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Seagrass ecosystems, vital as primary producer habitats for maintaining high biodiversity and delivering numerous ecosystem services, face increasing threats from climate change, particularly marine heatwaves. This study introduces a pioneering methodology that integrates Dynamic Bayesian Networks of ecosystem resilience with climate projections, aiming to enhance our understanding of seagrass responses to extreme climate events. We developed cutting-edge metrics for measuring shoot density and biomass in terms of population and site extinction, presented as annual ratios relative to their respective baselines. These metrics include associated uncertainties and projected recovery times. This innovative approach was applied in a case study focusing on Zostera muelleri in Gladstone Harbour, Australia. Utilising five downscaled climate models with a 10 km resolution, our study encompasses a range of Shared Socioeconomic Pathways and emissions trajectories, offering a comprehensive perspective on potential future scenarios. Our findings reveal significant variations in seagrass resilience and recovery times across different climate scenarios, accompanied by varying degrees of uncertainty. For instance, under the optimistic SSP1-1.9 scenario, seagrass demonstrated a capacity for recovery heat stress, with shoot density ratios improving from 0.2 (90% Prediction Interval 0.219, 0.221) in 2041 to 0.5 (90% PI 0.198, 1.076) by 2044. However, this scenario also highlighted potential site extinction risks, with recovery gaps spanning 12 to 18 years. In contrast, the more pessimistic SSP5-8.5 scenario revealed a significant decline in seagrass health, with shoot density ratios decreasing from 0.42 (90% PI 0.226, 0.455) in 2041 to just 0.2 (90% PI 0.211, 0.221) in 2048, and no recovery observed after 2038. This study, through its novel integration of climate models, Dynamic Bayesian Networks, and Monte Carlo methods, offers a groundbreaking approach to ecological forecasting, significantly enhancing seagrass resilience assessment and supporting climate adaptation strategies under changing climatic conditions. This methodology holds great potential for application across various sites and future climate scenarios, offering a versatile tool for integrating Dynamic Bayesian Networks ecosystem models.

Reports on treatment characteristics and long term outcomes for severe burns are scarce, while the need to compare outcomes of novel treatment modalities to standard of care is increasing. Our national database on burn treatment enabled analysis of patient as well as treatment characteristics during acute treatment and following reconstructive procedures. Furthermore, outcome data of longitudinal scar assessments were analysed from a single burn centre database. Acute and reconstructive data were analysed for patients admitted to the three Dutch burn centres with total body surface area burned of ≥ 20% TBSA. Long term outcome was analysed from a single centre scar database, both for a period of 2009-2019. Treatment characteristics from 396 surviving acute burn patients were analysed. Surgical treatment was required in 89.6% of these patients and 110 patients (27.8%) needed reconstructive surgery in the years after the burn incident, with a mean of 4.4 reconstructive procedures per patient. Main indications were contractures (70.5%) and arms (45.0%) and head and neck region (41.2%) were most frequently affected. Techniques used for reconstructive corrections were predominantly excision, release and flaps (54.7%), followed by skin transplants (32.4%). Scar quality was significantly worse in patients with more severe burns compared to those with TBSA < 20% during prolonged times. These data provide insight into health care utilization, treatment characteristics and outcomes in severely burned patients. These real-world data can guide future development of improved treatment strategies for at risk patients as well as anatomical locations.

BackgroundAnticholinergic burden has been associated with adverse outcomes such as falls. To date, no gold standard measure has been identified to assess anticholinergic burden, and no conclusion has been drawn on which of the different measure algorithms best predicts falls in older patients from general practice. This study compared the ability of five measures of anticholinergic burden to predict falls. To account for patients' individual susceptibility to medications, the added predictive value of typical anticholinergic symptoms was further quantified in this context.Methods and findingsTo predict falls, models were developed and validated based on logistic regression models created using data from two German cluster-randomized controlled trials. The outcome was defined as "≥ 1 fall" vs. "no fall" within a 6-month follow-up period. Data from the RIME study (n = 1,197) were used in model development, and from PRIMUM (n = 502) for external validation. The models were developed step-wise in order to quantify the predictive ability of anticholinergic burden measures, and anticholinergic symptoms. In the development set, 1,015 patients had complete data and 188 (18.5%) experienced ≥ 1 fall within the 6-month follow-up period. The overall predictive value of the five anticholinergic measures was limited, with neither the employed anticholinergic variable (binary / count / burden), nor dose-dependent or dose-independent measures differing significantly in their ability to predict falls. The highest c-statistic was obtained using the German Anticholinergic Burden Score (0.73), whereby the optimism-corrected c-statistic was 0.71 after interval validation using bootstrapping and 0.63 in the external validation. Previous falls and dizziness / vertigo had the strongest prognostic value in all models.ConclusionsThe ability of anticholinergic burden measures to predict falls does not appear to differ significantly, and the added value they contribute to risk classification in fall-prediction models is limited. Previous falls and dizziness / vertigo contributed most to model performance.

Vascular dementia (VaD) accounts for 15–20% of all dementia cases. It is a syndrome of acquired cognitive impairment with a complex pathophysiological basis. A novel herbal formulation (Sailuotong; SLT) consisting of Panax ginseng C.A Mey, Ginkgo biloba L and Crocus sativus L extracts was developed to treat VaD. Preclinical animal studies found significant improvements in memory and in pathogenic biochemical parameters. Appropriate safety of SLT was shown in acute and chronic toxicity studies, and early clinical trials of SLT demonstrated enhancements in cognition in VaD patients. A fully powered study with a long intervention period is needed to confirm the efficacy and safety of this novel intervention. A rigorous phase III clinical trial was developed with the aim of recruiting 238 patients diagnosed with mild to moderate probable VaD, or VaD mixed with Alzheimer’s disease (where cerebrovascular disease is the clinical dominant contributor to dementia, abbreviated as CVD+AD). Using a permuted block strategy, participants will be randomly allocated to receive SLT (120 mg bd) or placebo capsules for an intervention period of 52 weeks and will be followed-up for an additional 13 weeks. The primary outcome measures are the Vascular Dementia Assessment Scale-cognitive subscale and Alzheimer’s Disease Cooperative Study-Activities of Daily Living scale. Secondary outcome measures include the Clinician’s Interview Based Impression of Change-Plus, CLOX, EXIT-25, Neuropsychiatric Inventory-Clinician rating scale, and Dementia Quality of Life questionnaire. Safety is assessed through adverse event reports and liver, renal, and coagulation studies. Primary and secondary outcome measures will be compared between treatment and placebo groups, using intention to treat and per protocol analyses. We hypothesise that a 52-week treatment of SLT will be clinically effective and well tolerated in participants with VaD or AD+CVD. This project will provide vital efficacy and safety data for this novel treatment approach to VaD.

