BACKGROUND:: External volume expansion (EVE) by suction has been proposed to improve the survival of fat grafting by preparing the recipient site. In previous experimental work, EVE demonstrated the capacity to stimulate cell proliferation, vessel remodeling and adipogenesis. This study investigated possible mechanisms underlying these observed changes.
METHODS:: A miniaturized EVE device was applied to the dorsum of mice for 2 hours. Hypoxia during stimulation was assessed with pimonidazole hydrochloride, and tissue perfusion was measured for up to 2 days via hyperspectral imaging. Treated tissues were evaluated by microscopy for the presence of edema, inflammation, the effects on cell proliferation and vessel remodeling.
RESULTS:: EVE-treated tissues were grossly expanded with 2 hours of stimulation, developing a macroscopic swelling that slowly regressed over the course of hours following stimulus cessation. This gross swelling was reflective of histological signs of intense edema, persistent for at least 1 hour post-EVE. Tissues were hypoxic during stimulation, and hyperspectral imaging demonstrated decreased tissue content of both oxygenated- and deoxygenated-hemoglobin in the first hour after EVE release. The onset of inflammation was already apparent by the end of stimulation, and remained elevated through two days post-EVE. At this time point epidermal and dermal cell proliferation, as well as vascular density, were significantly increased.
CONCLUSIONS:: EVE sets in motion various mechanisms including mechanical stimulation, edema, ischemia and inflammation that over distinct time periods maintain an environment conducive to cell proliferation and angiogenesis, which can be elicited even by a single 2 hour EVE cycle.
Long-term synaptic plasticity involves changes in the expression and membrane insertion of cell-surface proteins. Interestingly, the mRNAs encoding many cell-surface proteins are localized to dendrites, but whether dendritic protein synthesis is required for activity-induced surface expression of specific proteins is unknown. Herein, we used microfluidic devices to demonstrate that dendritic protein synthesis is necessary for activity-induced insertion of GluN2A-containing NMDA receptors in rat hippocampal neurons. Furthermore, visualization of activity-induced local translation of GluN2A mRNA and membrane insertion of GluN2A protein in dendrites was directly observed and shown to depend on a 3' untranslated region cytoplasmic polyadenylation element and its associated translation complex. These findings uncover a novel mechanism for cytoplasmic polyadenylation element-mediated posttranscriptional regulation of GluN2A mRNA to control NMDA receptor surface expression during synaptic plasticity.
Mouse, but not Human STING, Binds and Signals in Response to the Vascular Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid
Vascular disrupting agents such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA) represent a novel approach for cancer treatment. DMXAA has potent antitumor activity in mice and, despite significant preclinical promise, failed human clinical trials. The antitumor activity of DMXAA has been linked to its ability to induce type I IFNs in macrophages, although the molecular mechanisms involved are poorly understood. In this study, we identify stimulator of IFN gene (STING) as a direct receptor for DMXAA leading to TANK-binding kinase 1 and IFN regulatory factor 3 signaling. Remarkably, the ability to sense DMXAA was restricted to murine STING. Human STING failed to bind to or signal in response to DMXAA. Human STING also failed to signal in response to cyclic dinucleotides, conserved bacterial second messengers known to bind and activate murine STING signaling. Collectively, these findings detail an unexpected species-specific role for STING as a receptor for an anticancer drug and uncover important insights that may explain the failure of DMXAA in clinical trials for human cancer.
BACKGROUND: Heart failure is a highly prevalent, morbid, and costly disease with a poor long-term prognosis. Evidence-based therapies utilized over the past 2 decades hold the promise of improved outcomes, yet few contemporary studies have examined survival trends in patients with acute heart failure. The primary objective of this population-based study was to describe trends in short- and long-term survival in patients hospitalized with acute decompensated heart failure (ADHF). A secondary objective was to examine patient characteristics associated with decreased long-term survival.
METHODS AND RESULTS: We reviewed the medical records of 9748 patients hospitalized with ADHF at all 11 medical centers in central Massachusetts during 1995, 2000, 2002, and 2004. Patients hospitalized with ADHF were more likely to be elderly and to have been diagnosed with multiple comorbidities in 2004 compared with 1995. Over this period, survival was significantly improved in-hospital, and at 1, 2, and 5 years postdischarge. Five-year survival rates increased from 20% in 1995 to 29% in 2004. Although survival improved substantially over time, older patients and patients with chronic kidney disease, chronic obstructive pulmonary disease, anemia, low body mass index, and low blood pressures had consistently lower postdischarge survival rates than patients without these comorbidities.
