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Abstract Background Few data are available regarding the epidemiology of invasive aspergillosis (IA) in ICU patients. The aim of this study was to examine epidemiology and economic outcomes (length of stay, hospital costs) among ICU patients with IA who lack traditional risk factors for IA, such as cancer, transplants, neutropenia or HIV infection. Methods Retrospective cohort study using Premier Inc. Perspective™ US administrative hospital database (2005–2008). Adults with ICU stays and aspergillosis (ICD-9 117.3 plus 484.6) who received initial antifungal therapy (AF) in the ICU were included. Patients with traditional risk factors (cancer, transplant, neutropenia, HIV/AIDS) were excluded. The relationship of antifungal therapy and co-morbidities to economic outcomes were examined using Generalized linear models. Results From 6,424 aspergillosis patients in the database, 412 (6.4%) ICU patients with IA were identified. Mean age was 63.9 years and 53% were male. Frequent co-morbidities included steroid use (77%), acute respiratory failure (76%) and acute renal failure (41%). In-hospital mortality was 46%. The most frequently used AF was voriconazole (71% received at least once). Mean length of stay (LOS) was 26.9 days and mean total hospital cost was $76,235. Each 1 day lag before initiating AF therapy was associated with 1.28 days longer hospital stay and 3.5% increase in costs (p Conclusions Invasive aspergillosis in ICU patients is associated with high mortality and hospital costs. Antifungal timing impacts economic outcomes. These findings underscore the importance of timely diagnosis, appropriate treatment, and consideration of Aspergillus as a potential etiology in ICU patients.

Abstract Background Ecosystem-based approaches for adaptation (EbA) integrate the use of biodiversity and ecosystem services into an overall strategy for helping people adapt to climate change. To date, insight into these approaches has often been based on reports from isolated anecdotal case studies. Although these are informative, and provide evidence that people are using ecosystems to adapt, they provide rather limited insight in terms of measuring and evaluating the effectiveness of EbA, especially when compared with technical or structural adaptation interventions. The body of scientific evidence indicating how effective such approaches are is lacking in some aspects. Where evidence does exist it is often dispersed across a range of related fields, such as natural resource management, disaster risk reduction and agroecology. To date, there has been little attempt to systematically assemble and analyse this evidence. Therefore, the current state of evidence regarding the merits or otherwise of EbA is unknown and it has not been possible to identify prevailing knowledge gaps to inform research and analysis, which will enable policymakers to compare EbA with other adaptation options. Methods This protocol details the methodology to be used to conduct a systematic map of peer-reviewed published journal papers and a limited selection of grey literature, to give a methodical overview of the state of the evidence base for EbA effectiveness, and to identify the current knowledge gaps. It addresses the following question: What is the state of the evidence base regarding the ability of ecosystem-based approaches for adaptation to help people adapt to the impacts of climate change?

Abstract Background To examine adiponectin levels in people with Latent Autoimmune Diabetes in Adults using a matched pair case control study. Findings Patients with LADA (n = 64), were matched for sex with type 2 diabetic and non-diabetic controls. A matched paired T-test was used to examine average adiponectin levels in the LADA patients' versus controls. The average adiponectin level for the LADA patients was 9.96 μg/ml compared to 6.4 μg/ml for Type 2 matched controls and 9.6 μg/ml for non-diabetic controls. Mean difference for the LADA-type 2 comparison was calculated after data was log transformed and showed a difference of 1.58 μg/ml (95%CI: 1.28-1.95, p = 0.0001). There was no significant difference between LADA and non-diabetic controls (p = 0.54). Conclusions Adiponectin levels are higher among people with LADA compared to those with type 2 diabetes and are equivalent to levels seen in non-diabetic controls. This suggests that risk of complications in LADA, as with type 1 diabetes may be related more to glycaemic control rather than to factors of the metabolic syndrome.

Abstract Background Latent autoimmune diabetes in adults [LADA] is a type 1 diabetes that is slowly developing. This means many people are treated as having type 2 diabetes at diagnosis as they are adults who are not immediately insulin dependent. LADA can be distinguished from type 2 diabetes by antibody tests. Patients who are antibody positive have an autoimmune reaction which is similar to that of type 1 diabetes and is not found in type 2 diabetes. We would like to examine the best way of treating LADA in the early phase of the conditions, with tablets (similar to type 2 diabetes) or with insulin (similar to type 1 diabetes). Methods/design This is an open parallel group prospective randomised trial. Participants need to have a GAD antibody test results of 101 WHO units or more and a diagnosis of diabetes not requiring insulin at diagnosis. Participants will need to have been diagnosed within 12 months and not treated with insulin at study entry. They will be randomised to receive either insulin (NovoMix 30) or tablets (diet treated followed by metformin followed by glitazone (with or without metformin) followed by insulin). Primary outcome assessment will be for change in HbA1c and change in fasting C-peptide over 24 months. Secondary outcome measures will include Quality of life, GAD antibody levels, adverse events, inflammatory markers, insulin resistance, and markers of the metabolic syndrome. Discussion This study seeks the best treatment for early LADA in terms of maintaining glycaemic control and maintaining natural insulin production. Trial registration ISRCTN63815121

