The central nonsense-mediated mRNA decay (NMD) regulator, Upf1, selectively targets nonsense-containing mRNAs for rapid degradation. In yeast, Upf1 preferentially associates with mRNAs that are NMD substrates, but the mechanism of its selective retention on these mRNAs has yet to be elucidated. Previously, we demonstrated that Upf1 associates with 40S ribosomal subunits. Here, we define more precisely the nature of this association using conventional and affinity-based purification of ribosomal subunits, and a two-hybrid screen to identify Upf1-interacting ribosomal proteins. Upf1 coimmunoprecipitates specifically with epitope-tagged 40S ribosomal subunits, and Upf1 association with high-salt washed or puromycin-released 40S subunits was found to occur without simultaneous eRF1, eRF3, Upf2, or Upf3 association. Two-hybrid analyses and in vitro binding assays identified a specific interaction between Upf1 and Rps26. Using mutations in domains of UPF1 known to be crucial for its function, we found that Upf1:40S association is modulated by ATP, and Upf1:Rps26 interaction is dependent on the N-terminal Upf1 CH domain. The specific association of Upf1 with the 40S subunit is consistent with the notion that this RNA helicase not only triggers rapid decay of nonsense-containing mRNAs, but may also have an important role in dissociation of the premature termination complex.
A mutation in the stalk of the newcastle disease virus hemagglutinin-neuraminidase (HN) protein prevents triggering of the F protein despite allowing efficient HN-F complex formation
Newcastle disease virus (NDV)-induced membrane fusion requires formation of a complex between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins. Substitutions for NDV HN stalk residues A89, L90, and L94 block fusion by modulating formation of the HN-F complex. Here, we demonstrate that a nearby L97A substitution, though previously shown to block fusion, allows efficient HN-F complex formation and likely acts by preventing changes in the HN stalk required for triggering of the bound F protein.
Female genital cosmetic surgery is surgery performed on a woman within a normal range of variation of human anatomy. The issues are heightened by a lack of long-term and substantive evidence-based literature, conflict of interest from personal financial gain through performing these procedures, and confusion around macroethical and microethical domains. It is a source of conflict and controversy globally because the benefit and harm of offering these procedures raise concerns about harmful cultural views, education, and social vulnerability of women with regard to both ethics and human rights. The rights issues of who is defining normal female anatomy and function, as well as the economic vulnerability of women globally, bequeath the profession a greater responsibility to ensure that there is adequate health and general education-not just among patients but broadly in society-that there is neither limitation nor interference in the decision being made, and that there are no psychological disorders that could be influencing such choices. Published by Elsevier Ireland Ltd. All rights reserved.
Many of the pathogens that cause human infectious diseases do not infect rodents or other mammalian species. Small animal models that allow studies of the pathogenesis of these agents and evaluation of drug efficacy are critical for identifying ways to prevent and treat human infectious diseases. Immunodeficient mice engrafted with functional human cells and tissues, termed 'humanized' mice, represent a critical pre-clinical bridge for in vivo studies of human pathogens. Recent advances in the development of humanized mice have allowed in vivo studies of multiple human infectious agents providing novel insights into their pathogenesis that was otherwise not possible.
The authors of 4 National Institutes of Health-sponsored antibiotic treatment trials of patients with persistent unexplained symptoms despite previous antibiotic treatment of Lyme disease determined that retreatment provides little if any benefit and carries significant risk. Two groups recently provided an independent reassessment of these trials and concluded that prolonged courses of antibiotics are likely to be helpful. We have carefully considered the points raised by these groups, along with our own critical review of the treatment trials. On the basis of this analysis, the conclusion that there is a meaningful clinical benefit to be gained from retreatment of such patients with parenteral antibiotic therapy cannot be justified.