BackgroundHalf the US population uses drugs with anticholinergic properties. Their potential harms may outweigh their benefits. Amitriptyline is among the most frequently prescribed anticholinergic medicinal products, is used for multiple indications, and rated as strongly anticholinergic. Our objective was to explore and quantify (anticholinergic) adverse drug reactions (ADRs) in patients taking amitriptyline vs. placebo in randomized controlled trials (RCTs) involving adults and healthy individuals.MethodsWe searched electronic databases from their inception until 09/2022, and clinical trial registries from their inception until 09/2022. We also performed manual reference searches. Two independent reviewers selected RCTs with ≥100 participants of ≥18 years, that compared amitriptyline (taken orally) versus placebo for all indications. No language restrictions were applied. One reviewer extracted study data, ADRs, and assessed study quality, which two others verified. The primary outcome was frequency of anticholinergic ADRs as a binary outcome (absolute number of patients with/without anticholinergic ADRs) in amitriptyline vs. placebo groups.ResultsTwenty-three RCTs (mean dosage 5mg to 300mg amitriptyline/day) and 4217 patients (mean age 40.3 years) were included. The most frequently reported anticholinergic ADRs were dry mouth, drowsiness, somnolence, sedation, fatigue, constitutional, and unspecific anticholinergic ADRs. Random-effects meta-analyses showed anticholinergic ADRs had a higher odd's ratio for amitriptyline versus placebo (OR = 7.41; [95% CI, 4.54 to 12.12]). Non-anticholinergic ADRs were as frequent for amitriptyline as placebo. Meta-regression analysis showed anticholinergic ADRs were not dose-dependent.DiscussionThe large OR in our analysis shows that ADRs indicative of anticholinergic activities can be attributed to amitriptyline. The low average age of participants in our study may limit the generalizability of the frequency of anticholinergic ADRs in older patients. A lack of dose-dependency may reflect limited reporting of the daily dosage when the ADRs occurred. The exclusion of small studies (RegistrationPROSPERO: CRD42020111970.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of problematic infections in individuals with predisposing conditions. Infections can be treated with colistin but some isolates are resistant to this antibiotic. To better understand the genetic basis of resistance, we experimentally evolved 19 independent resistant mutants from the susceptible laboratory strain PAO1. Whole genome sequencing identified mutations in multiple genes including phoQ and pmrB that have previously been associated with resistance, pitA that encodes a phosphate transporter, and carB and eno that encode enzymes of metabolism. Individual mutations were engineered into the genome of strain PAO1. Mutations in pitA, pmrB and phoQ increased the minimum inhibitory concentration (MIC) for colistin 8-fold, making the bacteria resistant. Engineered pitA/phoQ and pitA/pmrB double mutants had higher MICs than single mutants, demonstrating additive effects on colistin susceptibility. Single carB and eno mutations did not increase the MIC suggesting that their effect is dependent on the presence of other mutations. Many of the resistant mutants had increased susceptibility to β-lactams and lower growth rates than the parental strain demonstrating that colistin resistance can impose a fitness cost. Two hundred and fourteen P. aeruginosa isolates from a range of sources were tested and 18 (7.8%) were colistin resistant. Sequence variants in genes identified by experimental evolution were present in the 18 resistant isolates and may contribute to resistance. Overall our results identify pitA mutations as novel contributors to colistin resistance and demonstrate that resistance can reduce fitness of the bacteria.

Background The increasing prevalence of cardiovascular diseases (CVDs) in developing countries like Bangladesh has been linked to progressive urbanisation. Comparisons of rural and urban populations often find a higher prevalence of CVD risk factors in the urban population, but rural-to-urban migrants might have different CVD risk profiles than either rural or urban residents. This study aimed to describe differences in CVD risk factors between migrants and non-migrants siblings and to determine whether acculturation factors were associated with CVD risk factors among migrants. Methods Using a sibling-pair comparative study, 164 male migrant who migrated from Pirganj rural areas to Dhaka City and their rural siblings (total N = 328) were assessed by interview, anthropometric measurement, blood pressure and blood samples. Comparisons were made using linear or logistic mixed effects models. Findings Physical inactivity, inadequate intake of fruit and vegetables and possible existence of a mental health disorder had 3.3 (1.73; 6.16), 4.3 (2.32; 7.92) and 2.9 (1.37; 6.27) times higher odds among migrants than their rural siblings, respectively. Migrants watched television on average 20 minutes (95% CI 6.17–35.08 min/day) more per day than the rural sibling group whereas PUFA intake, fruit and vegetable and fish intake of the migrants were -5.3 gm/day (-6.91; -3.70), -21.6 serving/week (-28.20; -15.09), -14.1 serving/week (-18.32; -9.87), respectively, lower than that of the rural siblings. No significant difference was observed for other variables. After adjusting, the risk of physical inactivity, inadequate fruit and vegetable intake, a mental health disorder and low HDL were significantly higher in migrants than in rural siblings and tended to be higher for each increasing tertile of urban life exposure. Conclusion The findings suggest that migration from rural-to-urban environment increases CVD risk which exacerbate with time spent in urban area due to acculturation. This study gives new insights into the increased CVD risk related with migration and urbanization in Bangladesh.

Peer-grouping is used in many sectors for organisational learning, policy implementation, and benchmarking. Clustering provides a statistical, data-driven method for constructing meaningful peer groups, but peer groups must be compatible with business constraints such as size and stability considerations. Additionally, statistical peer groups are constructed from many different variables, and can be difficult to understand, especially for non-statistical audiences. We developed methodology to apply business constraints to clustering solutions and allow the decision-maker to choose the balance between statistical goodness-of-fit and conformity to business constraints. Several tools were utilised to identify complex distinguishing features in peer groups, and a number of visualisations are developed to explain high-dimensional clusters for non-statistical audiences. In a case study where peer group size was required to be small (≤ 100 members), we applied constrained clustering to a noisy high-dimensional data-set over two subsequent years, ensuring that the clusters were sufficiently stable between years. Our approach not only satisfied clustering constraints on the test data, but maintained an almost monotonic negative relationship between goodness-of-fit and stability between subsequent years. We demonstrated in the context of the case study how distinguishing features between clusters can be communicated clearly to different stakeholders with substantial and limited statistical knowledge.

The Blue Iguana Recovery Programme maintains a captive breeding and head-starting program for endangered Grand Cayman blue iguanas (Cyclura lewisi) on Grand Cayman, Cayman Islands. In May 2015, program staff encountered two lethargic wild Grand Cayman blue iguanas within the Queen Elizabeth II Botanic Park (QEIIBP). Spiral-shaped bacteria were identified on peripheral blood smears from both animals, which molecular diagnostics identified as a novel Helicobacter species (provisionary name Helicobacter sp. GCBI1). Between March 2015 and February 2017, 11 Grand Cayman blue iguanas were identified with the infection. Two of these were found dead and nine were treated; five of the nine treated animals survived the initial infection. Phylogenetic analysis of the 16S rRNA gene suggests Helicobacter sp. GCBI1 is most closely related to Helicobacter spp. in chelonians. We developed a Taqman qPCR assay specific for Helicobacter sp. GCBI1 to screen tissue and/or blood samples from clinical cases, fecal and cloacal samples from clinically healthy Grand Cayman blue iguanas, including previously infected and recovered iguanas, and iguanas housed adjacent to clinical cases. Fecal and/or cloacal swab samples were all negative, suggesting that Grand Cayman blue iguanas do not asymptomatically carry this organism nor shed this pathogen per cloaca post infection. Retrospective analysis of a 2014 mortality event affecting green iguanas (Iguana iguana) from a separate Grand Cayman location identified Helicobacter sp. GCBI1 in two of three cases. The source of infection and mode of transmission are yet to be confirmed. Analysis of rainfall data reveal that all infections occurred during a multi-year dry period, and most occurred shortly after the first rains at the end of seasonal drought. Additionally, further screening has identified Helicobacter sp. GCBI1 from choanal swabs of clinically normal green iguanas in the QEIIBP, suggesting they could be asymptomatic carriers and a potential source of the pathogen.

BackgroundAs oesophageal cancer has short survival, it is likely pre-diagnosis health behaviours will have carry-over effects on post-diagnosis survival times. Cancer registry data sets do not usually contain pre-diagnosis health behaviours and so need to be augmented with data from external health surveys. A new algorithm is introduced and tested to augment cancer registries with external data when one-to-one data linkage is not available.MethodsThe algorithm is to use external health survey data to impute pre-diagnosis health behaviour for cancer patients, estimate misclassification errors in these imputed values and then fit misclassification corrected Cox regression to quantify the association between pre-diagnosis health behaviour and post-diagnosis survival. Data from US cancer registries and a US national health survey are used in testing the algorithm.ResultsIt is demonstrated that the algorithm works effectively on simulated smoking data when there is no age confounding. But age confounding does exist (risk of death increases with age and most health behaviours change with age) and interferes with the performance of the algorithm. The estimate of the hazard ratio (HR) of pre-diagnosis smoking was HR = 1.32 (95% CI 0.82,2.68) with HR = 1.93 (95% CI 1.08,7.07) in the squamous cell sub-group and pre-diagnosis physical activity was protective of survival with HR = 0.25 (95% CI 0.03, 0.81). But the method failed for less common behaviours (such as heavy drinking).ConclusionsFurther improvements in the I2C2 algorithm will permit enrichment of cancer registry data through imputation of new variables with negligible risk to patient confidentiality, opening new research opportunities in cancer epidemiology.