Abstract Background Microalbuminuria and subsequent progression to proteinuria and nephropathy is associated with increased oxidative stress, increased inflammatory cytokines and increased cardiovascular (CVD) risk. The common functional IL-6 -174G>C gene variant is also associated with elevated levels of inflammatory cytokines and CVD risk. Methods The aim of this study was to examine the association between the IL-6 -174G>C gene variant with plasma total antioxidant status (TAOS) in 552 subjects with type 2 diabetes in relation to urinary protein excretion. Results In subjects free from CVD, there was a significant interaction between urinary protein excretion (normoalbuminuria/ microalbuminuria/proteinuria) and the -174C allele (compared to -174GG) in determining plasma TAOS (p value for interaction = 0.03). In the -174C allele carriers there was a significant association between plasma TAOS and urinary protein excretion: normalbuminuria v microalbuminuria v proteinuria: 44.30% ± 11.32 vs. 39.74% ± 14.83 vs. 37.93% ± 16.42, ANOVA p = 0.025. In those with CVD, no interaction or association was observed with the -174C allele (p = 0.246). Conclusion The IL-6 -174G>C gene variant is associated with differences in plasma oxidative stress in response to altered protein excretion in subjects with type 2 diabetes.

Background: Epigenetic modifications, including DNA methylation (DNAm), can play a role in the biological embedding of early-life adversity (ELA) through serotonergic mechanisms. The current study examines methylation of the CpG island in the promoter region of the stress-responsive serotonin transporter gene (SLC6A4) and is the first to jointly assess how it is influenced by ELA severity, timing, and type-specifically, deprivation and threat., Methods: We use data from 627 Youth Emotion Project study participants, recruited from two US high schools. Using adjusted linear regressions, we analyze DNA collected in early adulthood from 410 participants and ELA based on interviewer-rated responses from concurrent Childhood Trauma Interviews, adjusting for survey-measured covariates., Results: ELA robustly predicted mean CpG island SLC6A4 DNAm percent across 71 CpG sites. Each additional major-severity ELA event was associated with a 0.121-percentage-point increase (p<0.001), equating to a 0.177 standard deviation (sd) higher DNAm level (95 % CI: 0.080, 0.274) with each 1-sd higher adversity score. When modeled separately, both childhood and adolescent ELA predicted SLC6A4 DNAm. When modeled jointly, adolescent ELA was most strongly predictive, and child adversity remained significantly associated with DNAm through indirect associations via adolescent adversity. Additionally, the ELA-SLC6A4 DNAm association may vary by adversity type. Across separate models for childhood and adolescent exposures, deprivation coefficients are positive and statistically significant. Meanwhile, threat coefficients are positive and not significantly significant but do not statistically differ from deprivation coefficients. In models including all ELA dimensions, one major adolescent deprivation event is associated with a 0.222-percentage-point increased SLC6A4 DNAm (p<0.05), or a 1-sd higher deprivation score with a 0.157-sd increased DNAm., Conclusion: Results further implicate epigenetic modification on serotonergic neurotransmission via DNAm in the downstream sequelae of ELA-particularly adolescent deprivation-and support preventive interventions in adolescence to mitigate biological embedding., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024. Published by Elsevier Ltd.)

Landmark studies have shown decreased coherence between different emotion response systems (e.g., physiology and facial expressions) in people with psychosis. However, while there is good evidence to suggest broad signs of affective dysfunction (e.g., blunting of facial expression) in the critical clinical high-risk (CHR) state, it is not clear whether these signs fit into a broader pattern of decoupling. This is in part due to there being no studies to date with this population that include a dyadic interaction. The current laboratory-based dyadic interaction study examined whether there is decreased coherence in CHR between autonomic physiology, as indexed by heart rate, and facial expressions of emotion, assessed by automated facial expressions analysis. The study included 145 individuals consisting of 34 CHR-partner and 41 control-partner pairs who completed clinical interviews and engaged in three naturalistic 10-min conversations while their physiology and expressions were continuously monitored. Compared to controls, CHR youth showed decreased coherence between heart rate and positive ( t = 4.09) and negative ( t = -7.90) facial expressions. Across CHR and control youth, greater severity of psychosis risk symptoms was related to lower coherence between heart rate and positive ( t = 3.97-11.69) and neutral expressions ( t = 0.06-4.98), and a change in the direction of the relationship between heart rate and negative expression intensity ( t = 7.88-10.60). These findings provide the first evidence for changes in coherence between physiology and facial expressions of emotion in CHR individuals, with larger changes in coherence relating to greater general psychotic-like symptom severity. This evidence may be leveraged to identify targets for early diagnosis and intervention. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Putting feelings into words is often thought to be beneficial. Few studies, however, have examined associations between natural emotion word use and cardiovascular reactivity. This laboratory-based study examined emotion word use (i.e., from computerized text analysis) and cardiovascular reactivity (i.e., interbeat interval changes from baseline) across two interaction contexts (i.e., conflict and positive conversations) in 49 mixed-sex married couples (age: M = 43.11, SD = 9.20) from diverse socioeconomic backgrounds. We focused on both frequency (i.e., relative proportion of emotion words) and diversity (i.e., relative proportion of unique emotion words) of emotion words. Data were collected between 2015 and 2017 and analyzed treating both partners and conversations as repeated measures, resulting in 196 observations overall (four per dyad). Findings showed that (a) when spouses used more negative emotion words (especially anger), they showed higher cardiovascular reactivity. This finding was robust when controlling for covariates; generalized across gender, interaction contexts, and socioeconomic status. Moreover, (b) when spouses used a more diverse negative emotion vocabulary, they showed higher cardiovascular reactivity, but this was not robust when controlling for negative emotion word frequency. Associations between (c) positive emotion word use and cardiovascular reactivity were not statistically significant. Verbalizing negative emotions thus seems to go along with higher cardiovascular reactivity, at least in the short term. Replication is needed across other relationship types, genders, and sexual orientations. These findings highlight emotion word use as an indicator of cardiovascular reactivity, which has implications for the identification of potential health risks that emerge during marital interactions. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Purpose: Poor sleep is associated with short-term dysregulation of mood and is a risk factor for major depressive disorder (MDD). This study examines whether objectively measured sleep in late adolescence prospectively predicts major depressive episode (MDE) onset in early adulthood as well as whether daily affect mediates this association., Methods: The present study draws on subjective and objective sleep data, ecological momentary assessment, and diagnostic data from the longitudinal Youth Emotion Project to examine whether: a) short sleep predicts dysregulated ecological momentary assessment-measured mood the next day; b) sleep predicts depressive episodes over the subsequent 5 years; and c) dysregulated daily moods mediate the associations between short sleep and later MDD. Fixed effects, logistic regression, and formal mediation analyses were employed., Results: Our results showed that nights with less sleep are followed by days with more negative affect; short sleep predicted MDEs over the subsequent 5 years (adjusting for prior MDD); and negative affect mediates the relationship between short sleep and later MDEs., Discussion: Overall, our findings show sleep to be an important risk factor and hence a promising point of intervention for improving mood and reducing the risk of future MDEs in adolescents and early adults., (Copyright © 2023 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.)

