Zebrafish fully regenerate lost bone, including after fin amputation, through a process mediated by dedifferentiated, lineage-restricted osteoblasts. Mechanisms controlling the osteoblast regenerative program from its initiation through reossification are poorly understood. We show that fin amputation induces a Wnt/β-catenin-dependent epithelial to mesenchymal transformation (EMT) of osteoblasts in order to generate proliferative Runx2+ preosteoblasts. Localized Wnt/β-catenin signaling maintains this progenitor population toward the distal tip of the regenerative blastema. As they become proximally displaced, preosteoblasts upregulate sp7 and subsequently mature into re-epithelialized Runx2−/sp7+ osteoblasts that extend preexisting bone. Auto-crine bone morphogenetic protein (BMP) signaling promotes osteoblast differentiation by activating sp7 expression and counters Wnt by inducing Dickkopf-related Wnt antagonists. As such, opposing activities of Wnt and BMP coordinate the simultaneous demand for growth and differentiation during bone regeneration. This hierarchical signaling network model provides a conceptual framework for understanding innate bone repair and regeneration mechanisms and rationally designing regenerative therapeutics.
Transgenic zebrafish research has provided valuable insights into gene functions and cell behaviors directing vertebrate development, physiology, and disease models. Most approaches use constitutive transgene expression and therefore do not provide control over the timing or levels of transgene induction. We describe an inducible gene expression system that uses new tissue-specific zebrafish transgenic lines that express the Gal4 transcription factor fused to the estrogen-binding domain of the human estrogen receptor. We show these Gal4-ERT driver lines confer rapid, tissue-specific induction of UAS-controlled transgenes following tamoxifen exposure in both embryos and adult fish. We demonstrate how this technology can be used to define developmental windows of gene function by spatiotemporal-controlled expression of constitutively active Notch1 in embryos. Given the array of existing UAS lines, the modular nature of this system will enable many previously intractable zebrafish experiments.
It is important to monitor alcohol use in the care of liver disease patients, but patient self-report can be unreliable. We therefore evaluated the performance of urine ethyl glucuronide (EtG) and ethyl sulfate (EtS) in detecting alcohol use in the days preceding a clinical encounter.
Subjects (n=120) were recruited at a university-based Hepatology clinic or during hospitalization. Alcohol consumption was ascertained by validated self-report measures. Urine EtG (cutoff 100 ng/mL) and EtS (cutoff 25 ng/mL) concentrations were assayed by a contracted laboratory using tandem mass spectrometry. The sensitivity and specificity of each biomarker in the detection of drinking during the 3 and 7 days preceding the clinic visit were determined, as well as the influence of liver disease severity on these results.
Urine EtG (sensitivity 76%, specificity 93%) and urine EtS (sensitivity 82%, specificity 86%) performed well in identifying recent drinking, and liver disease severity does not affect biomarker performance. After elimination of one false negative self-report, urine EtG > 100 ng/mL was 100% specific for drinking within the past week, whereas 9% of the subjects without evidence of alcohol drinking for at least one week had EtS > 25 ng/mL.
Urine EtG and EtS can objectively supplement the detection of recent alcohol use in patients with liver disease. Additional research may determine optimal methods for integrating these tests into clinical care.
alcohol drinking; biological markers; liver diseases; ethyl glucuronide; ethyl sulfate
Aims: Hair ethyl glucuronide (EtG) is a promising biomarker of moderate-to-heavy alcohol consumption and may have utility in detecting and monitoring alcohol use in clinical populations where alcohol use is of particular importance. This study evaluated the relationship between hair EtG and drinking in patients with liver disease. Methods: The subjects (n = 200) were patients with liver disease who presented for care at a university medical center. Alcohol use during the 3 months preceding participation in the study was assessed, and a sample of hair was obtained for EtG testing. Classification of drinking status (any drinking or averaging at least 28 g per day) by hair EtG was evaluated, as well as the effects of liver disease severity and demographic and hair care factors. Results: The area under the receiver operating characteristic curve for detecting an average of 28 g or more per day during the prior 90 days was 0.93. The corresponding sensitivity and specificity of hair EtG ≥8 pg/mg for averaging at least 28 g of ethanol per day were 92 and 87%, respectively. Cirrhosis and gender may have a modest influence on the relationship between drinking and hair EtG. Conclusion: Hair EtG was highly accurate in differentiating subjects with liver disease averaging at least 28 g of ethanol per day from abstainers and lighter drinkers.
