Adipose tissue lipogenesis is paradoxically impaired in human obesity, promoting ectopic triglyceride (TG) deposition, lipotoxicity, and insulin resistance. We previously identified mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a sterile 20 protein kinase reported to be upstream of c-Jun NH2-terminal kinase (JNK) signaling, as a novel negative regulator of insulin-stimulated glucose transport in adipocytes. Using full-genome microarray analysis we uncovered a novel role for Map4k4 as a suppressor of lipid synthesis. We further report here the surprising finding that Map4k4 suppresses adipocyte lipogenesis independently of JNK. Thus, while Map4k4 silencing in adipocytes enhances the expression of lipogenic enzymes, concomitant with increased conversion of (14)C-glucose and (14)C-acetate into TGs and fatty acids, JNK1 and JNK2 depletion causes the opposite effects. Furthermore, high expression of Map4k4 fails to activate endogenous JNK, while Map4k4 depletion does not attenuate JNK activation by tumor necrosis factor alpha. Map4k4 silencing in cultured adipocytes elevates both the total protein expression and cleavage of sterol-regulated element binding protein-1 (Srebp-1) in a rapamycin-sensitive manner, consistent with Map4k4 signaling via mechanistic target of rapamycin complex 1 (mTORC1). We show Map4k4 depletion requires Srebp-1 upregulation to increase lipogenesis and further show that Map4k4 promotes AMP-protein kinase (AMPK) signaling and the phosphorylation of mTORC1 binding partner raptor (Ser792) to inhibit mTORC1. Our results indicate that Map4k4 inhibits adipose lipogenesis by suppression of Srebp-1 in an AMPK- and mTOR-dependent but JNK-independent mechanism.
Synthetic oligodeoxynucleotides containing suppressive TTAGGG motifs inhibit AIM2 inflammasome activation
Synthetic oligodeoxynucleotides (ODNs) comprised of the immunosuppressive motif TTAGGG block TLR9 signaling, prevent STAT1 and STAT4 phosphorylation and attenuate a variety of inflammatory responses in vivo. In this study, we demonstrate that such suppressive ODN abrogate activation of cytosolic nucleic acid-sensing pathways. Pretreatment of dendritic cells and macrophages with the suppressive ODN-A151 abrogated type I IFN, TNF-alpha, and ISG induction in response to cytosolic dsDNA. In addition, A151 abrogated caspase-1-dependent IL-1beta and IL-18 maturation in dendritic cells stimulated with dsDNA and murine CMV. Inhibition was dependent on A151's phosphorothioate backbone, whereas substitution of the guanosine residues for adenosine negatively affected potency. A151 mediates these effects by binding to AIM2 in a manner that is competitive with immune-stimulatory DNA and as a consequence prevents AIM2 inflammasome complex formation. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases.
A rare case of right ventricular outflow tract (RVOT) primary spindle cell sarcoma with intraoperative TEE images is reported below.
Despite effective vaccines, influenza remains a major global health threat due to the morbidity and mortality caused by seasonal epidemics, as well as the 2009 pandemic. Also of profound concern are the rare but potentially catastrophic transmissions of avian influenza to humans, highlighted by a recent H7N9 influenza outbreak. Murine and human studies reveal that the clinical course of influenza is the result of a combination of both host and viral genetic determinants. While viral pathogenicity has long been the subject of intensive efforts, research to elucidate host genetic determinants, particularly human, is now in the ascendant, and the goal of this review is to highlight these recent insights.