Emotional acceptance is thought to play an important role in protecting mental health. However, few studies have examined emotional acceptance among older adults who may experience declines in functioning, including executive functioning. The present laboratory-based study examined whether emotional acceptance and (to determine specificity) detachment and positive reappraisal moderated links between executive functioning and mental health symptoms in a sample of healthy older adults. Emotion regulation strategies were measured using questionnaire-based measures (using established questionnaires) as well as performance-based measures (instructing individuals to use emotional acceptance, detachment, and positive reappraisal in response to sad film clips). Executive functioning was measured using a battery of working memory, inhibition, and verbal fluency tasks. Mental health symptoms were measured using questionnaires to assess anxiety and depressive symptoms. Results showed that (a) emotional acceptance moderated the link between executive functioning and mental health such that lower executive functioning predicted higher levels of anxiety and depressive symptoms at low but not at high levels of emotional acceptance. Moderation effects tended to be (b) stronger for emotional acceptance compared to the other emotion regulation strategies (though not all comparisons were statistically significant). Findings were (c) robust when controlling for age, gender, and education for questionnaire-based (but not performance-based) emotional acceptance. These findings contribute to the literature on emotion regulation specificity and highlight the mental health benefits of emotional acceptance in the face of low executive functioning. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Parent-child relationships are hotbeds of emotion and play a key role in mental health. The present proof-of-concept study examined facial expressions of emotion during adolescent-parent interactions and links with internalizing mental health symptoms. Neutral, negative, and positive facial expressions were objectively measured in 28 parent-adolescent dyads during three 10-min dyadic interactions. Internalizing mental health symptoms were measured using anxiety and depressive symptom questionnaires. Data were analyzed using actor-partner interdependence modeling. Results revealed that higher levels of (a) adolescents' neutral facial expressions as well as (b) parents' negative facial expressions were associated with higher levels of adolescents' mental health symptoms. Findings did not support a robust link between (c) positive expressions and mental health symptoms. Together, these results demonstrate the utility of facial expressions of emotion during parent-child interactions as behavioral correlates of adolescents' internalizing mental health symptoms, highlight the need for replication with larger samples, and suggest directions for future research. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

When confronted with an emotion prototype (e.g., loss), individuals may experience not only target emotions (e.g., sadness), but also nontarget emotions (emotions that are atypical or incongruent with an emotion prototype; e.g., gratitude in response to loss). What are the cognitive correlates of nontarget emotions? Drawing from models of emotion generation, the present laboratory-based study examined associations between aspects of executive functioning (i.e., working memory, inhibition, verbal fluency) and the subjective experience of positive and negative nontarget emotions in response to sad and awe film clips in 129 healthy older adults. Findings showed that (a) lower working memory was associated with higher levels of positive and negative nontarget (but not target) emotions in response to sad and awe film clips. Moreover, (b) associations were specific to working memory and not found for other aspects of executive functioning. Associations were (c) robust when accounting for age, gender, education, target emotion and physiological arousal (except for negative nontarget emotions in response to the sad film clips). Finally, (d) findings were driven by awe, happiness, calm, and gratitude for the sad film clips and disgust, fear, sadness, compassion, happiness, love, and excitement for the awe film clips. Overall, these findings show a link between lower working memory function and elevated nontarget emotional experiences in late life. Directions for future research are discussed. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