This paper examines the market for initial coin offerings (ICOs). ICOs are smart contracts based on blockchain technology that are designed for entrepreneurs to raise external finance by issuing tokens without an intermediary. Unlike existing mechanisms for early-stage finance, tokens potentially provide investors with rapid opportunities thanks to liquid trading platforms. The marketability of tokens offers novel insights into entrepreneurial finance, which I explore in this paper. First, I document that investors earn on average 8.2% on the first day of trading. However, about 40% of all ICOs destroy investor value on the first day of trading. Second, I explore the determinants of market outcomes and find that management quality and the ICO profile are positively correlated with the funding amount and returns, whereas highly visionary projects have a negative effect. Among the 21% of all tokens that get delisted from a major exchange platform, highly visionary projects are more likely to fail, which investors anticipate. Third, I explore the sensitivity of the ICO market to adverse industry events such as China's ban of ICOs, the hack of leading ledgers, and the marketing ban on FaceBook. I find that the ICO market is highly susceptible to such environmental shocks, resulting in substantial welfare losses for investors.

Canine distemper virus (CDV) is a multi-host pathogen that can cause significant mortality in domestic, wild terrestrial and marine mammals. It is a major conservation threat in some endangered species. Infection can result in severe respiratory disease and fatal encephalitis. Diagnosis and disease monitoring in wildlife, and differentiation of CDV from rabies (a life-threatening zoonotic disease that can produce similar neurologic signs), would benefit from the availability of a portable, point-of-care (POC) diagnostic test. We therefore developed a quantitative RT-PCR assay for CDV using shelf-stable, lyophilized reagents and target-specific primers and probes for use with the handheld Biomeme two3™ qPCR thermocycler. Biomeme's extraction methodology, lyophilized reagents, and thermocycler were compared to our standard laboratory-based methods to assess sensitivity, efficiency and overall test performance. Results using a positive control plasmid for CDV showed comparable sensitivity (detection of 50 copies) and PCR efficiency between the two platforms, and CDV detection was similar between platforms when tested using a modified live CDV vaccine. Significantly higher Ct values (average Ct = 5.1 cycles) were observed using the Biomeme platform on known CDV positive animal samples. CDV detection using the Biomeme platform was similar in 25 of 26 samples from suspect CDV cases when compared to standard virology laboratory testing. One false positive was observed that was negative upon retest. The Biomeme methodology can be adapted for detection of specific targets, and this portable technology saves time by eliminating the need for local or international sample transport for laboratory-based diagnostics. However, results of our testing suggest that decreased diagnostic sensitivity (higher Ct values) relative to laboratory-based methods was observed using animal samples, so careful validation and optimization are essential. Portable qPCR platforms can empower biologists and wildlife health professionals in remote and low-resource settings, which will greatly improve our understanding of CDV disease ecology and associated conservation threats in wildlife.

OBJECTIVE:In breast cancer patients, treatment at the end of life accounts for a major share of medical spending. However, little is known about the variability of cost trajectories between patients. This study aims to identify underlying latent groups of advanced breast cancer patients with similar cost trajectories over the last year before death. METHODS:Data from deceased advanced breast cancer patients, diagnosed between 2010 and 2017, were retrieved from the Southeast Netherlands Advanced Breast Cancer (SONABRE) Registry. Costs of hospital care over the last twelve months before death were analyzed, and the variability of longitudinal patterns between patients were explored using group-based trajectory modeling. Descriptive statistics and multinomial logistic regression were applied to investigate differences between the identified latent groups. RESULTS:We included 558 patients. Over the last twelve months before death, mean hospital costs were €2,255 (SD = €492) per month. Costs increased over the last five months and reached a maximum of €3,614 in the last month of life, driven by hospital admissions, while spending for medication declined over the last three months of life. Based on patients' individual cost trajectories, we identified six latent groups with distinct longitudinal patterns, of which only two showed a marked increase in costs over the last twelve months before death. Latent groups were constituted of heterogeneous patients, and clinical characteristics explained membership only to a limited extent. CONCLUSIONS:The average costs of advanced breast cancer patients increased towards the end of life. However, we uncovered several latent groups of patients with divergent cost trajectories, which did not reflect the overall increasing trend. The mechanisms underlying the variability in cost trajectories warrants further research.

[This corrects the article DOI: 10.1371/journal.pone.0223827.].

Multiple tumors in patients are frequently diagnosed, either synchronous or metachronous. The distinction between a second primary and a metastasis is important for treatment. Chromosomal DNA copy number aberrations (CNA) patterns are highly unique to specific tumors. The aim of this study was to assess genome-wide CNA-patterns as method to identify clonally related tumors in a prospective cohort of patients with synchronous or metachronous tumors, with at least one intrapulmonary tumor. In total, 139 tumor pairs from 90 patients were examined: 35 synchronous and 104 metachronous pairs. Results of CNA were compared to histological type, clinicopathological methods (Martini-Melamed-classification (MM) and ACCP-2013-criteria), and, if available, EGFR- and KRAS-mutation analysis. CNA-results were clonal in 74 pairs (53%), non-clonal in 33 pairs (24%), and inconclusive in 32 pairs (23%). Histological similarity was found in 130 pairs (94%). Concordance between histology and conclusive CNA-results was 69% (74 of 107 pairs: 72 clonal and two non-clonal). In 31 of 103 pairs with similar histology, genetics revealed non-clonality. In two out of four pairs with non-matching histology, genetics revealed clonality. The subgroups of synchronous and metachronous pairs showed similar outcome for the comparison of histological versus CNA-results. MM-classification and ACCP-2013-criteria, applicable on 34 pairs, and CNA-results were concordant in 50% and 62% respectively. Concordance between mutation matching and conclusive CNA-results was 89% (8 of 9 pairs: six clonal and two non-clonal). Interestingly, in one patient both tumors had the same KRAS mutation, but the CNA result was non-clonal. In conclusion, although some concordance between histological comparison and CNA profiling is present, arguments exist to prefer extensive molecular testing to determine whether a second tumor is a metastasis or a second primary.

Floodplain wetland ecosystems respond dynamically to flooding, fire and geomorphological processes. We employed a combined geomorphological and environmental proxy approach to assess allochthonous and autochthonous macro-charcoal accumulation in the Macquarie Marshes, Australia, with implications for the reconstruction of fire regimes and environmental conditions in large, open-system wetlands. After accounting for fluvial macro-charcoal flux (1.05 ± 0.32 no. cm-2 a-1), autochthonous macro-charcoal in ~1 m deep sediment profiles spanning ~1.7 ka were highly variable and inconsistent between cores and wetlands (concentrations from 0 to 438 no. cm-3, mean accumulation rates from 0 to 3.86 no. cm-2 a-1). A positive correlation existed between the number of recent fires, satellite-observed ignition points, and macro-charcoal concentrations at the surface of the wetlands. Sedimentology, geochemistry, and carbon stable isotopes (δ13C range -15 to -25 ‰) were similar in all cores from both wetlands and varied little with depth. Application of macro-charcoal and other environmental proxy techniques is inherently difficult in large, dynamic wetland systems due to variations in charcoal sources, sediment and charcoal deposition rates, and taphonomic processes. Major problems facing fire history reconstruction using macro-charcoal records in these wetlands include: (1) spatial and temporal variations in fire activity and ash and charcoal products within the wetlands, (2) variations in allochthonous inputs of charcoal from upstream sources, (3) tendency for geomorphic dynamism to affect flow dispersal and sediment and charcoal accumulation, and (4) propensity for post-depositional modification and/or destruction of macro-charcoal by flooding and taphonomic processes. Recognition of complex fire-climate-hydrology-vegetation interactions is essential. High-resolution, multifaceted approaches with reliable geochronologies are required to assess spatial and temporal patterns of fire and to reconstruct in order to interpret wetland fire regimes.