Unlike humans, some vertebrate animals are able to completely regenerate damaged appendages and other organs. For example, adult zebrafish will regenerate the complex structure of an amputated caudal fin to a degree that the original and replacement fins are indistinguishable. The blastema, a mass of cells that uniquely forms following appendage amputation in regenerating animals, is the major source of regenerated tissue. However, the cell lineage(s) that contribute to the blastema and their ultimate contribution(s) to the regenerated fin have not been definitively characterized. It has been suggested that cells near the amputation site dedifferentiate forming multipotent progenitors that populate the blastema and then give rise to multiple cell types of the regenerated fin. Other studies propose that blastema cells are non-uniform populations that remain restricted in their potential to contribute to different cell lineages. We tested these models by using inducible Cre-lox technology to generate adult zebrafish with distinct, isolated groups of genetically labeled cells within the caudal fin. We then tracked populations of several cell types over the entire course of fin regeneration in individual animals. We found no evidence for the existence of multipotent progenitors. Instead, multiple cell types, including epidermal cells, intra-ray fibroblasts, and osteoblasts, contribute to the newly regenerated tissue while remaining highly restricted with respect to their developmental identity. Our studies further demonstrate that the regenerating fin consists of many repeating blastema “units” dedicated to each fin ray. These blastema each have an organized structure of lineage restricted, dedifferentiated cells that cooperate to regenerate the caudal fin.
regeneration; dedifferentiation; zebrafish; blastema; caudal fin
To summarize published data on pharmacologic treatments for alcohol dependence alone and in combination with brief psychosocial therapies that may be feasible for primary care and specialty medical settings.
We conducted electronic searches of published original research articles and reviews in MEDLINE, SCOPUS, CINAHL, Embase, and PsychINFO. In addition, hand searches of reference lists of review articles, supplemental searches of internet references and contacts with experts in the field were conducted. Randomized controlled studies published between January, 1960 and August, 2010 that met our inclusion/exclusion criteria were included.
A total of 85 studies, representing 18,937 subjects, met our criteria for inclusion. The evidence base for oral naltrexone (6% more days abstinent than placebo in the largest study) and topiramate (prescribed off-label) (e.g., 26.2% more days abstinent than placebo in a recent study) is positive but modest. Acamprosate shows modest efficacy with recently abstinent patients, with European studies showing better results than US ones. The evidence-base for disulfiram is equivocal. Depot naltrexone shows efficacy (25% greater reduction in rate of heavy drinking vs. placebo, in one of the largest studies) in a limited number of studies. Some studies suggest that patients do better with extensive psychosocial treatments added to medications while others show that brief support can be equally effective.
Although treatment effects are modest, medications for alcohol dependence, in conjunction with either brief support or more extensive psychosocial therapy, can be effective in primary and specialty care medical settings.
Alcoholism; naltrexone; acamprosate; disulfiram; topiramate
There is increasing emphasis on screening, brief intervention, and referral to treatment (SBIRT) for unhealthy alcohol use in the general hospital, as highlighted by new Joint Commission recommendations on SBIRT. However, the evidence supporting this approach is not as robust relative to primary care settings. This review is targeted to hospital-based clinicians and administrators who are responsible for generally ensuring the provision of high quality care to patients presenting with a myriad of conditions, one of which is unhealthy alcohol use. The review summarizes the major issues involved in caring for patients with unhealthy alcohol use in the general hospital setting, including prevalence, detection, assessment of severity, reduction in drinking with brief intervention, common acute management scenarios for heavy drinkers, and discharge planning. The review concludes with consideration of Joint Commission recommendations on SBIRT for unhealthy alcohol use, integration of these recommendations into hospital work flows, and directions for future research.
Alcohol drinking; Alcoholism; Hospitalization; Patient discharge
Vaccination against common pathogens, such as influenza, is recommended for SLE patients to decrease infections and improve health. However, most vaccination response reports are limited to evaluation of SLE patients with quiescent disease. This study focuses on understanding the clinical, serological, therapeutic, and demographic factors which influence the response to influenza vaccination in patients with a range of disease activities.