How individuals regulate emotions in the face of loss has important consequences for well-being and health, but we know little about which emotion regulation strategies are most effective for older adults for whom loss is ubiquitous. The present laboratory-based study examined effects of three emotion regulation strategies (i.e., detachment, positive reappraisal, or acceptance in response to film clips depicting loss) on subjective emotional experiences, physiology, and perceptions of emotion regulation success and motivation in healthy older adults (N = 129, age range = 64-83). Results showed that, first, detachment decreased emotional experiences across the board; positive reappraisal decreased negative and increased positive emotional experiences; while acceptance did not alter emotional experiences. Second, detachment decreased physiological arousal (driven by increases in interbeat interval and decreases in respiration rate) whereas positive reappraisal and acceptance did not alter physiological arousal compared with "just watch" trials. Third, individuals felt most successful and willing to put forth their best effort when implementing acceptance, while they felt least successful and least willing to put forth their best effort for positive reappraisal. These findings illuminate longstanding discussions regarding how individuals can best regulate emotions in the face of loss. They show that older adults can regulate their emotional experiences and (to a lesser extent) their physiology with detachment numbing emotional experiences and decreasing physiological arousal; positive reappraisal brightening emotional experiences; and acceptance resulting in the highest perceptions of success and motivation. Thus, each emotion regulation strategy appears to be most effective in specific domains for older adults. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Self-correction-a key feature distinguishing science from pseudoscience-requires that scientists update their beliefs in light of new evidence. However, people are often reluctant to change their beliefs. We examined belief updating in action by tracking research psychologists' beliefs in psychological effects before and after the completion of four large-scale replication projects. We found that psychologists did update their beliefs; they updated as much as they predicted they would, but not as much as our Bayesian model suggests they should if they trust the results. We found no evidence that psychologists became more critical of replications when it would have preserved their pre-existing beliefs. We also found no evidence that personal investment or lack of expertise discouraged belief updating, but people higher on intellectual humility updated their beliefs slightly more. Overall, our results suggest that replication studies can contribute to self-correction within psychology, but psychologists may underweight their evidentiary value., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: The COVID-19 pandemic has brought dramatic changes to the daily lives of U.S. adolescents, including isolation from friends and extended family, transition to remote learning, potential illness and death of loved ones, and economic distress. This study's purpose is to measure changes in adolescents' perceived stress and mood early in the pandemic., Methods: The present study drew from a racially and ethnically diverse sample of high school student participants in an ongoing intervention study in the Midwestern U.S., 128 of whom provided reports of their daily stress and mood both before (December 2017 to March 2020) and during (March-July 2020) the COVID-19 pandemic. We expected to see increases in perceived stress, declines in positive mood states, and increases in negative mood states, with larger impacts on individuals from households with lower parental education levels., Results: Multilevel models revealed increases in perceived stress primarily for adolescents from low/moderate education families during the pandemic. Impacts on mood states also diverged by education: adolescents from low/moderate education households reported feeling more ashamed, caring, and excited than before the pandemic, changes that were not shared by their peers from high education households. Although changes in mood that arose with the onset of the pandemic became less pronounced over time, increased levels of home- and health-related stress stayed high for low/moderate education adolescents., Conclusions: During the COVID-19 period, we observed disparate impacts on adolescents according to household education level, with more dramatic and negative changes in the emotional well-being of adolescents from low/moderate education households., (Copyright © 2021 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.)

Stress during pregnancy affects maternal health and well-being, as well as the health and well-being of the next generation, in part through the hypothalamic-pituitary-adrenal (HPA) axis. Although most studies have focused solely on proximal experiences (i.e., during the pregnancy) as sources of prenatal stress, there has been a recent surge in studies that examine maternal early life adversity as a source of stress system dysregulation during pregnancy. The current study of 178 pregnant women examined the association of economic and life stress experienced during two time periods (i.e., childhood and pregnancy) with maternal HPA axis activity during the third trimester of pregnancy. Findings indicated that a current annual income of less than $15,000 and greater childhood disadvantage were associated with a flatter diurnal cortisol slope. Childhood maltreatment, particularly sexual abuse, was associated with a higher cortisol awakening response (CAR), even when controlling for recent adversity. We found some evidence that past adversity moderates the relationship between current adversity and diurnal cortisol, specifically for economic adversity and waking cortisol. Overall, our findings indicate that early life stressors play an important and underappreciated role in shaping stress biology during pregnancy., (© 2020 Wiley Periodicals LLC.)

Memory decline is a concern for aging populations across the globe. Positive affect plays an important role in healthy aging, but its link with memory decline has remained unclear. In the present study, we examined associations between positive affect (i.e., feeling enthusiastic, attentive, proud, active) and memory (i.e., immediate and delayed recall), drawing from a 9-year longitudinal study of a national sample of 991 middle-age and older U.S. adults. Results revealed that positive affect was associated with less memory decline across 9 years when analyses controlled for age, gender, education, depression, negative affect, and extraversion. Findings generalized across another measure that assessed additional facets of positive affect, across different (but not all) facets of positive affect and memory, and across age, gender, and education; findings did not emerge for negative affect. Reverse longitudinal associations between memory and positive affect were not significant. Possible pathways linking positive affect and memory functioning are discussed.