RNA interference (RNAi) has considerable potential as a therapeutic strategy, but the development of efficient in vivo RNA delivery methods remains challenging. To this end, we designed and synthesized chemically modified interfering nanoparticles (iNOPs) composed of functionalized poly-l-lysine dendrimers modified with reducible spacers to facilitate release of small interfering RNAs (siRNAs) in vivo. We show that the novel siRNA-iNOP complexes mediate efficient gene-specific RNAi in cultured cells and in mice, where they display enhanced tissue-targeting capabilities. At a clinically feasible dose of 1 mg kg-1, apolipoprotein B (apoB) siRNA-iNOP complexes achieved approximately 40-45% reduction of liver apoB mRNA and plasma apoB protein levels within 48 h of administration to mice, without apparent toxicity. Collectively, these findings demonstrate that siRNA delivery by the modified reducible iNOPs can provide a clinically significant and potentially tissue-specific new approach for RNAi therapy.
INTRODUCTION: Rheumatoid arthritis (RA) is a systemic disease and the most prevalent of all autoimmune disorders. Here we review recent advances in the development and availability of biologic agents with a focus on monoclonal antibody or smaller formats of targeted engineered therapeutics including novel, non-antibody-based therapeutics.
AREAS COVERED: Today an array of biologics blocking either proinflammatory cytokines or lymphocyte activation/survival are available that enable a substantial improvement over conventional disease-modifying antirheumatic drugs (DMARDs). We review the engineering process of antibody-based biologics, their preclinical and clinical application, and current efforts to treat RA by interfering with B-cell function (notable targets covered are CD20, CD38, B-cell activating factor, transmembrane activator and calcium-modulating and cyclophilin interactor), with T-cell function (CD3, CD4, CD28), with bone erosion (RANKL), and with cytokines or growth factors (tumor necrosis factor, interleukin-1 [IL-1], IL-6, IL-17, VEGF). Future treatment choices might encompass the blockade or modulation of danger-associated molecular patterns such as HMGB1, pattern recognition receptors, messenger RNAs or noncoding RNAs, histone acetylation, and inflammasome components.
EXPERT OPINION: Although current therapies can reduce the signs and symptoms of RA for many patients, the quest for a cure (or a more complete blockade of the structural damage) in RA is still ongoing and will need treatment approaches, which are not exclusively confined to blocking a particular cytokine, receptor, or autoreactive B or T cell involved in disease progression. To this end exciting treatment alternatives and drug targets are on the horizon that may become available to patients in the future.
An amphiphilic degradable polymer/hydroxyapatite composite with enhanced handling characteristics promotes osteogenic gene expression in bone marrow stromal cells
Electrospun polymer/hydroxyapatite (HA) composites combining biodegradability with osteoconductivity are attractive for skeletal tissue engineering applications. However, most biodegradable polymers such as poly(lactic acid) (PLA) are hydrophobic and do not blend with adequate interfacial adhesion with HA, compromising the structural homogeneity, mechanical integrity and biological performance of the composite. To overcome this challenge, we combined a hydrophilic polyethylene glycol (PEG) block with poly(d,l-lactic acid) to improve the adhesion of the degradable polymer with HA. The amphiphilic triblock copolymer PLA-PEG-PLA (PELA) improved the stability of HA-PELA suspension at 25wt.% HA content, which was readily electrospun into HA-PELA composite scaffolds with uniform fiber dimensions. HA-PELA was highly extensible (failure strain>200% vs. 100 degrees for HA-PLA), and exhibited an 8-fold storage modulus increase (unlike deterioration for HA-PLA) upon hydration, owing to the favorable interaction between HA and PEG. HA-PELA also better promoted osteochondral lineage commitment of bone marrow stromal cells in unstimulated culture and supported far more potent osteogenic gene expression upon induction than HA-PLA. We demonstrate that the chemical incorporation of PEG is an effective strategy to improve the performance of degradable polymer/HA composites for bone tissue engineering applications.