Blood specimens and disease activity information were collected from seventy-two SLE patients at baseline and 2, 6 and 12 weeks after influenza vaccination. Influenza-specific antibody responses were assessed for antibody concentration (Bmax), relative affinity (Ka), and hemagglutination inhibition (HAI). Using a cumulative score, the subjects were evenly divided into high and low responders. Autoantibody levels were evaluated at each time-point by immunofluorescence and standard ELISAs.
Low responders to the vaccine were more likely to have hematologic criteria (p=0.009), exhibit more ACR criteria (p=0.05), and be on concurrent prednisone treatment (p=0.04). Interestingly, European American patients were more likely to be low responders than African Americans (p = 0.03). Following vaccination, low responders were more likely to experience disease flares (p=0.01) and to have increased ANA titers (p = 0.04). Baseline serum interferon alpha activity was significantly higher in patients that experienced a flare after vaccination compared to a matched group of patients that did not flare (p= 0.04).
Ancestral background, prednisone treatment, hematological criteria and evidence of increased disease flares were associated with low antibody responses to influenza vaccination in SLE patients.
It had previously been suggested that individuals with cirrhosis may have a pattern of transferrin glycosylation that interferes with the interpretation of carbohydrate-deficient transferrin (CDT) testing for heavy alcohol use. The goal of this case series was to evaluate the prevalence of liver disease among individuals with poor resolution of transferrin glycoforms by high performance liquid chromatography.
We reviewed the electronic medical records of 35 consecutive patients with poor chromatographic resolution of disialotransferrin from trisialotransferrin, and recorded information on diagnosed liver disease, liver function testing, and other factors.
Thirty of the 35 subjects with poor chromatographic resolution of the transferrin glycoforms had sufficient data in the medical record for some estimation of liver function. Of these 30 subjects, 25 had previously diagnosed liver pathology. Of the remaining 5 subjects, 2 had liver imaging results suggestive of benign tumor; the remaining 3 had mildly elevated bilirubin and aminotransferase activity, and low albumin.
Liver abnormalities, but not necessarily cirrhosis, are common in individuals with poor chromatographic separation of transferrin glycoforms, which might lead to false positive results on CDT testing. However, the chromatographic-based assay can detect this issue, minimizing the reporting of false positives, but not necessarily assisting in valid detection of heavy drinking.
liver disease; carbohydrate-deficient transferrin
Both aging and loss of sex steroids have adverse effects on skeletal homeostasis, but whether and how they may influence each others negative impact on bone remains unknown. We report herein that both female and male C57BL/6 mice progressively lost strength (as determined by load-to-failure measurements) and bone mineral density in the spine and femur between the ages of 4 and 31 months. These changes were temporally associated with decreased rate of remodeling as evidenced by decreased osteoblast and osteoclast numbers and decreased bone formation rate; as well as increased osteoblast and osteocyte apoptosis, increased reactive oxygen species levels, and decreased glutathione reductase activity and a corresponding increase in the phosphorylation of p53 and p66shc, two key components of a signaling cascade that are activated by reactive oxygen species and influences apoptosis and lifespan. Exactly the same changes in oxidative stress were acutely reproduced by gonadectomy in 5-month-old females or males and reversed by estrogens or androgens in vivo as well as in vitro.We conclude that the oxidative stress that underlies physiologic organismal aging in mice may be a pivotal pathogenetic mechanism of the age-related bone loss and strength. Loss of estrogens or androgens accelerates the effects of aging on bone by decreasing defense against oxidative stress.
Fetal alcohol disorders are preventable, but self-reported alcohol consumption can be misleading and impede effective treatment. Biomarkers represent an alternative method for assessing alcohol use, and this study evaluated the relationship between blood phosphatidylethanol (PEth) and alcohol use in a sample of reproductive age women.