The microbial community making up the gut microbiota can profoundly influence intestinal homeostasis and immune system development, and is believed to influence the development of complex diseases including type 1 diabetes (T1D). T1D susceptible nonobese diabetic (NOD) mice have been shown to harbor a distinct microbiota to disease-protected mice. We hypothesized that the T1D susceptible genetic background of NOD mice would be resistant to the introduction of a C57BL/6-derived microbiota. NOD and C57BL/6 mice were cohoused either continually from birth, from birth until weaning or from weaning onwards, allowing transfer of microbiota between the mice. Cohousing NOD with C57BL/6 mice from before birth, resulted in moderate changes to the gut microbiota, whereas initiating cohousing at weaning only led to minimal changes. Terminating cohousing at weaning reduced the changes in the microbiota composition. However, diabetes onset was not significantly delayed and there was no reduction in intestinal inflammation or the proportion of regulatory T cells in the cohoused NOD mice. However, insulin but not islet-specific glucose-6-phosphatase catalytic subunit-related protein-specific CD8 + T cells were reduced by cohousing suggesting an epitope-specific modulation of the autoreactive response by the gut microbiota. These results suggest that the T1D susceptible genetic background of the NOD mouse was resistant to the introduction of a C57BL/6-derived microbiota., (© 2018 Australasian Society for Immunology Inc.)

Purpose: This study assessed the: (1) effect of an LGBTQI + health equity curriculum (eQuality) on implicit attitudes among first (M1) and second year (M2) medical students and (2) utility of dedicated time to explore implicit bias., Method: Implicit biases were assessed at baseline using implicit association tests (IAT) for all M2s and a random sample of first years (M1A). These students were then debriefed on strategies to mitigate bias. Following eQuality, all M1 and M2s completed post-intervention IATs. The remaining first years (M1B) were then debriefed. Paired sample t-tests assessed differences between pre/post. Independent sample t-tests assessed differences in post-IATs between M1 groups., Results: IATs indicated preferences for "Straight," "White," and "Thin" at both pre and post. M2s demonstrated statistically significant improvements pre to post for sexuality (p = 0.01) and race (p = 0.03). There were significant differences in post-intervention IAT scores between M1As who received the IAT and debriefing prior to eQuality and M1Bs for sexuality (p = 0.002) and race (p = 0.046). There were no significant changes for weight., Conclusion: eQuality reduced implicit preference for "Straight" and "White." Differences in M1 post-intervention IAT scores between groups suggest dedicating time to debrief implicit attitudes enhances bias mitigation.

Following publication of the original article [1] it came to the attention of the Production Editor that Figs. 1 and 2 had not been replaced with the newly revised figures supplied by the authors (the originals being unusable due to poor quality image and text).

Background: Dysbiosis of the gut microbiota has been implicated in the pathogenesis of many autoimmune conditions including type 1 diabetes (T1D). It is unknown whether changes in the gut microbiota observed in T1D are due to environmental drivers, genetic risk factors, or both. Here, we have performed an analysis of associations between the gut microbiota and T1D genetic risk using the non-obese diabetic (NOD) mouse model of T1D and the TwinsUK cohort., Results: Through the analysis of five separate colonies of T1D susceptible NOD mice, we identified similarities in NOD microbiome that were independent of animal facility. Introduction of disease protective alleles at the Idd3 and Idd5 loci (IL2, Ctla4, Slc11a1, and Acadl) resulted in significant alterations in the NOD microbiome. Disease-protected strains exhibited a restoration of immune regulatory pathways within the gut which could also be reestablished using IL-2 therapy. Increased T1D disease risk from IL-2 pathway loci in the TwinsUK cohort of human subjects resulted in some similar microbiota changes to those observed in the NOD mouse., Conclusions: These findings demonstrate for the first time that type 1 diabetes-associated genetic variants that restore immune tolerance to islet antigens also result in functional changes in the gut immune system and resultant changes in the microbiota.

Multisample, nonindexed pooling combined with next-generation sequencing (NGS) was used to discover RET proto-oncogene sequence variation within a cohort known to be unaffected by multiple endocrine neoplasia type 2 (MEN2). DNA samples (113 Caucasians, 23 persons of other ethnicities) were amplified for RET intron 9 to intron 16 and then divided into 5 pools of <30 samples each before library prep and NGS. Two controls were included in this study, a single sample and a pool of 50 samples that had been previously sequenced by the same NGS methods. All 59 variants previously detected in the 50-pool control were present. Of the 61 variants detected in the unaffected cohort, 20 variants were novel changes. Several variants were validated by high-resolution melting analysis and Sanger sequencing, and their allelic frequencies correlated well with those determined by NGS. The results from this unaffected cohort will be added to the RET MEN2 database.