Nicotinic acetylcholine receptors containing the alpha6 subunit contribute to ethanol activation of ventral tegmental area dopaminergic neurons
Nicotine and alcohol are often co-abused suggesting a common mechanism of action may underlie their reinforcing properties. Both drugs acutely increase activity of ventral tegmental area (VTA) dopaminergic (DAergic) neurons, a phenomenon associated with reward behavior. Recent evidence indicates that nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels activated by ACh and nicotine, may contribute to ethanol-mediated activation of VTA DAergic neurons although the nAChR subtype(s) involved has not been fully elucidated. Here we show that expression and activation of nAChRs containing the alpha6 subunit contribute to ethanol-induced activation of VTA DAergic neurons. In wild-type (WT) mouse midbrain sections that contain the VTA, ethanol (50 or 100 mM) significantly increased firing frequency of DAergic neurons. In contrast, ethanol did not significantly increase activity of VTA DAergic neurons in mice that do not express CHRNA6, the gene encoding the alpha6 nAChR subunit (alpha6 knock-out (KO) mice). Ethanol-induced activity in WT slices was also reduced by pre-application of the alpha6 subtype-selective nAChR antagonist, alpha-conotoxin MII[E11A]. When co-applied, ethanol potentiated the response to ACh in WT DAergic neurons; whereas co-application of ACh and ethanol failed to significantly increase activity of DAergic neurons in alpha6 KO slices. Finally, pre-application of alpha-conotoxin MII[E11A] in WT slices reduced ethanol potentiation of ACh responses. Together our data indicate that alpha6-subunit containing nAChRs may contribute to ethanol activation of VTA DAergic neurons. These receptors are predominantly expressed in DAergic neurons and known to be critical for nicotine reinforcement, providing a potential common therapeutic molecular target to reduce nicotine and alcohol co-abuse.
Generation of organized anterior foregut epithelia from pluripotent stem cells using small molecules
Anterior foregut endoderm (AFE) gives rise to therapeutically relevant cell types in tissues such as the esophagus, salivary glands, lung, thymus, parathyroid and thyroid. Despite its importance, reports describing the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation have mainly focused on the Nkx2.1(+) lung and thyroid lineages. Here, we describe a novel protocol to derive a subdomain of AFE, identified by expression of Pax9, from PSCs using small molecules and defined media conditions. We generated a reporter PSC line for isolation and characterization of Pax9(+) AFE cells, which when transplanted in vivo, can form several distinct complex AFE-derived epithelia, including mucosal glands and stratified squamous epithelium. Finally, we show that the directed differentiation protocol can be used to generate AFE from human PSCs. Thus, this work both broadens the range of PSC-derived AFE tissues and creates a platform enabling the study of AFE disorders.
BACKGROUND: Although debate continues on US healthcare and insurance reform, data are lacking on the effect of insurance on community-level cancer outcomes. Therefore, the objective of the present study was to examine the association of insurance and cancer outcomes.
MATERIALS AND METHODS: The US Census Bureau Current Population Survey, Small Area Health Insurance Estimates (2000) were used for the rates of uninsurance. Counties were divided into tertiles according to the uninsurance rates. The data were compared with the cancer incidence and survival for patients residing in counties captured by the Surveillance, Epidemiology, and End Results database (2000-2006). Aggregate patient data were collected of US adults (aged >/=18 y) diagnosed with the following cancers: pancreatic, esophageal, liver or bile duct, lung or bronchial, ovarian, colorectal, breast, prostate, melanoma, and thyroid. The outcomes included the stage at diagnosis, surgery, and survival. Univariate tests and proportional hazards were calculated.
RESULTS: The US uninsurance rate was 14.2%, and the range for the Surveillance, Epidemiology, and End Results counties was 8.3%-24.1%. Overall, patients from lower uninsurance rate counties demonstrated longer median survival. Adjusting for patient characteristics and cancer stage (for each cancer), the patients in the higher uninsurance rate counties demonstrated greater mortality (8%-15% increased risk on proportional hazards). The county uninsurance rate was associated with the stage at diagnosis for all cancers, except pancreatic and esophageal, and was also associated with the likelihood of being recommended for cancer-directed surgery (for all cancers).