Glucan particles (GPs) are hollow porous Saccharomyces cerevisiae cell walls that are treated so that they are composed primarily of beta-1,3-d-glucans. Our previous studies showed that GPs can serve as an effective vaccine platform. Here, we characterize CD4(+) T-cell and antibody responses in immunized mice as a function of antigen (ovalbumin) encapsulation, antigen dose, particle numbers, time, immunization schedule, and trapping methods. Although we found that GPs served as an effective adjuvant when admixed with free antigens for IgG1 antibody production, stronger CD4(+) T-cell and IgG2c antibody responses were stimulated when antigens were encapsulated inside GPs, suggesting that the GP platform acts as both an adjuvant and a delivery system. Vigorous T-cell and antibody responses were stimulated even at submicrogram antigen doses, as long as the number of GPs was kept at 5 x 10(7) particles per immunization. One prime and one boost were sufficient to elicit robust immune responses. In addition, strong antigen-specific antibody and T-cell responses prevailed up to 20 months following the last immunization, including those of gamma interferon (IFN-gamma), interleukin 17A (IL-17A), and dual IFN-gamma/IL-17A-secreting CD4(+) T cells. Finally, robust immune responses were observed using generally recognized as safe (GRAS) materials (alginate and calcium, with or without chitosan) to trap antigens within GPs. Thus, these studies demonstrate that antigens encapsulated into GPs make an effective vaccine platform that combines adjuvanticity and antigen delivery to elicit strong durable immune responses at relatively low antigen doses using translationally relevant formulations.
SUMMARY: Unlike DNA, RNA abundances can vary over several orders of magnitude. Thus, identification of RNA-protein binding sites from high-throughput sequencing data presents unique challenges. Although peak identification in ChIP-Seq data has been extensively explored, there are few bioinformatics tools tailored for peak calling on analogous datasets for RNA-binding proteins. Here we describe ASPeak (abundance sensitive peak detection algorithm), an implementation of an algorithm that we previously applied to detect peaks in exon junction complex RNA immunoprecipitation in tandem experiments. Our peak detection algorithm yields stringent and robust target sets enabling sensitive motif finding and downstream functional analyses.
AVAILABILITY: ASPeak is implemented in Perl as a complete pipeline that takes bedGraph files as input. ASPeak implementation is freely available at https://sourceforge.net/projects/as-peak under the GNU General Public License. ASPeak can be run on a personal computer, yet is designed to be easily parallelizable. ASPeak can also run on high performance computing clusters providing efficient speedup. The documentation and user manual can be obtained from http://master.dl.sourceforge.net/project/as-peak/manual.pdf.
Organophosphate (OP) pesticide poisoning is a global health problem with over 250,000 deaths per year. OPs affect neuronal signaling through acetylcholine (Ach) neurotransmission via inhibition of acetylcholinesterase (AChE), leading to accumulation of Ach at the synaptic cleft and excessive stimulation at post-synaptic receptors. Mortality due to OP agents is attributed to respiratory dysfunction, including central apnea. Cholinergic circuits are integral to many aspects of the central control of respiration, however it is unclear which mechanisms predominate during acute OP intoxication. A more complete understanding of the cholinergic aspects of both respiratory control as well as neural modification of pulmonary function is needed to better understand OP-induced respiratory dysfunction. In this article, we review the physiologic mechanisms of acute OP exposure in the context of the known cholinergic contributions to the central control of respiration. We also discuss the potential central cholinergic contributions to the known peripheral physiologic effects of OP intoxication.
OBJECTIVE: To assess whether parent training in behavioral intervention, combined with a 16-session nutrition and activity education program, would improve weight loss relative to nutrition and activity education alone in adolescents and young adults with Down syndrome.
STUDY DESIGN: Twenty-one patients with Down syndrome aged 13-26 years with a body mass index > /= 85th percentile were enrolled and randomized to a 6-month nutrition and activity education intervention (n = 10) or to a nutrition and activity education+behaviorial intervention (n = 11), and followed for 6 months after the active intervention period (1-year follow-up). The primary outcome measure was body weight; secondary outcomes included percentage body fat by bioelectric impedance; intake of fruits, vegetables, and energy-dense low-nutrient snack food (treats) by 3-day food record; and moderate/vigorous physical activity by accelerometry.