Multi-sample pooling and Illumina Genome Analyzer (GA) sequencing allows high throughput sequencing of multiple samples to determine population sequence variation. A preliminary experiment, using the RET proto-oncogene as a model, predicted ≤ 30 samples could be pooled to reliably detect singleton variants without requiring additional confirmation testing. This report used 30 and 50 sample pools to test the hypothesized pooling limit and also to test recent protocol improvements, Illumina GAIIx upgrades, and longer read chemistry. The SequalPrep(TM) method was used to normalize amplicons before pooling. For comparison, a single 'control' sample was run in a different flow cell lane. Data was evaluated by variant read percentages and the subtractive correction method which utilizes the control sample. In total, 59 variants were detected within the pooled samples, which included all 47 known true variants. The 15 known singleton variants due to Sanger sequencing had an average of 1.62 ± 0.26% variant reads for the 30 pool (expected 1.67% for a singleton variant [unique variant within the pool]) and 1.01 ± 0.19% for the 50 pool (expected 1%). The 76 base read lengths had higher error rates than shorter read lengths (33 and 50 base reads), which eliminated the distinction of true singleton variants from background error. This report demonstrated pooling limits from 30 up to 50 samples (depending on error rates and coverage), for reliable singleton variant detection. The presented pooling protocols and analysis methods can be used for variant discovery in other genes, facilitating molecular diagnostic test design and interpretation.

Determination of sequence variation within a genetic locus to develop clinically relevant databases is critical for molecular assay design and clinical test interpretation, so multisample pooling for Illumina genome analyzer (GA) sequencing was investigated using the RET proto-oncogene as a model. Samples were Sanger-sequenced for RET exons 10, 11, and 13-16. Ten samples with 13 known unique variants ("singleton variants" within the pool) and seven common changes were amplified and then equimolar-pooled before sequencing on a single flow cell lane, generating 36 base reads. For comparison, a single "control" sample was run in a different lane. After alignment, a 24-base quality score-screening threshold and 3; read end trimming of three bases yielded low background error rates with a 27% decrease in aligned read coverage. Sequencing data were evaluated using an established variant detection method (percent variant reads), by the presented subtractive correction method, and with SNPSeeker software. In total, 41 variants (of which 23 were singleton variants) were detected in the 10 pool data, which included all Sanger-identified variants. The 23 singleton variants were detected near the expected 5% allele frequency (average 5.17%+/-0.90% variant reads), well above the highest background error (1.25%). Based on background error rates, read coverage, simulated 30, 40, and 50 sample pool data, expected singleton allele frequencies within pools, and variant detection methods; >or=30 samples (which demonstrated a minimum 1% variant reads for singletons) could be pooled to reliably detect singleton variants by GA sequencing.

Premature infants receiving chronic total parenteral nutrition (TPN) due to feeding intolerance develop intestinal atrophy and reduced nutrient absorption. Although providing the intestinal trophic hormone glucagon-like peptide-2 (GLP-2) during chronic TPN improves intestinal growth and morphology, it is uncertain whether GLP-2 enhances absorptive function. We placed catheters in the carotid artery, jugular and portal veins, duodenum, and a portal vein flow probe in piglets before providing either enteral formula (ENT), TPN or a coinfusion of TPN plus GLP-2 for 6 days. On postoperative day 7, all piglets were fed enterally and digestive functions were evaluated in vivo using dual infusion of enteral ((13)C) and intravenous ((2)H) glucose, in vitro by measuring mucosal lactase activity and rates of apical glucose transport, and by assessing the abundances of sodium glucose transporter-1 (SGLT-1) and glucose transporter-2 (GLUT2). Both ENT and GLP-2 pigs had larger intestine weights, longer villi, and higher lactose digestive capacity and in vivo net glucose and galactose absorption compared with TPN alone. These endpoints were similar in ENT and GLP-2 pigs except for a lower intestinal weight and net glucose absorption in GLP-2 compared with ENT pigs. The enhanced hexose absorption in GLP-2 compared with TPN pigs corresponded with higher lactose digestive and apical glucose transport capacities, increased abundance of SGLT-1, but not GLUT-2, and lower intestinal metabolism of [(13)C]glucose to [(13)C]lactate. Our findings indicate that GLP-2 treatment during chronic TPN maintains intestinal structure and lactose digestive and hexose absorptive capacities, reduces intestinal hexose metabolism, and may facilitate the transition to enteral feeding in TPN-fed infants.

Crystals of phase II K2Cr2O7, potassium dichromate, space group P1 , grown from aqueous solution undergo a first-order transition to phase I, space group reportedly P21/n, at a phase-transition temperature, TPT, of 544 (2) K on first heating; the corresponding transition on cooling is at 502 (2) K. The endotherm on subsequent heatings occurs reproducibly at TPT = 531 (2) K. Mass loss between ca 531 and 544 K, identified as included water, is rapid and continues more slowly to higher temperatures for a total loss of ca 0.20%. The higher TPT on first heating is associated with the increasing pressure of superheated water occupying inclusion defects. The latent diagonal glide plane in phase II allows the structure of phase I to be inferred. The triclinic structure at 296 K has been independently redetermined. Normal probability analysis shows high consistency between the resulting and previous atomic coordinates, but with uncertainties reduced by a factor of ca 2. The earlier uncertainties are systematically underestimated by a comparable factor. The structure of phase IIb, space group A2/a on transposing axes, was determined at ca 300 K by Krivovichev et al. [Acta Cryst. (2000), C56, 629-630]. The first-order transition between phases I and II arises from the ca 60 degrees relative rotation of terminal O atoms in each tetrahedron as the n glide plane is gained or lost. A transition between phases IIb and I, also of first order, is likely but not between phases II and IIb. An intermediate phase may exist between phases IIb and I.

