for dengue virus (DENV) by reverse transcriptase-polymerase chain reaction (RT-PCR) and IgM enzyme-linked immunosorbent assay (ELISA). Severe dengue was defined as severe plasma leakage or shock, severe bleeding, or organ involvement at presentation, during hospitalization, or follow-up. RESULTS: Of 1089 dengue patients identified, 281 (26%) were severe. Compared to those with nonsevere dengue, patients with severe dengue were more often aged 10-19 years (55% vs 40%, P < .001) and hospitalized (87% vs 30%, P < .001). Severe plasma leakage or shock was more common among children aged 0-9 (59%) or 10-19 years (86%) than adults (49%) (P < .01). Severe bleeding was less common among 10-19 year olds (24%) compared to 0-9 year olds (45%) and adults (52%; P < .01). CONCLUSIONS: Severe plasma leakage was the most common presentation among children, highlighting important differences from adults. Vaccination against dengue could help prevent severe dengue among children in Puerto Rico.

Human iPSC-derived Down syndrome astrocytes display genome-wide perturbations in gene expression, an altered adhesion profile, and increased cellular dynamics.

Down syndrome (DS), caused by the triplication of human chromosome 21, leads to significant alterations in brain development and is a major genetic cause of intellectual disability. While much is known about changes to neurons in DS, the effects of trisomy 21 on non-neuronal cells such as astrocytes are poorly understood. Astrocytes are critical for brain development and function, and their alteration may contribute to DS pathophysiology. To better understand the impact of trisomy 21 on astrocytes, we performed RNA-sequencing on astrocytes from newly produced DS human induced pluripotent stem cells (hiPSCs). While chromosome 21 genes were upregulated in DS astrocytes, we found consistent up- and down-regulation of genes across the genome with a strong dysregulation of neurodevelopmental, cell adhesion and extracellular matrix molecules. ATAC (assay for transposase-accessible chromatin)-seq also revealed a global alteration in chromatin state in DS astrocytes, showing modified chromatin accessibility at promoters of cell adhesion and extracellular matrix genes. Along with these transcriptomic and epigenomic changes, DS astrocytes displayed perturbations in cell size and cell spreading as well as modifications to cell-cell and cell-substrate recognition/adhesion, and increases in cellular motility and dynamics. Thus, triplication of chromosome 21 is associated with genome-wide transcriptional, epigenomic and functional alterations in astrocytes that may contribute to altered brain development and function in DS.

C-Reactive Protein Testing to Guide Antibiotic Prescribing for COPD Exacerbations.

BACKGROUND: Point-of-care testing of C-reactive protein (CRP) may be a way to reduce unnecessary use of antibiotics without harming patients who have acute exacerbations of chronic obstructive pulmonary disease (COPD). METHODS: We performed a multicenter, open-label, randomized, controlled trial involving patients with a diagnosis of COPD in their primary care clinical record who consulted a clinician at 1 of 86 general medical practices in England and Wales for an acute exacerbation of COPD. The patients were assigned to receive usual care guided by CRP point-of-care testing (CRP-guided group) or usual care alone (usual-care group). The primary outcomes were patient-reported use of antibiotics for acute exacerbations of COPD within 4 weeks after randomization (to show superiority) and COPD-related health status at 2 weeks after randomization, as measured by the Clinical COPD Questionnaire, a 10-item scale with scores ranging from 0 (very good COPD health status) to 6 (extremely poor COPD health status) (to show noninferiority). RESULTS: A total of 653 patients underwent randomization. Fewer patients in the CRP-guided group reported antibiotic use than in the usual-care group (57.0% vs. 77.4%; adjusted odds ratio, 0.31; 95% confidence interval [CI], 0.20 to 0.47). The adjusted mean difference in the total score on the Clinical COPD Questionnaire at 2 weeks was -0.19 points (two-sided 90% CI, -0.33 to -0.05) in favor of the CRP-guided group. The antibiotic prescribing decisions made by clinicians at the initial consultation were ascertained for all but 1 patient, and antibiotic prescriptions issued over the first 4 weeks of follow-up were ascertained for 96.9% of the patients. A lower percentage of patients in the CRP-guided group than in the usual-care group received an antibiotic prescription at the initial consultation (47.7% vs. 69.7%, for a difference of 22.0 percentage points; adjusted odds ratio, 0.31; 95% CI, 0.21 to 0.45) and during the first 4 weeks of follow-up (59.1% vs. 79.7%, for a difference of 20.6 percentage points; adjusted odds ratio, 0.30; 95% CI, 0.20 to 0.46). Two patients in the usual-care group died within 4 weeks after randomization from causes considered by the investigators to be unrelated to trial participation. CONCLUSIONS: CRP-guided prescribing of antibiotics for exacerbations of COPD in primary care clinics resulted in a lower percentage of patients who reported antibiotic use and who received antibiotic prescriptions from clinicians, with no evidence of harm. (Funded by the National Institute for Health Research Health Technology Assessment Program; PACE Current Controlled Trials number, ISRCTN24346473.).

Clinical Metagenomic Sequencing for Species Identification and Antimicrobial Resistance Prediction in Orthopedic Device Infection.

Diagnosis of orthopedic device-related infection is challenging, and causative pathogens may be difficult to culture. Metagenomic sequencing can diagnose infections without culture, but attempts to detect antimicrobial resistance (AMR) determinants using metagenomic data have been less successful. Human DNA depletion may maximize the amount of microbial DNA sequence data available for analysis. Human DNA depletion by saponin was tested in 115 sonication fluid samples generated following revision arthroplasty surgery, comprising 67 where pathogens were detected by culture and 48 culture-negative samples. Metagenomic sequencing was performed on the Oxford Nanopore Technologies GridION platform. Filtering thresholds for detection of true species versus contamination or taxonomic misclassification were determined. Mobile and chromosomal genetic AMR determinants were identified in Staphylococcus aureus-positive samples. Of 114 samples generating sequence data, species-level positive percent agreement between metagenomic sequencing and culture was 50/65 (77%; 95% confidence interval [CI], 65 to 86%) and negative percent agreement was 103/114 (90%; 95% CI, 83 to 95%). Saponin treatment reduced the proportion of human bases sequenced in comparison to 5-µm filtration from a median (interquartile range [IQR]) of 98.1% (87.0% to 99.9%) to 11.9% (0.4% to 67.0%), improving reference genome coverage at a 10-fold depth from 18.7% (0.30% to 85.7%) to 84.3% (12.9% to 93.8%). Metagenomic sequencing predicted 13/15 (87%) resistant and 74/74 (100%) susceptible phenotypes where sufficient data were available for analysis. Metagenomic nanopore sequencing coupled with human DNA depletion has the potential to detect AMR in addition to species detection in orthopedic device-related infection. Further work is required to develop pathogen-agnostic human DNA depletion methods, improving AMR determinant detection and allowing its application to other infection types.