RESULTS: At 6 months, mean body weight in the nutrition and activity education+behavioral intervention group was 3.2 kg lower than that in the nutrition and activity education group (95% CI, 1.0-5.5 kg; P = .005). Mean group differences were sustained at 1 year (3.6 kg; 95% CI, 1.4-5.9 kg; P = .002). At 6 months, moderate/vigorous physical activity time increased by an average of 18 minutes/day compared with baseline in the nutrition and activity education+behavioral intervention group (P = .01) and decreased by 7 minutes/day in the nutrition and activity education group (P = .30). These changes were largely maintained at 1 year, but were not statistically significant. Vegetable intake in the nutrition and activity education+behavioral intervention group exceeded that in the nutrition and activity education group by a mean of 1.6 servings at 1 year (P = .009), but not at 6 months. No between-group differences were observed for percentage body fat or consumption of fruits or treats.
CONCLUSION: Parent-supported behavioral intervention appears to be a successful adjunct to a 6-month nutrition education intervention in achieving weight loss in adolescents and young adults with Down syndrome.
Value in healthcare can be defined as the ratio of the benefits that accrue to patients from treatment received to the dollars spent providing that treatment. It is widely believed that healthcare in the United States is moving from a volume-driven paradigm to a system that increasingly rewards value in healthcare delivery. How can orthopaedic surgeons measure the value and the quality of care they deliver to their patients?
Dichlorvos exposure to the Kolliker-fuse nuclei is sufficient but not necessary for OP induced apnea
Patients exposed to organophosphate (OP) compounds demonstrate a central apnea. The Kolliker-fuse nuclei (KF) are cholinergic nuclei in the brainstem involved in central respiratory control. We hypothesize that exposure of the KF is both necessary and sufficient for OP induced central apnea. We performed an animal study of acute OP exposure. Anesthetized and spontaneously breathing Wistar rats (n=24) were exposed to a lethal dose of dichlorvos using three experimental models. Experiment 1 (n=8) involved systemic OP poisoning using subcutaneous (SQ) 2,2-dichlorovinyl dimethyl phosphate (dichlorvos) at 100mg/kg or 3x LD50. Experiment 2 (n=8) involved isolated poisoning of the KF using stereotactic microinjections of dichlorvos (625mug in 50mul) into the KF. Experiment 3 (n=8) involved systemic OP poisoning with isolated protection of the KF using SQ dichlorvos (100mg/kg) and stereotactic microinjections of organophosphatase A (OpdA), an enzyme that degrades dichlorvos. Respiratory and cardiovascular parameters were recorded continuously. Animals were followed post exposure for 1h or until death. There was no difference in respiratory depression between animals with SQ dichlorvos and those with dichlorvos microinjected into the KF. Despite differences in amount of dichlorvos (100mg/kg vs. 1.8mg/kg) and method of exposure (SQ vs. CNS microinjection), 10min following dichlorvos both groups (SQ vs. microinjection respectively) demonstrated a similar percent decrease in respiratory rate (51.5 vs. 72.2), minute ventilation (49.2 vs. 68.8) and volume of expired gas (17.5 vs. 0.0). Animals with OpdA exposure to the KF during systemic OP exposure demonstrated less respiratory depression, compared to SQ dichlorvos alone (p < 0.04). No animals with SQ dichlorvos survived past 25min post exposure, compared to 50% of animals with OpdA exposure to the KF. In conclusion, exposure of the KF is sufficient but not necessary for OP induced apnea. Protection of the KF during systemic OP exposure mitigates OP induced apnea.
Cellular stress response and innate immune signaling: integrating pathways in host defense and inflammation
Extensive research in the past decade has identified innate immune recognition receptors and intracellular signaling pathways that culminate in inflammatory responses. Besides its role in cytoprotection, the importance of cell stress in inflammation and host defense against pathogens is emerging. Recent studies have shown that proteins in cellular stress responses, including the heat shock response, ER stress response, and DNA damage response, interact with and regulate signaling intermediates involved in the activation of innate and adaptive immune responses. The effect of such regulation by cell stress proteins may dictate the inflammatory profile of the immune response during infection and disease. In this review, we describe the regulation of innate immune cell activation by cell stress pathways, present detailed descriptions of the types of stress response proteins and their crosstalk with immune signaling intermediates that are essential in host defense, and illustrate the relevance of these interactions in diseases characteristic of aberrant immune responses, such as chronic inflammatory diseases, autoimmune disorders, and cancer. Understanding the crosstalk between cellular stress proteins and immune signaling may have translational implications for designing more effective regimens to treat immune disorders.