Alcohol use was estimated by validated self-report methods in 80 non-pregnant women ages 18 to 35. PEth was measured by a contracted laboratory using a liquid chromatography-tandem mass spectrometry assay. Regression methods appropriate for the distribution of PEth were used to define its relationship to alcohol consumption during the prior 2 weeks and explore the effects of drinking patterns on this association. Receiver operating characteristic analysis was used to estimate the sensitivity of PEth for various drinking levels at 95% specific cutoffs.
PEth had a positive linear association with grams of alcohol consumed (p<0.001), and was detectable in 93% of subjects consuming an average of 2 or more drinks per day. The relationship between total alcohol consumption and PEth may be stronger in women with recent heavy drinking days. The relationship between drinking and PEth varied considerably between individuals, and sensitivity for a certain amount of drinking was low at a highly specific cutoff concentration.
PEth is a highly sensitive indicator of moderate and heavy alcohol consumption in reproductive age women and may complement the use of self-report alcohol screens when additional objective markers of alcohol use are desirable. However, choosing a highly valid cutoff concentration for PEth to differentiate various levels of alcohol consumption may not be feasible.
phosphatidylethanol; alcohol drinking; women; biomarker
Aims: The goal of this preliminary study was to evaluate the relationship between blood phosphatidylethanol (PEth) and recent drinking in patients with liver disease and hypertension. Methods: Twenty-one patients with liver disease and 21 patients with essential hypertension were recruited at an academic medical center. Alcohol consumption was estimated using validated self-report methods, and blood PEth was measured by HPLC-MS/MS at a contracted laboratory. Nonparametric comparisons were made between abstainers/light drinkers, moderate drinkers consuming between 1 and 3 drinks per day, and those drinking above this level. Regression methods were used to estimate the effects of liver disease, gender, and age on the relationship between PEth and alcohol use, and to estimate the strength of the linear relationship between PEth and drinking. Results: PEth differed significantly between the three drinking groups (P < 0.001). The relationship between PEth and alcohol did not differ between hypertension and liver disease patients (P = 0.696), nor by gender and age. While there was substantial variability between subjects in the PEth concentration given a similar level of reported drinking, the amount of ethanol consumed was strongly associated with the PEth concentration (P < 0.001). Conclusion: Results support PEth measurement by HPLC-MS/MS as a promising marker of past 1- to 2-week moderate to heavy alcohol consumption in patients with and without liver disease. PEth appears useful for differentiating abstinence or light drinking from moderate to heavy consumption, but may have limited utility for differentiating moderate from heavy alcohol use.
Brief alcohol intervention may improve outcomes for injury patients with hazardous drinking but is less effective with increased severity of alcohol involvement. This study evaluated a brief method for detecting problem drinking in minor trauma patients and differentiating hazardous drinkers from those with more severe alcohol problems.
Subjects included 60 minor trauma patients in an academic urban emergency department (ED) who had consumed any amount of alcohol in the prior month. Screening and risk stratification involved the use of a heavy-drinking-day screening item and the Rapid Alcohol Problems Screen (RAPS). We compared the heavy-drinking-day item to past-month alcohol use, as obtained by validated self-reporting methods, and measured the percentage of carbohydrate-deficient transferrin (%CDT) to assess the accuracy of self-reporting. The Alcohol Dependence Scale (ADS) was administered to gauge the severity of alcohol involvement and compared to the RAPS.
Eighty percent of the subjects endorsed at least one heavy drinking day in the past year, and all patients who exceeded recommended weekly drinking limits endorsed at least one heavy drinking day. Among those with at least one heavy drinking day, 58% had a positive RAPS result. Persons with no heavy drinking days (n=12) had a median ADS of 0.5 (range 0 to 3). RAPS-negative persons with heavy drinking days (n=20) had a median ADS of 2 (range 0 to 8). RAPS-positive persons with heavy drinking days (n=28) had a median ADS of 8 (range 1 to 43).
A heavy-drinking-day item is useful for detecting hazardous drinking patterns, and the RAPS is useful for differentiating more problematic drinkers who may benefit from referral from those more likely to respond to a brief intervention. This represents a time-sensitive approach for risk-stratifying non-abstinent injury patients prior to ED discharge.