The land iguanas, Conolophus pallidus and Conolophus subcristatu are large and charismatic lizards endemic to the Galápagos archipelago, but little information exists on their normal health parameters. The former is restricted to Santa Fe island, while C. subcristatus inhabits the islands of the central and western region of the archipelago. Both species are classified as vulnerable by the IUCN Red List of Threatened Species. As part of a population health assessment authorized by the Galápagos National Park, wild adult iguanas from three islands (North Seymour, South Plazas, and Santa Fe) were captured in July 2018. Data from a single C. subcristatus X Amblyrhynchus cristatus hybrid captured on South Plazas is also included. We analyzed blood samples drawn from 52 healthy wild adult land iguanas captured on three islands. An iSTAT portable blood analyzer was used to obtain values for pH, lactate, pO2, pCO2, HCO3-, sO2%, hematocrit, packed cell volume (PCV), hemoglobin Na, K, iCa, and glucose. Standard laboratory hematology techniques were employed for PCV determination; resulting values were also compared to the hematocrit values generated by the iSTAT. Body temperature, heart rate, respiratory rate, and body measurements were also recorded and compared to previously published data for the marine iguana (Amblyrhynchus cristatus), which shares a common ancestor with the land iguana. The data reported here provide preliminary baseline values that may be useful in comparisons between captive and wild populations, between wild populations, and in detecting changes in health status among Galápagos land iguanas affected by anthropogenic threats, climate change, or natural disturbances.

With the use of high-density multi-electrode recording devices, electrophysiological signals resulting from action potentials of individual neurons can now be reliably detected on multiple adjacent recording electrodes. Spike sorting assigns these signals to putative neural sources. However, until now, spike sorting can only be performed after completion of the recording, preventing true real time usage of spike sorting algorithms. Utilizing the unique propagation patterns of action potentials along axons detected as high-fidelity sequential activations on adjacent electrodes, together with a convolutional neural network-based spike detection algorithm, we introduce RT-Sort (Real Time Sorting), a spike sorting algorithm that enables the sorted detection of action potentials within 7.5ms±1.5ms (mean±STD) after the waveform trough while the recording remains ongoing. RT-Sorts true real-time spike sorting capabilities enable closed loop experiments with latencies comparable to synaptic delay times. We show RT-Sorts performance on both Multi-Electrode Arrays as well as Neuropixels probes to exemplify RT-Sorts functionality on different types of recording hardware and electrode configurations.

Metastasis is the most dreaded outcome after a breast cancer diagnosis, and little is known regarding what triggers or promotes breast cancer to spread distally, or how to prevent or eradicate metastasis effectively. Bilateral breast cancers are an uncommon form of breast cancers. In our study, a percentage of bilateral breast cancers were clonally related based on copy number variation profiling. Whole exome sequencing and comparative sequence analysis revealed that a limited number of somatic mutations were acquired in this breast-to-breast metastasis that might promote breast cancer distant spread. One somatic mutation acquired was SIVA-D160N that displayed pro-metastatic phenotypes in vivo and in vitro. Over-expression of SIVA-D160N promoted migration and invasion of human MB-MDA-231 breast cancer cells in vitro, consistent with a dominant negative interfering function. When introduced via tail vein injection, 231 cells over-expressing SIVA-D160N displayed enhanced distant spread on IVIS imaging. Over-expression of SIVA-D160N promoted invasion and anchorage independent growth of mouse 4T1 breast cancer cells in vitro. When introduced orthotopically via mammary fat pad injection in syngeneic Balb/c mice, over-expression of SIVA-D160N in 4T1 cells increased orthotopically implanted mammary gland tumor growth as well as liver metastasis. Clonally related bilateral breast cancers represented a novel system to investigate metastasis and revealed a role of SIVA-D160N in breast cancer metastasis. Further characterization and understanding of SIVA function, and that of its interacting proteins, may elucidate mechanisms of breast cancer metastasis, providing clinically useful biomarkers and therapeutic targets.

Past research on interspecies communication has shown that animals can be trained to use Augmentative Interspecies Communication (AIC) devices, such as soundboards, to make simple requests of their caretakers. The recent uptake in AIC devices by hundreds of pet owners around the world offers a novel opportunity to investigate whether AIC is possible with owner-trained family dogs. To answer this question, we carried out two studies to test pet dogs ability to recognise and respond appropriately to food-related, play-related, and outside-related words on their soundboards. One study was conducted by researchers, and the other by citizen scientists who followed the same procedure. Further, we investigated whether these behaviours depended on the identity of the person presenting the word (unfamiliar person or dogs owner) and the mode of its presentation (spoken or produced by a pressed button). We find that dogs produced contextually appropriate behaviours for both play-related and outside-related words regardless of the identity of the person producing them and the mode in which they were produced. Therefore, pet dogs can be successfully taught by their owners to associate words recorded onto soundboard buttons to their outcomes in the real world, and they respond appropriately to these words even when they are presented in the absence of any other cues, such as the owners body language.

INTRODUCTION: Sepsis is a major cause of morbidity and mortality worldwide. In the updated, 2016 Sepsis-3 criteria, sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, where organ dysfunction can be represented by an increase in the Sequential Organ Failure Assessment (SOFA) score of 2 points or more. We sought to apply the Sepsis-3 criteria to characterise the septic cohort in the Amsterdam University Medical Centres database (Amsterdam UMCdb). METHODS: We examined adult intensive care unit (ICU) admissions in the Amsterdam UMCdb, which contains de-identified data for patients admitted to a mixed surgical-medical ICU at a tertiary academic medical centre in the Netherlands. We operationalised the Sepsis-3 criteria, defining organ dysfunction as an increase in the SOFA score of 2 points or more, while infection was defined as a new course of antibiotics or an escalation in antibiotic therapy, with at least one antibiotic given intravenously. Patients with sepsis were determined to be in septic shock if they additionally required the use of vasopressors and had a lactate level >2 mmol/L. RESULTS: We identified 18,221 ICU admissions from 16,408 patients in our cohort. There were 6,312 unique sepsis episodes, of which 30.2% met the criteria for septic shock. A total of 4,911/6,312 sepsis (77.8%) episodes occurred on ICU admission. Forty-seven percent of emergency medical admissions and 36.7% of emergency surgical admissions were for sepsis. Overall, there was a 12.5% ICU mortality rate; patients with septic shock had a higher ICU mortality rate (38.4%) than those without shock (11.4%). CONCLUSIONS: We successfully operationalised the Sepsis-3 criteria to the Amsterdam UMCdb, allowing the characterization and comparison of sepsis epidemiology across different centres.

BACKGROUND: Esophagectomy is a complex oncologic operation associated with high rates of postoperative complications. While respiratory and septic complications have been well-defined, the implications of acute kidney injury (AKI) remain unclear. Using a nationally representative database, we aimed to characterize the association of AKI with mortality, resource use, and 30-day readmission. METHODS: All adults undergoing elective esophagectomy with a diagnosis of esophageal or gastric cancer were identified in the 2010-2019 Nationwide Readmissions Database. Study cohorts were stratified based on presence of AKI. Multivariable regressions and Royston-Parmar survival analysis were used to evaluate the independent association between AKI and outcomes of interest. RESULTS: Of an estimated 40,438 patients, 3,210 (7.9%) developed AKI. Over the 10-year study period, the incidence of AKI increased from 6.4% to 9.7%. Prior radiation/chemotherapy and minimally invasive operations were associated with reduced odds of AKI, whereas public insurance coverage and concurrent infectious and respiratory complications had greater risk of AKI. After risk adjustment, AKI remained independently associated with greater odds of in-hospital mortality (AOR: 4.59, 95% CI: 3.62-5.83) and had significantly increased attributable costs ($112,000 vs $54,000) and length of stay (25.7 vs 13.3 days) compared to patients without AKI. Furthermore, AKI demonstrated significantly increased hazard of 30-day readmission (hazard ratio: 1.16, 95% CI: 1.01-1.32). CONCLUSIONS: AKI after esophagectomy is associated with greater risk of mortality, hospitalization costs, and 30-day readmission. Given the significant adverse consequences of AKI, careful perioperative management to mitigate this complication may improve quality of esophageal surgical care at the national level.