CONCLUSIONS: Health insurance coverage at a community level appears to influence survival for patients with cancer. Additional investigations are needed to examine whether individual versus community associations exist and how best to surmount barriers to cancer care.
The role of Bruton's tyrosine kinase in the development and BCR/TLR-dependent activation of AM14 rheumatoid factor B cells
The protein kinase Btk has been implicated in the development, differentiation, and activation of B cells through its role in the BCR and TLR signaling cascades. These receptors and in particular, the BCR and either TLR7 or TLR9 also play a critical role in the activation of autoreactive B cells by RNA- or DNA-associated autoantigens. To explore the role of Btk in the development of autoreactive B cells, as well as their responses to nucleic acid-associated autoantigens, we have now compared Btk-sufficient and Btk-deficient mice that express a prototypic RF BCR encoded by H- and L-chain sdTgs. These B cells bind autologous IgG2a with low affinity and only proliferate in response to IgG2a ICs that incorporate DNA or RNA. We found that Btk-sufficient RF(+) B cells mature into naive FO B cells, all of which express the Tg BCR, despite circulating levels of IgG2a. By contrast, a significant proportion of Btk-deficient RF(+) B cells acquires a MZ or MZ precursor phenotype. Remarkably, despite the complete inability of RF(+) Xid/y B cells to respond to F(ab')2 anti-IgM, RF(+) Xid/y B cells could respond well to autoantigen-associated ICs. These data reveal unique features of the signaling cascades responsible for the activation of autoreactive B cells.
A germline point mutation in Runx1 uncouples its role in definitive hematopoiesis from differentiation
Definitive hematopoiesis requires the master hematopoietic transcription factor Runx1, which is a frequent target of leukemia-related chromosomal translocations. Several of the translocation-generated fusion proteins retain the DNA binding activity of Runx1, but lose subnuclear targeting and associated transactivation potential. Complete loss of these functions in vivo resembles Runx1 ablation, which causes embryonic lethality. We developed a knock-in mouse that expresses full-length Runx1 with a mutation in the subnuclear targeting cofactor interaction domain, Runx1(HTY350-352AAA). Mutant mice survive to adulthood, and hematopoietic stem cell emergence appears to be unaltered. However, defects are observed in multiple differentiated hematopoietic lineages at stages where Runx1 is known to play key roles. Thus, a germline mutation in Runx1 reveals uncoupling of its functions during developmental hematopoiesis from subsequent differentiation across multiple hematopoietic lineages in the adult. These findings indicate that subnuclear targeting and cofactor interactions with Runx1 are important in many compartments throughout hematopoietic differentiation. Elsevier Inc. All rights reserved.
Thalamocortical connectivity plays a vital role in brain function. The anatomy and function of thalamocortical networks have been extensively studied in animals by numerous invasive techniques. Non-invasively mapping thalamocortical networks in humans has also been demonstrated by utilizing resting-state functional magnetic resonance imaging (rsfMRI). However, success in simultaneously imaging multiple thalamocortical networks in animals is rather limited. This is largely due to the profound impact of anesthesia used in most animal experiments on functional connectivity measurement. Here we have employed an awake animal imaging approach to systematically map thalamocortical connectivity for multiple thalamic nuclei in rats. Seed-based correlational analysis demonstrated robust functional connectivity for each thalamic nucleus in the cortex, and the cortical connectivity profiles revealed were in excellent accordance with the known thalamocortical anatomical connections. In addition, partial correlation analysis was utilized to further improve the spatial specificity of thalamocortical connectivity. Taken together, these findings have provided important evidence supporting the validity of rsfMRI measurement in awake animals. More importantly, the present study has made it possible to non-invasively investigate the function, neuroplasticity and mutual interactions of thalamocortical networks in animal models.