The role of adaptation in molecular evolution has been contentious for decades. Here, we shed light on the adaptive potential in Saccharomyces cerevisiae by presenting systematic fitness measurements for all possible point mutations in a region of Hsp90 under four environmental conditions. Under elevated salinity, we observe numerous beneficial mutations with growth advantages up to 7% relative to the wild type. All of these beneficial mutations were observed to be associated with high costs of adaptation. We thus demonstrate that an essential protein can harbor adaptive potential upon an environmental challenge, and report a remarkable fit of the data to a version of Fisher's geometric model that focuses on the fitness trade-offs between mutations in different environments. Evolution.
We developed a method called residue contact frequency (RCF), which uses the complex structures generated by the protein-protein docking algorithm ZDOCK to predict interface residues. Unlike interface prediction algorithms that are based on monomers alone, RCF is binding partner specific. We evaluated the performance of RCF using the area under the precision-recall (PR) curve (AUC) on a large protein docking Benchmark. RCF (AUC = 0.44) performed as well as meta-PPISP (AUC = 0.43), which is one of the best monomer-based interface prediction methods. In addition, we test a support vector machine (SVM) to combine RCF with meta-PPISP and another monomer-based interface prediction algorithm Evolutionary Trace to further improve the performance. We found that the SVM that combined RCF and meta-PPISP achieved the best performance (AUC = 0.47). We used RCF to predict the binding interfaces of proteins that can bind to multiple partners and RCF was able to correctly predict interface residues that are unique for the respective binding partners. Furthermore, we found that residues that contributed greatly to binding affinity (hotspot residues) had significantly higher RCF than other residues.
The Cellient automated cell block system is useful in the differential diagnosis of atypical glandular cells in Papanicolaou tests
BACKGROUND: Atypical glandular cells (AGC) is a very important diagnosis in gynecological cytology. In the current study, the authors investigated the usefulness of Cellient cell blocks (CB) for characterizing AGC on Papanicolaou (Pap) tests.
METHODS: A total of 148 patients with an AGC diagnosis based on Pap tests by cytotechnologists and referred to cytopathologists were studied. Among these patients, there were 68 patients with CB preparations and 80 patients with Pap tests only (TP-AGC group). Follow-up results by Pap tests or biopsies were obtained in 117 of 148 patients. The median follow-up was 13 months (range, 1 month-36 months).
RESULTS: Of the 68 patients with CBs, 31 (46%) were reclassified as negative for dysplasia or low-grade intraepithelial lesion; 30 patients (44%) retained a diagnosis of AGC (CB-AGC group); and 7 patients (10%) were given specific diagnoses of high-grade intraepithelial lesion (3 patients), endocervical adenocarcinoma in situ (1 patient), and invasive adenocarcinoma (3 patients). On follow-up, the CB-AGC group was found to have a significantly lower rate of negative/low-grade squamous intraepithelial lesion diagnoses compared with the TP-AGC group (55% vs 85%; P = .006). The CB-AGC group had a significantly higher rate of endocervical or endometrial adenocarcinoma compared with the TP-AGC group (36% vs 8%; P = .003) at the time of follow-up. The rates of high-grade squamous intraepithelial lesion were not found to be statistically different between these 2 groups (9% vs 7%; P = .66).
CONCLUSIONS: The Cellient CB is a useful technique to further categorize a diagnosis of AGC on Pap tests. Using the Cellient CB system, the pathologist has the ability to improve the diagnostic accuracy of AGC so that unnecessary colposcopic evaluation or biopsies can be avoided.
Effect of fesoterodine in vulnerable elderly subjects with urgency incontinence: a double-blind, placebo controlled trial
PURPOSE: We evaluated the efficacy and safety of flexible dose fesoterodine in medically complex vulnerable elderly subjects with urgency urinary incontinence.