Lipids play very important roles in lung biology, mainly reducing the alveolar surface tension at the air-liquid interface thereby preventing end-expiratory collapse of the alveoli. In the present study we performed an extensive quantitative lipidomic analysis of mouse lung to provide the i) total lipid quantity, ii) distribution pattern of the major lipid classes, iii) composition of individual lipid species and iv) glycerophospholipid distribution pattern according to carbon chain length (total number of carbon atoms) and degree of unsaturation (total number of double bonds). We analysed and quantified 160 glycerophospholipid species, 24 sphingolipid species, 18 cholesteryl esters and cholesterol from lungs of a) newborn (P1), b) 15-day-old (P15) and c) 12-week-old adult mice (P84) to understand the changes occurring during postnatal pulmonary development. Our results revealed an increase in total lipid quantity, correlation of lipid class distribution in lung tissue and significant changes in the individual lipid species composition during postnatal lung development. Interestingly, we observed significant stage-specific alterations during this process. Especially, P1 lungs showed high content of monounsaturated lipid species; P15 lungs exhibited myristic and palmitic acid containing lipid species, whereas adult lungs were enriched with polyunsaturated lipid species. Taken together, our study provides an extensive quantitative lipidome of the postnatal mouse lung development, which may serve as a reference for a better understanding of lipid alterations and their functions in lung development and respiratory diseases associated with lipids.

BACKGROUND:First line chemotherapy is effective in 75 to 80% of patients with metastatic colorectal cancer (mCRC). We studied whether microRNA (miR) expression profiles can predict treatment outcome for first line fluoropyrimidine containing systemic therapy in patients with mCRC. METHODS:MiR expression levels were determined by next generation sequencing from snap frozen tumor samples of 88 patients with mCRC. Predictive miRs were selected with penalized logistic regression and posterior forward selection. The prediction co-efficients of the miRs were re-estimated and validated by real-time quantitative PCR in an independent cohort of 81 patients with mCRC. RESULTS:Expression levels of miR-17-5p, miR-20a-5p, miR-30a-5p, miR-92a-3p, miR-92b-3p and miR-98-5p in combination with age, tumor differentiation, adjuvant therapy and type of systemic treatment, were predictive for clinical benefit in the training cohort with an AUC of 0.78. In the validation cohort the addition of the six miR signature to the four clinicopathological factors demonstrated a significant increased AUC for predicting treatment response versus those with stable disease (SD) from 0.79 to 0.90. The increase for predicting treatment response versus progressive disease (PD) and for patients with SD versus those with PD was not significant. in the validation cohort. MiR-17-5p, miR-20a-5p and miR-92a-3p were significantly upregulated in patients with treatment response in both the training and validation cohorts. CONCLUSION:A six miR expression signature was identified that predicted treatment response to fluoropyrimidine containing first line systemic treatment in patients with mCRC when combined with four clinicopathological factors. Independent validation demonstrated added predictive value of this miR-signature for predicting treatment response versus SD. However, added predicted value for separating patients with PD could not be validated. The clinical relevance of the identified miRs for predicting treatment response has to be further explored.

Despite the important functions of PPARγ in various cell types of the lung, PPARγ-deficiency in club cells induces only mild emphysema. Peroxisomes are distributed in a similar way as PPARγ in the lung and are mainly enriched in club and AECII cells. To date, the effects of PPARγ-deficiency on the overall peroxisomal compartment and its metabolic alterations in pulmonary club cells are unknown. Therefore, we characterized wild-type and club cell-specific PPARγ knockout-mice lungs and used C22 cells to investigate the peroxisomal compartment and its metabolic roles in the distal airway epithelium by means of 1) double-immunofluorescence labelling for peroxisomal proteins, 2) laser-assisted microdissection of the bronchiolar epithelium and subsequent qRT-PCR, 3) siRNA-transfection of PPARγand PPRE dual-luciferase reporter activity in C22 cells, 4) PPARg inhibition by GW9662, 5) GC-MS based lipid analysis. Our results reveal elevated levels of fatty acids, increased expression of PPARα and PPRE activity, a strong overall upregulation of the peroxisomal compartment and its associated gene expression (biogenesis, α-oxidation, β-oxidation, and plasmalogens) in PPARγ-deficient club cells. Interestingly, catalase was significantly increased and mistargeted into the cytoplasm, suggestive for oxidative stress by the PPARγ-deficiency in club cells. Taken together, PPARα-mediated metabolic induction and proliferation of peroxisomes via a PPRE-dependent mechanism could compensate PPARγ-deficiency in club cells.

In many taxa, individual social traits appear to be consistent across time and context, thus meeting the criteria for animal personality. How these differences are maintained in response to changes in population density is unknown, particularly in large mammals, such as ungulates. Using a behavioral reaction norm (BRN) framework, we examined how among- and within-individual variation in social connectedness, measured using social network analyses, change as a function of population density. We studied a captive herd of elk (Cervus canadensis) separated into a group of male elk and a group of female elk. Males and females were exposed to three different density treatments and we recorded social associations between individuals with proximity-detecting radio-collars fitted to elk. We constructed social networks using dyadic association data and calculated three social network metrics reflective of social connectedness: eigenvector centrality, graph strength, and degree. Elk exhibited consistent individual differences in social connectedness across densities; however, they showed little individual variation in their response to changes in density, i.e., individuals oftentimes responded plastically, but in the same manner to changes in density. Female elk had highest connectedness at an intermediate density. In contrast, male elk increased connectedness with increasing density. Whereas this may suggest that the benefits of social connectedness outweigh the costs of increased competition at higher density for males, females appear to exhibit a threshold in social benefits (e.g. predator detection and forage information). Our study illustrates the importance of viewing social connectedness as a density-dependent trait, particularly in the context of plasticity. Moreover, we highlight the need to revisit our understanding of density dependence as a population-level phenomenon by accounting for consistent individual differences not only in social connectedness, but likely in other ecological processes (e.g., predator-prey dynamics, mate choice, disease transfer).

NCKX5 is an ion exchanger expressed mostly in pigment cells; however, the functional role for this protein in melanogenesis is not clear. A variant allele of SLC24A5, the gene encoding NCKX5, has been shown to correlate with lighter skin pigmentation in humans, indicating a key role for SLC24A5 in determining human skin colour. SLC24A5 expression has been found to be elevated in melanoma. Knockdown analyses have shown SLC24A5 to be important for pigmentation, but to date the function of this ion exchanger in melanogenesis has not been fully established. Our data suggest NCKX5 may have an alternative activity that is key to its role in the regulation of pigmentation. Here Xenopus laevis is employed as an in vivo model system to further investigate the function of NCKX5 in pigmentation. SLC24A5 is expressed in the melanophores as they differentiate from the neural crest and develop in the RPE of the eye. Morpholino knockdown and rescue experiments were designed to elucidate key residues and regions of the NCKX5 protein. Unilateral morpholino injection at the 2 cell stage resulted in a reduction of pigmentation in the eye and epidermis of one lateral side of the tadpole. Xenopus and human SLC24A5 can rescue the morpholino effects. Further rescue experiments including the use of ion exchange inactive SLC24A5 constructs raise the possibility that full ion exchanger function of NCKX5 may not be required for rescue of pigmentation.

Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied.ST from 56 patients included in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP) and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA), parvovirus associated arthritis, reactive arthritis and RA), disease outcome (resolving vs persistent) and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers.We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables.Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.