Noncontrast CT of the head is a widely used noninvasive investigation for a variety of acute and chronic neurological conditions. Since CT head without contrast is usually the first and often the only investigation in the emergency room for many neurological symptoms, it is imperative to detect subtle vascular changes, which in many patients can be life-saving. The vascular abnormalities may present with increased density and/or size of the vessels, filling defects, and be associated with parenchymal and bony changes. In this article, we present examples of several vascular pathologies which can be identified on the noncontrast CT of the head, and learn imaging and interpretation techniques to help recognize what often are nebulous changes. While some of the findings are diagnostic by themselves and others subtle, any suspicious abnormality should be followed with dedicated vascular imaging such as CT/MR angiogram, venogram, or catheter angiogram for confirmation and better characterization.
BACKGROUND AND OBJECTIVES: Homeless women are at high risk of drug and alcohol dependence and may receive less opportunity for treatment. Our objective was to examine the association between experiencing homelessness and motivation to change drug or alcohol use.
METHODS: Women (n = 154) participants in a study of substance dependence at an urban medical center (69 with some homeless days in the last 90 days; 85 continuously housed at baseline) completed six items rating motivation to change alcohol or drug use (ie, importance, readiness, and confidence) at baseline and in 3-, 6-, and 12-month follow-up interviews. Unadjusted and longitudinal analyses controlling for covariates (eg, demographics, insurance status, substance use consequences, mental health status, and participation in treatment) were conducted.
RESULTS: There were no significant differences between women experiencing homeless days versus continuously housed women in the odds of reporting high motivation to change alcohol or drug use, either in unadjusted baseline analyses or longitudinal analyses adjusted for covariates. Covariates that were significantly associated with high importance, readiness or confidence to change behavior were higher life time consequences of substance use, and participation in 12-step programs.
DISCUSSION AND CONCLUSIONS: The findings suggest that clinicians should not make assumptions that homeless women have low motivation to change their substance use.
SCIENTIFIC SIGNIFICANCE AND FUTURE DIRECTIONS: The same opportunities for addiction treatment should be offered to homeless as to housed women.
“Microaggressions” refer to those brief occurrences and encounters that subtly reinforce systems of power and privilege. Libraries and information organizations are not immune to these microaggressions. They exist in our library catalogues, archives, research, professional organizations and interpersonal interactions. The authors explore biases in librarianship, particularly in bibliographic metadata, then present tools to engage librarians as well as faculty and graduate students.
Weight-based discrimination and medication adherence among low-income African Americans with hypertension: how much of the association is mediated by self-efficacy?
OBJECTIVES: Much of the excessive morbidity and mortality from cardiovascular disease among African Americans results from low adherence to anti-hypertensive medications. Therefore, we examined the association between weight-based discrimination and medication adherence.
METHODS: We used cross-sectional data from low-income African Americans with hypertension. Ordinal logistic regression estimated the odds of medication non-adherence in relation to weight-based discrimination adjusted for age, sex, education, income, and weight.
RESULTS: Of all participants (n = 780), the mean (SD) age was 53.7 (9.9) years and the mean (SD) weight was 210.1 (52.8) lbs. Reports of weight-based discrimination were frequent (28.2%). Weight-based discrimination (but not weight itself) was associated with medication non-adherence (OR: 1.94; 95% CI: 1.41-2.67). A substantial portion 38.9% (95% CI: 19.0%-79.0%) of the association between weight-based discrimination and medication non-adherence was mediated by medication self-efficacy.
CONCLUSION: Self-efficacy is a potential explanatory factor for the association between reported weight-based discrimination and medication non-adherence. Future research should develop and test interventions to prevent weight-based discrimination at the societal, provider, and institutional levels.
The Accuracy of Recalled versus Measured Pre-Pregnancy Weight for the Calculation of Pre-Pregnancy Body Mass Index
Background: In 2009, the Institute of Medicine (IOM) published gestational weight gain (GWG) guidelines with the goal of optimizing maternal and fetal outcomes. GWG recommendations are specific to pre-pregnancy body mass index (BMI): 28-40 lbs for underweight (UW; BMI2), 25-35 lbs for normal weight (NW; 18.5≤BMI/m2), 15-25 lbs for overweight (OW; 25 ≤BMI/m2), and 11-20 lbs for obese (OB; BMI≥30 kg/m2) women. With upwards of 50% of pregnancies in the U.S. unplanned, measured pre-pregnancy weight is often unavailable in clinical and research settings. Evaluating the accuracy of recalled pre-pregnancy weight early in prenatal care is important in order to establish accuracy of pre-pregnancy BMI calculations in order to counsel about GWG accurately.