The long-horned beetle, Dectes texanus LeConte (Coleoptera: Cerambycidae), is a stem-boring pest of soybeans, Glycine max (L.) Merrill (Fabales: Fabaceae). Soybean stems and stubble were collected from 131 counties in Arkansas, Mississippi, Missouri, and Tennessee and dissected to determine D. texanus infestation rates. All states sampled had D. texanus present in soybeans. Data from Tennessee and Arkansas showed sample infestations of D. texanus averaging nearly 40%. Samples from Missouri revealed higher infestation in the twelve southeastern counties compared to the rest of the state. Data from Mississippi suggested that D. texanus is not as problematic there as in Arkansas, Missouri, and Tennessee. Infestation rates from individual fields varied greatly (0–100%) within states. In Tennessee, second crop soybeans (i.e. soybeans planted following winter wheat) had lower infestations than full season soybeans. A map of pest distribution is presented that documents the extent of the problem, provides a baseline from which changes can be measured, contributes data for emergency registration of pesticides for specific geographic regions, and provides useful information for extension personnel, crop scouts, and growers.
Glycine max; distribution
BACKGROUND: Liver cirrhosis mortality differs by ethnicity in the United States. Some studies suggest alcohol sensitivity may contribute to this finding. This analysis evaluated if alcohol-associated changes in aspartate aminotransferase (AST) and alanine transaminase (ALT) differed by ethnicity among heavy drinkers. METHODS: Subjects included 1691 subjects from Project MATCH, a multicenter alcohol use disorders treatment trial. Changes in AST and ALT over 15 months were modeled as functions of ethnicity, age, gender, time, study site and alcohol use. The main focus was on ethnic differences in changes in transaminase activity occurring with changes in alcohol use. RESULTS: At all levels of alcohol consumption AST was lower in non-Hispanic whites relative to African Americans and Mexican Americans. Changes in AST associated with changes in alcohol use did not vary by ethnicity. ALT significantly differed only between Mexican Americans and non-Hispanic whites. Similar to AST, alcohol-associated ALT change did not differ by ethnicity. CONCLUSIONS: Among individuals with alcohol use disorders participating in a treatment trial, the effect of alcohol drinking on transaminase activity did not vary by ethnicity. However, in the general population, alcohol may still interact with other factors in mediating ethnic differences in cirrhosis mortality.
The Immune Epitope Database and Analysis Resource (IEDB) is dedicated to capturing, housing and analyzing complex immune epitope related data .
To identify and extract relevant data from the scientific literature in an efficient and accurate manner, novel processes were developed for manual and semi-automated annotation.
Formalized curation strategies enable the processing of a large volume of context-dependent data, which are now available to the scientific community in an accessible and transparent format. The experiences described herein are applicable to other databases housing complex biological data and requiring a high level of curation expertise.
TPX2, a microtubule-associated protein, is required downstream of Ran-GTP to induce spindle assembly. TPX2 activity appears to be tightly regulated during the cell cycle, and we report here one molecular mechanism for this regulation. We found that TPX2 protein levels are cell cycle regulated, peaking in mitosis and declining sharply during mitotic exit. TPX2 is degraded in mitotic extracts, as well as in HeLa cells exiting from mitosis. This instability depends, both in vitro and in vivo, on the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. In a reconstituted system, TPX2 is efficiently ubiquitinated by APC/C that has been activated by Cdh1. Two discrete elements in TPX2 are required for recognition by APC/CCdh1: a KEN box and a novel element in amino acids 1 to 86. Interestingly, the latter element, which has no known APC/C recognition motifs, is required for the ubiquitination of TPX2 by APC/CCdh1 in vitro and for its degradation in vivo. We conclude that APC/CCdh1 controls the stability of TPX2, thereby ensuring accurate regulation of the spindle assembly in the cell cycle.
To evaluate racial and ethnic disparity in blood pressure and cholesterol measurement and to analyze factors associated with any observed disparity.
Cross-sectional analysis of the household component of the 1996 Medical Expenditure Panel Survey.
Representative sample of the U.S. non-institutionalized population age 21 or older.
Prevalence of self-reported blood pressure measurement within 2 years and cholesterol measurement within 5 years were calculated by race/ethnicity. Logistic regression was used to adjust for health insurance status, having a usual source of care, health status, and socioeconomic and demographic factors. Odds ratios and 95% confidence intervals (95% CIs) from the logistic regression were converted to prevalence ratios to estimate relative risk (RR).