Participants in clinical trials frequently fail to appreciate key differences between research and clinical care. This phenomenon, known as therapeutic misconception, undermines informed consent to clinical research, but to date there have been no effective interventions to reduce it and concerns have been expressed that to do so might impede recruitment. We determined whether a scientific reframing intervention reduces therapeutic misconception without significantly reducing willingness to participate in hypothetical clinical trials.This prospective randomized trial was conducted from 2015 to 2016 to test the efficacy of an informed consent intervention based on scientific reframing compared to a traditional informed consent procedure (control) in reducing therapeutic misconception among patients considering enrollment in hypothetical clinical trials modeled on real-world studies for one of five disease categories. Patients with diabetes mellitus, hypertension, coronary artery disease, head/neck cancer, breast cancer, and major depression were recruited from medical clinics and a clinical research volunteer database. The primary outcomes were therapeutic misconception, as measured by a validated, ten-item Therapeutic Misconception Scale (range = 10-50), and willingness to participate in the clinical trial.154 participants completed the study (age range, 23-87 years; 92.3% white, 56.5% female); 74 (48.1%) had been randomized to receive the experimental intervention. Therapeutic misconception was significantly lower (p = 0.004) in the scientific reframing group (26.4, 95% CI [23.7 to 29.1] compared to the control group (30.9, 95% CI [28.4 to 33.5], and remained so after controlling for education (p = 0.017). Willingness to participate in the hypothetical trial was not significantly different (p = 0.603) between intervention (52.1%, 95% CI [40.2% to 62.4%]) and control (56.3%, 95% CI [45.3% to 66.6%] groups.An enhanced educational intervention augmenting traditional informed consent led to a meaningful reduction in therapeutic misconception without a statistically significant change in willingness to enroll in hypothetical clinical trials. Additional study of this intervention is required in real-world clinical trials.

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a 'phenotype of interest' (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with 'Fuzzy-Spreader'-like morphologies were also identified through a visual screen. The second, a 'mutant of interest' (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid.

OBJECTIVES:Hematoma quality (especially the fibrin matrix) plays an important role in the bone healing process. Here, we investigated the effect of interleukin-1 beta (IL-1β) on fibrin clot formation from platelet-poor plasma (PPP). METHODS:Five-milliliter of rat whole-blood samples were collected from the hepatic portal vein. All blood samples were firstly standardized via a thrombelastograph (TEG), blood cell count, and the measurement of fibrinogen concentration. PPP was prepared by collecting the top two-fifths of the plasma after centrifugation under 400 × g for 10 min at 20°C. The effects of IL-1β cytokines on artificial fibrin clot formation from PPP solutions were determined by scanning electronic microscopy (SEM), confocal microscopy (CM), turbidity, and clot lysis assays. RESULTS:The lag time for protofibril formation was markedly shortened in the IL-1β treatment groups (243.8 ± 76.85 in the 50 pg/mL of IL-1β and 97.5 ± 19.36 in the 500 pg/mL of IL-1β) compared to the control group without IL-1β (543.8 ± 205.8). Maximal turbidity was observed in the control group. IL-1β (500 pg/mL) treatment significantly decreased fiber diameters resulting in smaller pore sizes and increased density of the fibrin clot structure formed from PPP (P < 0.05). The clot lysis assay revealed that 500 pg/mL IL-1β induced a lower susceptibility to dissolution due to the formation of thinner and denser fibers. CONCLUSION:IL-1β can significantly influence PPP fibrin clot structure, which may affect the early bone healing process.

OBJECTIVE:To investigate whether exhaled breath analysis using an electronic nose can identify differences between inflammatory joint diseases and healthy controls. METHODS:In a cross-sectional study, the exhaled breath of 21 rheumatoid arthritis (RA) and 18 psoriatic arthritis (PsA) patients with active disease was compared to 21 healthy controls using an electronic nose (Cyranose 320; Smiths Detection, Pasadena, CA, USA). Breathprints were analyzed with principal component analysis, discriminant analysis, and area under curve (AUC) of receiver operating characteristics (ROC) curves. Volatile organic compounds (VOCs) were identified by gas chromatography and mass spectrometry (GC-MS), and relationships between breathprints and markers of disease activity were explored. RESULTS:Breathprints of RA patients could be distinguished from controls with an accuracy of 71% (AUC 0.75, 95% CI 0.60-0.90, sensitivity 76%, specificity 67%). Breathprints from PsA patients were separated from controls with 69% accuracy (AUC 0.77, 95% CI 0.61-0.92, sensitivity 72%, specificity 71%). Distinction between exhaled breath of RA and PsA patients exhibited an accuracy of 69% (AUC 0.72, 95% CI 0.55-0.89, sensitivity 71%, specificity 72%). There was a positive correlation in RA patients of exhaled breathprints with disease activity score (DAS28) and number of painful joints. GC-MS identified seven key VOCs that significantly differed between the groups. CONCLUSIONS:Exhaled breath analysis by an electronic nose may play a role in differential diagnosis of inflammatory joint diseases. Data from this study warrant external validation.

Little is known regarding the extent by which patients undergoing outpatient cataract surgery are at risk for postoperative hospitalization. We sought to determine the percentage of patients undergoing cataract surgery who were subsequently hospitalized, the patient characteristics associated with postoperative hospitalization, and the reasons for hospitalization.We identified all beneficiaries of a large U.S. managed care network age ≥40 years old who underwent ≥1 cataract surgery from 2001-2011. All enrollees who required inpatient hospitalization within 7, 14, 30, and 90 days following initial cataract surgery and the reasons for hospitalization were determined. Logistic regression was performed to assess factors that significantly impacted the odds of requiring postoperative hospitalization.Among the 64,981 patients who underwent cataract surgery, rates of hospitalization within 7, 14, 30, and 90 days were 0.3%, 0.5%, 1.3% and 4.2%, respectively. Among the 10,674 patients who had no major preexisting medical comorbidities, 0.1% were hospitalized within 7 days. The odds of hospitalization increased by 35% (OR = 1.35 [CI, 1.23-1.48]) with the presence of each additional comorbidity and by 14% with each additional hospitalization in the 3 years prior to cataract surgery (OR = 1.14 [CI, 1.10-1,18]). Those who were hospitalized in the 30 days prior to cataract surgery had 524% increased odds of being hospitalized within 7 days after cataract surgery (OR = 6.24, [CI, 3.37-11.57]) compared to those with no record of preoperative hospitalization. Postoperative hospitalizations were most commonly due to cardiovascular conditions, comprising over 25% of primary diagnoses associated with hospitalization.The risk of hospitalization after cataract surgery is low, and is very low among those with no major preexisting medical comorbidities. Opportunities may exist to limit comprehensive preoperative evaluation and testing to those who have serious pre-existing medical comorbidities.

Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45-150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6-8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for 24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals.

Here we present the results of a large-scale bioinformatics annotation of non-coding RNA loci in 48 avian genomes. Our approach uses probabilistic models of hand-curated families from the Rfam database to infer conserved RNA families within each avian genome. We supplement these annotations with predictions from the tRNA annotation tool, tRNAscan-SE and microRNAs from miRBase. We identify 34 lncRNA-associated loci that are conserved between birds and mammals and validate 12 of these in chicken. We report several intriguing cases where a reported mammalian lncRNA, but not its function, is conserved. We also demonstrate extensive conservation of classical ncRNAs (e.g., tRNAs) and more recently discovered ncRNAs (e.g., snoRNAs and miRNAs) in birds. Furthermore, we describe numerous "losses" of several RNA families, and attribute these to either genuine loss, divergence or missing data. In particular, we show that many of these losses are due to the challenges associated with assembling avian microchromosomes. These combined results illustrate the utility of applying homology-based methods for annotating novel vertebrate genomes.

We have previously shown that overweight may increase the risk of developing rheumatoid arthritis (RA) in autoantibody positive individuals. Adipose tissue could contribute to the development of RA by production of various bioactive peptides. Therefore, we examined levels of adipokines in serum and synovial tissue of subjects at risk of RA.Fifty-one individuals positive for immunoglobulin M rheumatoid factor (IgM-RF) and/or anti-citrullinated protein antibodies (ACPA), without arthritis, were included in this prospective study. Levels of adiponectin, vaspin, resistin, leptin, chemerin and omentin were determined in baseline fasting serum samples (n = 27). Synovial tissue was obtained by arthroscopy at baseline and we examined the expression of adiponectin, resistin and visfatin by immunohistochemistry.The development of clinically manifest arthritis after follow-up was associated with baseline serum vaspin levels (HR1.5 (95% CI 1.1 to 2.2); p = 0.020), also after adjustment for overweight (HR1.7 (95% CI 1.1 to 2.5); p = 0.016). This association was not seen for other adipokines. Various serum adipokine levels correlated with BMI (adiponectin r = -0.538, leptin r = 0.664; chemerin r = 0.529) and systemic markers of inflammation such as CRP levels at baseline (adiponectin r = -0.449, omentin r = -0.557, leptin r = 0.635, chemerin r = 0.619, resistin r = 0.520) and ESR (leptin r = 0.512, chemerin r = 0.708), p-value

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting "Colony Forming Units". To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.