The mechanisms of bile acid uptake have been studied with primary monolayer cultures of rat hepatocytes. Hepatocytes were incubated with taurocholic acid (TC), glycocholic acid (GC), cholic acid (CA), glycochenodeoxycholic acid (GCDC), chenodeoxycholic acid (CDCA), deoxycholic acid (DOCA), lithocholic acid (LCA), or cholylglycylhistamine (CCH), a neutral bile acid derivative for 10 s to 60 min in medium containing sodium chloride, sodium chloride with 1 mM ouabain, or choline chloride. Cells were washed free of radioactive tracer, cell-associated radioactivity was quantitated, and bile acid uptake rates, kinetic parameters of uptake, and steady-state bile acid content were calculated. Two mechanisms for bile acid uptake were identified. Uptake of TC, GC, CA, and GCDC occurred predominantly via a sodium-dependent, ouabain-suppressible saturable mechanism, presumably sodium-coupled transport. Estimates of apparent Km and Vmax for these bile acids were TC, 33 micro M and 0.36 nmol . min-1 . mg prot-1; GC, 18 micro M and 0.22 nmol . min-1 . mg prot-1; CA, 13 micro M and 0.10 nmol . min-1 . mg prot; and GCDC, 6 micro M and 0.21 nmol . min-1 . mg prot, respectively. Uptake via this sodium-coupled mechanism exhibited considerable substrate selectivity. It was enhanced by increased ring hydroxylation and amino acid conjugation and decreased by further conjugation with a neutral histamine group (CGH). In contrast, uptake of CDCA, DOCA, LCA, and CGH occurred primarily via a nonsaturable sodium-independent mechanism, possibly simple diffusion. This mechanism accounted for only a small portion of uptake of TC, GC, CA, and GCDC at low bile acid concentrations. Nonsaturable bile acid uptake rates appeared to correlate with decane-buffer partition coefficients and to be related to bile acid structure.

Chloride is the predominant inorganic anion in bile, and it has been proposed that active chloride transport, possibly via a sodium-coupled mechanism, may contribute to that portion of canalicular bile formation not directly related to bile acid transport (bile acid-dependent bile formation or BAIBF). We have therefore examined the anion specificity of BAIBF using the isolated perfused rat liver and have studied sodium-chloride flux coupling and the sodium dependence of intracellular chloride content using 22Na and 36Cl transport by cultured rat hepatocytes. BAIBF by the isolated rat liver was unaltered by replacement of chloride with nitrate or benzenesulfonate but was significantly reduced by replacement of chloride with sulfate or thiocyanate. In cultured hepatocytes, sodium entry rate was reduced when chloride in the incubation medium was replaced by cyclamate, benzenesulfonate, or sulfate and mannitol but was unaffected when chloride was replaced by nitrate, gluconate, or thiocyanate. Conversely, chloride entry rate was decreased when sodium was replaced with choline but was unaffected when sodium was replaced by lithium or when ouabain was added to the medium. Thus no consistent evidence of sodium-chloride flux coupling was observed. Steady-state exchangeable intracellular chloride in the cultured hepatocytes was unaffected by ouabain or by replacement of sodium with choline and was increased when sodium was replaced by lithium. These findings indicate that basal BAIBF exhibits no specific chloride requirement. Although they do not exclude the possible existence in rat liver of sodium-coupled chloride transport, they provide no evidence that such a mechanism accounts for a major portion either of chloride transport by individual rat hepatocytes or of basal BAIBF by intact rat liver.

The binding of epidermal growth factor (EGF) to its plasma membrane receptor results in the stimulation of a tyrosyl residue-specific protein kinase, which has been shown to be part of the receptor. The mechanism by which EGF binding give rise to the stimulation of kinase activity is not understood in detail; however, a number of recent studies have implicated receptor dimerization or oligomerization in this process. We prepared Triton X-100 extracts of A431 cells in which the concentration of EGF receptors was on the order of 10(-7) M. When samples of the extracts were incubated with or without EGF and then treated with the high-yield cross-linking reagent bis(sulfosuccinimidyl)suberate (BS3), covalent receptor dimers could be detected in high yield in samples that had been treated with both EGF and BS3, whereas only monomeric receptor was detected in untreated samples or in samples that had been treated with either EGF or BS3. The yield of receptor dimers trapped by cross-linking correlated with the stimulation of autophosphorylation by EGF and with the concentration of EGF present. EGF-induced receptor dimers were also efficiently cross-linked in highly purified receptor preparations, suggesting that EGF-induced dimerization is a process intrinsic to the receptor, requiring no additional accessory proteins.