Mexican Americans compared to non-Hispanic whites were less likely to have a blood pressure measurement (RR, 0.85; 95% CI, 0.81 to 0.89) or a cholesterol measurement (RR, 0.72; 95% CI, 0.65 to 0.78). Non-Hispanic blacks had blood pressure and cholesterol measurements similar to non-Hispanic whites. In a multivariate analysis, Mexican Americans had similar blood pressure measurements (RR, 0.97; 95% CI, 0.94 to 1.00) and cholesterol measurements (RR, 1.04; 95% CI, 0.99 to 1.08). The factors associated with the largest disparity were lack of health insurance, not having a usual source of care, and low education.
No disparity was found between non-Hispanic blacks and non-Hispanic whites in undergoing blood pressure and cholesterol measurement. Disparities in cardiovascular preventive services for Mexican Americans were associated with lack of health insurance and a usual source of care, but other demographic and socioeconomic factors were also important.
health services accessibility; health surveys; socioeconomic factors; minority groups; blood pressure; cholesterol
Glucocorticoids depress bone formation by inhibiting osteoblastogenesis and increasing osteoblast apoptosis. However, the role of bone resorption in the initial rapid phase of bone loss characteristic of glucocorticoid-induced osteoporosis is unexplained, and the reason for the efficacy of bisphosphonates in this condition remains unknown. We report that in murine osteoclast cultures, glucocorticoids prolonged the baseline survival of osteoclasts and antagonized bisphosphonate-induced caspase activation and apoptosis by a glucocorticoid receptor–mediated action. Consistent with the in vitro evidence, in a murine model of glucocorticoid-induced osteoporosis, the number of cancellous osteoclasts increased, even though osteoclast progenitor number was reduced. Moreover, in mice receiving both glucocorticoids and bisphosphonates, the expected proapoptotic effect of bisphosphonates on osteoclasts was abrogated, as evidenced by maintenance of osteoclast numbers and, additionally, loss of bone density. In contrast, bisphosphonate administration prevented glucocorticoid-induced osteoblast apoptosis. These results indicate that the early loss of bone with glucocorticoid excess is caused by extension of the life span of pre-existing osteoclasts, an effect not preventable by bisphosphonates. Therefore, the early beneficial effects of these agents must be due, in part, to prolonging the life span of osteoblasts.
Genetic screens for modifiers of activated Ras phenotypes have identified a novel protein, kinase suppressor of Ras (KSR), which shares significant sequence homology with Raf family protein kinases. Studies using Drosophila melanogaster and Caenorhabditis elegans predict that KSR positively regulates Ras signaling; however, the function of mammalian KSR is not well understood. We show here that two predicted kinase-dead mutants of KSR retain the ability to complement ksr-1 loss-of-function alleles in C. elegans, suggesting that KSR may have physiological, kinase-independent functions. Furthermore, we observe that murine KSR forms a multimolecular signaling complex in human embryonic kidney 293T cells composed of HSP90, HSP70, HSP68, p50CDC37, MEK1, MEK2, 14-3-3, and several other, unidentified proteins. Treatment of cells with geldanamycin, an inhibitor of HSP90, decreases the half-life of KSR, suggesting that HSPs may serve to stabilize KSR. Both nematode and mammalian KSRs are capable of binding to MEKs, and three-point mutants of KSR, corresponding to C. elegans loss-of-function alleles, are specifically compromised in MEK binding. KSR did not alter MEK activity or activation. However, KSR-MEK binding shifts the apparent molecular mass of MEK from 44 to >700 kDa, and this results in the appearance of MEK in membrane-associated fractions. Together, these results suggest that KSR may act as a scaffolding protein for the Ras-mitogen-activated protein kinase pathway.
Pulmonary artery banding can produce significant distortion of the branch pulmonary arteries including selective stenosis of one artery and only minor narrowing of the other. This may complicate total repair of the underlying anomaly. A successful technique is described for revising a severely stenotic orifice of the right pulmonary artery in truncus arteriosus.
A planned repository of immune epitope data with associated analysis tools should be a boon to vaccine development