INTRODUCTION:The cholinergic anti-inflammatory pathway can downregulate inflammation via the release of acetylcholine (ACh) by the vagus nerve. This neurotransmitter binds to the α7 subunit of nicotinic acetylcholine receptors (α7nAChR), expressed on macrophages and other immune cells. We tested the pharmacological and functional profile of two novel compounds, PMP-311 and PMP-072 and investigated their role in modulating collagen-induced arthritis (CIA) in mice. METHODS:Both compounds were characterized with binding, electrophysiological, and pharmacokinetic studies. For in vivo efficacy studies in the CIA model the compounds were administered daily by oral gavage from day 20 till sacrifice at day 34. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. RESULTS:Treatment with PMP-311 was effective in preventing disease onset, reducing clinical signs of arthritis, and reducing synovial inflammation and bone destruction. PMP-072 also showed a trend in arthritis reduction at all concentrations tested. The data showed that while both compounds bind to α7nAChR with high affinity, PMP-311 acts like a classical agonist of ion channel activity, and PMP-072 can actually act as an ion channel antagonist. Moreover, PMP-072 was clearly distinct from typical competitive antagonists, since it was able to act as a silent agonist. It synergizes with the allosteric modulator PNU-120596, and subsequently activates desensitized α7nAChR. However, PMP-072 was less efficacious than PMP-311 at both channel activation and desensitization, suggesting that both conducting and non-conducting states maybe of importance in driving an anti-inflammatory response. Finally, we found that the anti-arthritic effect can be observed despite limited penetration of the central nervous system. CONCLUSIONS:These data provide direct evidence that the α7nAChR in immune cells does not require typical ion channel activation to exert its antiinflammatory effects.

Previous studies have suggested the existence of enteropathy in cystic fibrosis (CF), which may contribute to intestinal function impairment, a poor nutritional status and decline in lung function. This study evaluated enterocyte damage and intestinal inflammation in CF and studied its associations with nutritional status, CF-related morbidities such as impaired lung function and diabetes, and medication use.Sixty-eight CF patients and 107 controls were studied. Levels of serum intestinal-fatty acid binding protein (I-FABP), a specific marker for enterocyte damage, were retrospectively determined. The faecal intestinal inflammation marker calprotectin was prospectively studied. Nutritional status, lung function (FEV1), exocrine pancreatic insufficiency (EPI), CF-related diabetes (CFRD) and use of proton pump inhibitors (PPI) were obtained from the medical charts.Serum I-FABP levels were elevated in CF patients as compared with controls (p

Despite the success of genome-wide association studies (GWAS) in detecting a large number of loci for complex phenotypes such as rheumatoid arthritis (RA) susceptibility, the lack of information on the causal genes leaves important challenges to interpret GWAS results in the context of the disease biology. Here, we genetically fine-map the RA risk locus at 19p13 to define causal variants, and explore the pleiotropic effects of these same variants in other complex traits. First, we combined Immunochip dense genotyping (n = 23,092 case/control samples), Exomechip genotyping (n = 18,409 case/control samples) and targeted exon-sequencing (n = 2,236 case/controls samples) to demonstrate that three protein-coding variants in TYK2 (tyrosine kinase 2) independently protect against RA: P1104A (rs34536443, OR = 0.66, P = 2.3 x 10(-21)), A928V (rs35018800, OR = 0.53, P = 1.2 x 10(-9)), and I684S (rs12720356, OR = 0.86, P = 4.6 x 10(-7)). Second, we show that the same three TYK2 variants protect against systemic lupus erythematosus (SLE, Pomnibus = 6 x 10(-18)), and provide suggestive evidence that two of the TYK2 variants (P1104A and A928V) may also protect against inflammatory bowel disease (IBD; P(omnibus) = 0.005). Finally, in a phenome-wide association study (PheWAS) assessing >500 phenotypes using electronic medical records (EMR) in >29,000 subjects, we found no convincing evidence for association of P1104A and A928V with complex phenotypes other than autoimmune diseases such as RA, SLE and IBD. Together, our results demonstrate the role of TYK2 in the pathogenesis of RA, SLE and IBD, and provide supporting evidence for TYK2 as a promising drug target for the treatment of autoimmune diseases.

Proof of concept for local gene therapy for the treatment of arthritis with immunomodulatory cytokine interferon beta (IFN-β) has shown promising results in animal models of rheumatoid arthritis (RA). For the treatment of RA patients, we engineered a recombinant adeno-associated serotype 5 vector (rAAV5) encoding human (h)IFN-β under control of a nuclear factor κB promoter (ART-I02).The potency of ART-I02 in vitro as well as biodistribution in vivo in arthritic animals was evaluated to characterize the vector prior to clinical application. ART-I02 expression and bioactivity after transduction was evaluated in fibroblast-like synoviocytes (FLS) from different species. Biodistribution of the vector after local injection was assessed in a rat adjuvant arthritis model through qPCR analysis of vector DNA. In vivo imaging was used to investigate transgene expression and kinetics in a mouse collagen induced arthritis model.Transduction of RA FLS in vitro with ART-I02 resulted in high expression levels of bioactive hIFN-β. Transduction of FLS from rhesus monkeys, rodents and rabbits with ART-I02 showed high transgene expression, and hIFN-β proved bioactive in FLS from rhesus monkeys. Transgene expression and bioactivity in RA FLS were unaltered in the presence of methotrexate. In vivo, vector biodistribution analysis in rats after intra-articular injection of ART-I02 demonstrated that the majority of vector DNA remained in the joint (>93%). In vivo imaging in mice confirmed local expression of rAAV5 in the knee joint region and demonstrated rapid detectable and sustained expression up until 7 weeks.These data show that hIFN-β produced by RA FLS transduced with ART-I02 is bioactive and that intra-articular delivery of rAAV5 drives expression of a therapeutic transgene in the joint, with only limited biodistribution of vector DNA to other tissues, supporting progress towards a phase 1 clinical trial for the local treatment of arthritis in patients with RA.

Every person carries a vast repertoire of CD4+ T-helper cells and CD8+ cytotoxic T cells for a healthy immune system. Somatic VDJ recombination at genomic loci that encode the T-cell receptor (TCR) is a key step during T-cell development, but how a single T cell commits to become either CD4+ or CD8+ is poorly understood. To evaluate the influence of TCR sequence variation on CD4+/CD8+ lineage commitment, we sequenced rearranged TCRs for both α and β chains in naïve T cells isolated from healthy donors and investigated gene segment usage and recombination patterns in CD4+ and CD8+ T-cell subsets. Our data demonstrate that most V and J gene segments are strongly biased in the naïve CD4+ and CD8+ subsets with some segments increasing the odds of being CD4+ (or CD8+) up to five-fold. These V and J gene associations are highly reproducible across individuals and independent of classical HLA genotype, explaining ~11% of the observed variance in the CD4+ vs. CD8+ propensity. In addition, we identified a strong independent association of the electrostatic charge of the complementarity determining region 3 (CDR3) in both α and β chains, where a positively charged CDR3 is associated with CD4+ lineage and a negatively charged CDR3 with CD8+ lineage. Our findings suggest that somatic variation in different parts of the TCR influences T-cell lineage commitment in a predominantly additive fashion. This notion can help delineate how certain structural features of the TCR-peptide-HLA complex influence thymic selection.