Objective: To examine differences in recalled versus measured pre-pregnancy weight and to examine factors associated with accuracy of recalled weights.
Methods: Medical record review of 1,998 randomly selected pregnancies. Eligible women received prenatal care in faculty and resident clinics at UMass Memorial Health Care (UMMHC), delivered between January 2007 and December 2012, and had available both: (1) a measured weight within one year of conception and (2) a pre-pregnancy weight self-reported at first prenatal visit. Data were obtained from the UMMHC paper or electronic prenatal record and the Allscripts EMR. We calculated the difference in weights as recalled pre-pregnancy weight minus most recent measured weight within one year of conception. Subjects were excluded if they received care at a non-faculty or non-resident practice, charts not available after three separate retrieval attempts, both weights of interest not available, or if measured weight occurred at a prenatal visit for a prior pregnancy. For women with more than one pregnancy during the study time frame, one was randomly selected for inclusion in the analytic data set.
Results: Of the 1,998 pregnancy charts reviewed, 400 records met eligibility criteria and were included in this analysis. Women were mean age 29.7 (SD: 6.2) years, 69.3% multigravida, 64.4% non-Hispanic white, 65.2% married, and 62.4% had a college or greater education. Based on recalled weight, 3.3% of women were underweight, 46.6% were normal weight, 25.9% overweight, and 24.2% obese. 63% received care in the faculty obstetric clinic. Recorded recalled weights were mean 2.4 (SD: 11.1) pounds lower than measured pre-pregnancy weight. This difference did not differ by age, location of care, pre-pregnancy BMI, marital status, race/ethnicity, primary language, gravity, education, or time between measured weight and conception, in unadjusted and adjusted models. For 88.7% of women, calculating pre-pregnancy BMI based on weight measured up to a year prior to conception or based on recalled pre-pregnancy weight reported at the first prenatal visit resulted in the same classification of pre-pregnancy BMI.
Conclusion: Prenatal care providers may calculate pre-pregnancy BMIs using recalled pre-pregnancy weights early in prenatal care and use such calculated BMIs to accurately provide GWG recommendations regardless of demographic variables, gravity, or location of care.
Myogenic Enhancers Regulate Expression of the Facioscapulohumeral Muscular Dystrophy-Associated DUX4 Gene
Facioscapulohumeral muscular dystrophy (FSHD) is linked to epigenetic dysregulation of the chromosome 4q35 D4Z4 macrosatellite. However, this does not account for the tissue specificity of FSHD pathology, which requires stable expression of an alternative full-length mRNA splice form of DUX4 (DUX4-fl) from the D4Z4 array in skeletal muscle. Here, we describe the identification of two enhancers, DUX4 myogenic enhancer 1 (DME1) and DME2 which activate DUX4-fl expression in skeletal myocytes but not fibroblasts. Analysis of the chromatin revealed histone modifications and RNA polymerase II occupancy consistent with DME1 and DME2 being functional enhancers. Chromosome conformation capture analysis confirmed association of DME1 and DME2 with the DUX4 promoter in vivo. The strong interaction between DME2 and the DUX4 promoter in both FSHD and unaffected primary myocytes was greatly reduced in fibroblasts, suggesting a muscle-specific interaction. Nucleosome occupancy and methylome sequencing analysis indicated that in most FSHD myocytes, both enhancers are associated with nucleosomes but have hypomethylated DNA, consistent with a permissive transcriptional state, sporadic occupancy, and the observed DUX4 expression in rare myonuclei. Our data support a model in which these myogenic enhancers associate with the DUX4 promoter in skeletal myocytes and activate transcription when epigenetically derepressed in FSHD, resulting in the pathological misexpression of DUX4-fl.