Transport of sodium, chloride, and taurocholate was studied in primary cultures of adult rat hepatocytes incubated in a balanced electrolyte solution containing 150 mM NaCl, various concentrations of taurocholate, and (22)Na, (36)Cl, [(3)H]taurocholate, and 3-O-[(3)H]methyl-D-glucose. Lithium chloride, choline chloride, or Na(2)SO(4) and mannitol were substituted isotonically for NaCl in selected studies. The steady-state intracellular concentrations of exchangeable sodium and chloride averaged 6.5 mM and 30.1 mM, respectively. Ouabain reversibly increased intracellular sodium concentration. Chloride entry rate was about double that of sodium. Unlike sodium entry, chloride entry rate increased nonlinearly with increasing extracellular concentration. Taurocholate entry exhibited both saturable and nonsaturable components; the former accounting for virtually all taurocholate uptake at concentrations comparable to those found in vivo. Taurocholate was actively concentrated by the cultured cells, with the steady-state intracellular-to-extracellular concentration ratio decreasing from over 50 to about 1 as extracellular taurocholate concentration was increased from 10 muM to 4 mM. Both the saturable uptake component and concentrative taurocholate transport were virtually abolished by substitution of choline or lithium for sodium or by addition of ouabain. Taurocholate entry rate first increased in a sigmoid fashion and then decreased as extracellular sodium concentration was increased from 0 to 150 mM. Sodium entry rate increased in the presence of added taurocholate with an average of one sodium ion accompanying each taurocholate molecule into the cell. These findings indicate that sodium and chloride differ strikingly in their mechanism and rate of entry into cultured rat hepatocytes and in their intracellular concentration. Moreover, hepatocytes concentrate taurocholate by a sodium-coupled mechanism with an apparently equimolar transport stoichiometry.

To characterize the transport mechanisms responsible for formation of canalicular bile, we have examined the effects of ion substitution on bile acid-dependent and bile acid-independent bile formation by the isolated perfused rat liver. Complete replacement of perfusate sodium with choline and lithium abolished taurocholate-induced choleresis and reduced biliary taurocholate output by greater than 70%. Partial replacement of perfusate sodium (25 of 128 mM) by choline reduced bile acid-independent bile formation by 30% and replacement of the remaining sodium (103 mM) by choline reduced bile acid-independent bile formation by an additional 64%. In contrast, replacement of the remaining sodium (103 mM) by lithium reduced bile acid-independent bile formation by only an additional 20%, while complete replacement of sodium (128 mM) by lithium reduced bile formation by only 17%, and lithium replaced sodium as the predominant biliary cation. Replacement of perfusate bicarbonate by Tricine, a zwitterionic amino acid buffer, decreased bile acid-independent bile formation by greater than or equal to 50% and decreased biliary bicarbonate output by approximately 60%, regardless of the accompanying cation. In separate experiments, replacement of sodium by lithium essentially abolished Na,K-ATPase activity measured either as ouabain-suppressible ATP hydrolysis in rat liver or kidney homogenates, or as ouabain-suppressible 86Rb uptake by cultured rat hepatocytes. These studies indicate that bile acid(taurocholate)-dependent bile formation by rat liver exhibits a specific requirement for sodium, a finding probably attributable to the role(s) of sodium in hepatic sodium-coupled taurocholate uptake and/or in maintenance of Na,K-ATPase activity. The surprising finding that bile acid-independent bile formation was substantially unaltered by complete replacement of sodium with the permeant cation lithium does not appear to be explained by Na,K-ATPase-mediated lithium transport. Although alternative interpretations exist, this observation is consistent with the hypothesis that much of basal bile acid-independent bile formation is attributable to an ion pump other than Na,K-ATPase, which directly or indirectly mediates bicarbonate transport.

The development of suberin lamellae in the hypodermis of Zea mays cv. LG 11 was observed by electron microscopy and the presence of suberin inferred from autoliuorescence and by Sudan black B staining in nodal (adventitious) and primary (seminal) root axes. Suberin lamellae were evident at a distance of 30-50 mm from the tip of roots growing at 20°C and became more prominent with distance from the tip. Both oxygen deficiency and growth at 13°C produced shorter roots in which the hypodermis was suberized closer to the root tip. There were no suberin lamellae in epidermal cells or cortical collenchyma adjacent to the hypodermis. Plasmodesmata were not occluded by the suberin lamellae: there were twice as many of them in the inner tangential hypodermal wall (1,14 μn -2 ) as in the junction between the epidermis and hypodermis (0.54 μm -2 ). Water uptake by seminal axes (measured by micropotometry) was greater at distances more than 100 mm from the root lip than in the apical zone where the hypodermis was unsuberized. In the more mature zones of roots grown at 13°C rates of water uptake were greater than in roots grown at 20°C even though hypodermal suberization was more marked. Sleeves of epidermal/hypodermal cells (plus some accessory collenchyma) were isolated from the basal 60 mm of nodal axes by enzymatic digestion (drisclase). The roots were either kept totally immersed in culture solution or had the basal 50 mm exposed to moist air above the solution surface. In both treatments the permeabilities to tritiated water and 86 Rb were low (circa 10 -5 mms -1 ) in sleeves isolated from the extreme base. In roots grown totally immersed, however, the permeability of sleeves increased 10 to 50-fold over a distance of 40 mm. In roots exposed to moist air the permeability remained at a low level until the point where the root entered the culture solution and then increased rapidly (> 50-fold in a distance of 8 mm). Growth of roots in oxygen depleted (5% O 2 ) solutions promoted the development of extensive cortical aerenchymas. These developments were not associated with any reduction in permeability of sleeves isolated from the basal 40 mm of the axis. It was concluded that the presence of suberin lamellae in hypodermal walls does not necessarily indicate low permeability of cells or tissues to water or solutes. The properties of the walls (lamellae?) can be greatly changed by exposure to moist air, perhaps due to increased oxygen availability.