Since high-density lipoprotein (HDL) has pro-endothelial and anti-thrombotic effects, a HDL recruiting stent may prevent restenosis. In the present study we address the functional characteristics of an apolipoprotein A-I (ApoA-I) antibody coating in vitro. Subsequently, we tested its biological performance applied on stents in vivo in rabbits.The impact of anti ApoA-I- versus apoB-antibody coated stainless steel discs were evaluated in vitro for endothelial cell adhesion, thrombin generation and platelet adhesion. In vivo, response to injury in the iliac artery of New Zealand white rabbits was used as read out comparing apoA-I-coated versus bare metal stents.ApoA-I antibody coated metal discs showed increased endothelial cell adhesion and proliferation and decreased thrombin generation and platelet adhesion, compared to control discs. In vivo, no difference was observed between ApoA-I and BMS stents in lumen stenosis (23.3±13.8% versus 23.3±11.3%, p=0.77) or intima surface area (0.81±0.62 mm2 vs 0.84±0.55 mm2, p=0.85). Immunohistochemistry also revealed no differences in cell proliferation, fibrin deposition, inflammation and endothelialization.ApoA-I antibody coating has potent pro-endothelial and anti-thrombotic effects in vitro, but failed to enhance stent performance in a balloon injury rabbit model in vivo.

Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone) electrospun scaffold (70:30 col/PCL) containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM), and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344) rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14%) over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold). Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration. Collectively these results suggest that microporous electrospun scaffolds pre-seeded with fibroblasts promote greater wound-healing than acellular scaffolds.

The transcription of four members of the Kunitz proteinase inhibitor (KPI) gene family of white clover (Trifolium repens L.), designated as Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5, was investigated at both local infection (roots) and systemic (leaf tissue) sites in white clover in response to infection with the clover root knot nematode (CRKN) Meloidogyne trifoliophila and the clover cyst nematode (CCN) Heterodera trifolii. Invasion by the CRKN resulted in a significant decrease in transcript abundance of Tr-KPI4 locally at both 4 days post-infection (dpi) and at 8 dpi, and an increase in transcription of Tr-KPI1 systemically at 8 dpi. In contrast, an increase in transcript abundance of all four Tr-KPI genes locally at 4 and 8 dpi, and an increase of Tr-KPI1, Tr-KPI2, and Tr-KPI5 at 8 dpi systemically was observed in response to infection with the CCN. Challenge of a resistant (R) genotype and a susceptible (S) genotype of white clover with the CCN revealed a significant increase in transcript abundance of all four Tr-KPI genes locally in the R genotype, while an increase in abundance of only Tr-KPI1, Tr-KPI2, and Tr-KPI5 was observed in the S genotype, and only at 4 dpi. The transcript abundance of a member of the1-AMINOCYCLOPROPANE-1-CARBOXYLATE (ACC) SYNTHASE gene family from white clover (Tr-ACS1) was significantly down-regulated locally in response to CRKN infection at 4 and 8 dpi and at 4 dpi, systemically, while abundance increased locally and systemically at 8 dpi in response to CCN challenge. Conversely, the abundance of the jasmonic acid (JA) signalling gene, CORONATINE-INSENSITIVE PROTEIN 1 from white clover (Tr-COI1) increased significantly at 8 dpi locally in response to CRKN infection, but decreased at 8 dpi in response to CCN infection. The significance of this differential regulation of transcription is discussed with respect to differences in infection strategy of the two nematode species.

Anthracnose caused by Colletotrichum lindemuthianum is a major disease of common bean (Phaseolus vulgaris) worldwide. Yellow beans are a major market class of common bean especially in eastern and southern Africa. The Yellow Bean Collection (YBC), which is comprised of 255 genotypes, and has not been used previously in genetic studies on anthracnose, is an excellent genetic resource for understanding the extent of anthracnose resistance and its genetic architecture in the yellow bean market class. The objectives of this study were i) evaluate the YBC for resistance to races 5, 19, 39, 51, 81, 183, 1050 and 1105 of C. lindemuthianum. and ii) conduct genome-wide association analysis to identify genomic regions and candidate genes associated with resistance to C. lindemuthianum. The YBC was genotyped with 72,866 SNPs, and genome-wide association analysis was conducted using Mixed Linear Model in TASSEL. Andean and Middle American genotypes with superior levels of resistance to the eight races were identified. YBC278 was the only one among 255 genotypes that was highly resistant to all eight races. Resistance to anthracnose in the YBC was controlled by major-effect loci on chromosomes Pv01, Pv03, Pv04, Pv05 and Pv07. The genomic region on Pv01, which overlapped with the Andean locus Co-1 provided resistance to races 81, 1050 and 1105. Significant SNPs for resistance to race 39 were identified on Pv02. The genomic region on Pv04, which overlapped with known major-effect loci Co-3, Co-15, Co-16, Co-y and Co-z, provided resistance to races 5, 19, 51 and 183. Novel genomic regions for resistance to race 39 were identified on Pv05 and Pv07. Plant resistance genes (R genes) with NB-ARC and LRR domains, which occurred in clusters, were identified as positional candidate genes for genomic regions on Pv02 and Pv04.

PURPOSE: Stunting is known to be heavily influenced by environmental factors, so the genetic contribution has received little attention. Here we report an exploration of genetic influences in stunted Zambian children with environmental enteropathy. METHOD: Children with stunting (LAZ < -2) were enrolled and given nutritional therapy. Those that were non-responsive to therapy were designated as cases, and children with good growth (LAZ > -1) from the same community as controls. Blood and stool samples were taken to measure biomarkers of intestinal inflammation, epithelial damage, and microbial translocation. Single nucleotide polymorphism array genotyping was carried out on saliva samples using the H3Africa consortium array. RESULTS: Genome wide associations were analysed in 117 cases and 41 controls. While no significant associations with stunting were observed at P

BACKGROUND: The prevalence of substance use in people with HIV (PWH) in the United States is higher than in the general population and is an important driver of HIV-related outcomes. We sought to assess if previously identified genetic associations that contribute to substance use are also observed in a population of PWH. METHODS: We performed genome-wide association studies (GWAS) of alcohol, smoking, and cannabis use phenotypes in a multi-ancestry population of 7,542 PWH from the Center for AIDS Research Network of Integrated Clinical Systems (CNICS). We conducted multi-ancestry GWAS for individuals of African (n = 3,748), Admixed American (n = 1,334), and European (n = 2,460) ancestry. Phenotype data were self-reported and collected using patient reported outcomes (PROs) and three questions from AUDIT-C, an alcohol screening tool. We analyzed nine phenotypes: 1) frequency of alcohol consumption, 2) typical number of drinks on a day when drinking alcohol, 3) frequency of five or more alcoholic drinks in a 30-day period, 4) smoking initiation, 5) smoking cessation, 6) cigarettes per day, 7) cannabis use initiation, 8) cannabis use cessation, 9) frequency of cannabis use during the previous 30 days. For each phenotype we considered a) variants previously identified as associated with a substance use trait and b) novel associations. RESULTS: We observed evidence for effects of previously reported single nucleotide polymorphisms (SNPs) related to alcohol (rs1229984, p = 0.001), tobacco (rs11783093, p = 2.22E-4), and cannabis use (rs2875907, p = 0.005). We also report two novel loci (19p13.2, p = 1.3E-8; and 20p11.21, p = 2.1E-8) associated with cannabis use cessation. CONCLUSIONS: Our analyses contribute to understanding the genetic bases of substance use in a population with relatively higher rates of use compared to the general population.

The All of Us (AoU) Research Program is making available one of the largest and most diverse collections of health data in the US to researchers. Using the All of Us database, we evaluated family and personal histories of five common types of cancer in 89,453 individuals, comparing these data to 24,305 participants from the 2015 National Health Interview Survey (NHIS). Comparing datasets, we found similar family cancer history (33%) rates, but higher personal cancer history in the AoU dataset (9.2% in AoU vs. 5.11% in NHIS), Methodological (e.g. survey-versus telephone-based data collection) and demographic variability may explain these between-data differences, but more research is needed.