MATERIALS AND METHODS: In this 12-week, randomized, double-blind, flexible dose, placebo controlled trial, subjects were community dwelling men and women 65 years old or older. Subjects had scores of 3 or more on the VES-13 (Vulnerable Elders Survey) and 20 or more on the MMSE (Mini-Mental State Examination), and 2 to 15 urgency urinary incontinence episodes and 8 or more micturitions per 24 hours on 3-day baseline diaries. Subjects randomized to fesoterodine received 4 mg once daily for 4 weeks and could then increase to 8 mg based on discussion with the investigator. Subjects receiving 8 mg could decrease the dose to 4 mg at any time (sham escalation and de-escalation for placebo). The primary outcome measure was change in daily urgency urinary incontinence episodes. Secondary outcomes included changes in other diary variables and patient reported quality of life measures. Safety evaluations included self-reported symptoms and post-void residual volume.
RESULTS: A total of 562 patients were randomized (mean age 75 years, 50.4% age 75 years or greater). Subjects had high rates of comorbidities, polypharmacy and functional impairment. At week 12 the fesoterodine group had significantly greater improvements in urgency urinary incontinence episodes per 24 hours (-2.84 vs -2.20, p = 0.002) and most other diary variables and quality of life, as well as a higher diary dry rate (50.8% vs 36.0%, p = 0.002). Adverse effects were generally similar to those of younger populations including risk of urinary retention.
CONCLUSIONS: To our knowledge this is the first antimuscarinic study in a community based, significantly older, medically complex elderly population with urgency urinary incontinence. Flexible dose fesoterodine significantly improved urgency urinary incontinence episodes and other outcomes vs placebo, and was generally well tolerated.
Published by Elsevier Inc. All rights reserved.
Decision-making process for conditions nominated to the recommended uniform screening panel: statement of the US Department of Health and Human Services Secretary's Advisory Committee on Heritable Disorders in Newborns and Children
PURPOSE: The US Secretary of Health and Human Services provides guidance to state newborn screening programs about which conditions should be included in screening (i.e., the "Recommended Uniform Screening Panel"). This guidance is informed by evidence-based recommendations from the Secretary's Advisory Committee on Heritable Disorders in Newborns and Children. This report describes the Advisory Committee's revised decision-making process for considering conditions nominated to the panel.
METHODS: An expert panel meeting was held in April 2012 to revise the decision matrix, which helps to guide the recommendation process. In January 2013, the Advisory Committee voted to adopt the revised decision matrix.
RESULTS: The revised decision matrix clarifies the approach to rating magnitude and certainty of the net benefit of screening to the population of screened newborns for nominated conditions, and now includes the consideration of the capability of state newborn screening programs for population-wide implementation by evaluating the feasibility and readiness of states to adopt screening for nominated conditions.
CONCLUSION: The revised decision matrix will bring increased quality, transparency, and consistency to the process of modifying the recommended uniform screening panel and will now allow formal evaluation of the challenges that state newborn screening programs face in adopting screening for new conditions.
Evidence for increasing usage of low-grade squamous intraepithelial lesion, cannot exclude high-grade squamous intraepithelial lesion (LSIL-H) Pap test interpretations
BACKGROUND: Pap test (PT) interpretations of low-grade squamous intraepithelial lesion (LSIL), cannot exclude high-grade squamous intraepithelial lesion (HSIL), or LSIL-H, are used in many laboratories; however monitoring its usage for quality assurance purposes is understudied.
METHODS: PTs from 2005 to 2010 were collected, and yearly frequencies of LSIL, HSIL, LSIL-H, and atypical squamous cells, cannot exclude HSIL (ASC-H) as a function of total PTs and total squamous intraepithelial lesions (SILs) were calculated. Two-year risk of cervical intraepithelial neoplasia 2 (CIN2) or worse (CIN2+) and CIN 3 or worse (CIN3+) was calculated.
RESULTS: A total of 352,220 PTs were identified including 17,301 abnormal PTs. LSIL-H usage increased from 2005 to 2010 (from 0.28% of total PTs in 2005 to 0.61% in 2010, P < .01; from 5.8% of total SILs in 2005 to 12% in 2010, P < .001). HSIL usage decreased significantly from 2005 to 2010 (from 0.7% of total PTs in 2005 to 0.48% in 2010, P = .048; from 14.5% of total SILs in 2005 to 9.5% in 2010, P < .01). Usage of LSIL and ASC-H did not change. Two-year risk of CIN2+ and CIN3+ for HSIL increased significantly from 2005 to 2010 (P < .01). Two-year risk of CIN2+ and CIN3+ for LSIL-H did not change significantly from 2005 to 2010.
CONCLUSIONS: The frequency of LSIL-H interpretations is significantly increasing at our institution, with a significant decrease in HSIL interpretations over the same period. Two-year risk of CIN2+ and CIN3+ for HSIL increased significantly as usage of LSIL-H increased and that of HSIL decreased. Laboratories using LSIL-H may benefit from monitoring its frequency to ensure its appropriate use. Cancer (Cancer Cytopathol) 2014;122:123-7.
(c) 2013 American Cancer Society.
Dynamic Regulation at the Neuronal Plasma Membrane: Novel Endocytic Mechanisms Control Anesthetic-Activated Potassium Channels and Amphetamine-Sensitive Dopamine Transporters: A Dissertation
Endocytic trafficking dynamically regulates neuronal plasma membrane protein presentation and activity, and plays a central role in excitability and plasticity. Over the course of my dissertation research I investigated endocytic mechanisms regulating two neuronal membrane proteins: the anesthetic-activated potassium leak channel, KCNK3, as well as the psychostimulant-sensitive dopamine transporter (DAT). My results indicate that KCNK3 internalizes in response to Protein Kinase C (PKC) activation, using a novel pathway that requires the phosphoserine binding protein, 14-3-3β, and demonstrates for the first time regulated KCNK3 channel trafficking in neurons. Additionally, PKC-mediated KCNK3 trafficking requires a non-canonical endocytic motif, which is shared exclusively between KCNK3 and sodium-dependent neurotransmitter transporters, such as DAT. DAT trafficking studies in intact ex vivo adult striatal slices indicate that DAT endocytic trafficking has both dynamin-dependent and –independent components. Moreover, DAT segregates into two populations at the neuronal plasma membrane: trafficking-competent and -incompetent. Taken together, these results demonstrate that novel, non-classical endocytic mechanisms dynamically control the plasma membrane presentation of these two important neuronal proteins.
Small RNAs and Argonautes Provide a Paternal Epigenetic Memory of Germline Gene Expression to Promote Thermotolerant Male Fertility: A Dissertation
During each life cycle, gametes must preserve and pass on both genetic and epigenetic information, making the germline both immortal and totipotent. In the male germline the dramatic morphological transformation of a germ cell through meiosis, into a sperm competent for fertilization, while retaining this information is an incredible example of cellular differentiation. This process of spermatogenesis is inherently thermosensitive in numerous metazoa ranging from worms to man. Here, I describe the role of two redundant AGO-class paralogs, ALG-3/4, and their small RNA cofactors, in promoting thermotolerant male fertility in Caenorhabditis elegans. alg-3/4 double mutants exhibit temperature dependent sterility resulting from defective spermiogenesis, the postmeiotic differentiation of haploid spermatids into spermatozoa competent for fertilization. The essential Argonaute CSR-1 functions with ALG-3/4 to positively regulate target genes required for spermiogenesis by promoting transcription via a small RNA positive feedback loop. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal loaded into CSR-1 to maintain transcriptionally active chromatin at genes required for spermiogenesis and to provide an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. The ALG-3/4 small RNA pathway also initiates silencing small RNA signals loaded into WAGO vii Argonautes, which function to posttranscripitonally silence their target mRNAs. Silencing WAGO/small RNA-complexes are present in sperm and presumably transmitted to offspring upon fertilization. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic activating and silencing signals in the form of Argonaute/small-RNA complexes, constituting a memory of gene expression in preceding generations.