CONCLUSION: Between 1995 and 2004, patients hospitalized with ADHF have become older and increasingly comorbid. Although there has been a significant improvement in survival among these patients, their long-term prognosis remains poor, as fewer than 1 in 3 patients hospitalized with ADHF in 2004 survived more than 5 years.
The histone acetyltransferase Rtt109 is the sole enzyme responsible for acetylation of histone H3 lysine 56 (H3K56) in fungal organisms. Loss of Rtt109 renders fungal cells extremely sensitive to genotoxic agents, and prevents pathogenesis in several clinically important species. Here, via a high throughput chemical screen of >300,000 compounds, we discovered a chemical inhibitor of Rtt109 that does not inhibit other acetyltransferase enzymes. This compound inhibits Rtt109 regardless of which histone chaperone cofactor protein (Asf1 or Vps75) is present, and appears to inhibit Rtt109 via a tight-binding, uncompetitive mechanism.
New generation of flow diverter (surpass) for unruptured intracranial aneurysms: a prospective single-center study in 37 patients
BACKGROUND AND PURPOSE: In patients harboring intracranial aneurysms, the major goal in treatment is to prevent bleeding. A new generation of an endoluminal device (Surpass Flow Diverter [Surpass]) was developed to reconstruct parent artery and occlude the aneurysm. We present our clinical and angiographic single-center experience.
METHODS: Patients with a wide range of complex unruptured aneurysms were treated with the Surpass placed in the parent artery and bridging the aneurysm. Clinical and angiographic follow-up were performed at 6 months. Data were prospectively collected.
RESULTS: Thirty seven patients (mean age, 56 years; range, 32-79), harboring 49 unruptured aneurysms were treated at our center. All except 1 patient were treated with a single device. Successful delivery of the device was achieved in all patients. All 35 nonbifurcation aneurysm necks were covered completely, whereas 14 bifurcation aneurysms were only partially covered. There was no major periprocedural morbidity or mortality. During follow-up, 4 patients (10.4%) experienced transient neurological deficit. One patient (3%) developed a minor stroke at 4-month follow-up with persistent neurological deficit. Twelve patients had neurological symptoms related to their aneurysm and 7 showed improvement of these symptoms during follow-up. At 6-month follow-up, 29 of 31 aneurysms studied that had complete neck coverage showed a complete occlusion (94%) including 1 case with a 95% to 100% occlusion, whereas 5 of the 10 bifurcation aneurysms were occluded.
CONCLUSIONS: Our study shows high safety and efficacy profile of a new generation endoluminal device in treatment of complex intracranial aneurysms. Long-term studies of treated bifurcation aneurysms are needed.
A negative transcriptional feedback loop generates circadian rhythms in Drosophila. PERIOD (PER) is a critical state-variable in this mechanism, and its abundance is tightly regulated. We found that the Drosophila homolog of ATAXIN-2 (ATX2)--an RNA-binding protein implicated in human neurodegenerative diseases--was required for circadian locomotor behavior. ATX2 was necessary for PER accumulation in circadian pacemaker neurons and thus determined period length of circadian behavior. ATX2 was required for the function of TWENTY-FOUR (TYF), a crucial activator of PER translation. ATX2 formed a complex with TYF and promoted its interaction with polyadenylate-binding protein (PABP). Our work uncovers a role for ATX2 in circadian timing and reveals that this protein functions as an activator of PER translation in circadian neurons.
An Institutional Review Board-approved protocol was used to quantify breast tissue inclusion in 52 women, under conditions simulating both craniocaudal (CC) and mediolateral oblique (MLO) views in mammography, dedicated breast CT in the upright subject position, and dedicated breast CT in the prone subject position. Using skin as a surrogate for the underlying breast tissue, the posterior aspect of the breast that is aligned with the chest-wall edge of the breast support in a screen-film mammography system was marked with the study participants positioned for CC and MLO views. The union of skin marks with the study participants positioned for CC and MLO views was considered to represent chest-wall tissue available for imaging with mammography and served as the reference standard. For breast CT, a prone stereotactic breast biopsy unit and a custom-fabricated barrier were used to simulate conditions during prone and upright breast CT, respectively. For the same breast marked on the mammography system, skin marks were made along the breast periphery that was just anterior to the apertures of the prone biopsy unit and the upright barrier. The differences in skin marks between subject positioning simulating breast CT (prone, upright) and mammography were quantified at six anatomic locations. For each location, at least one study participant had a skin mark from breast CT (prone, upright) posterior to mammography. However for all study participants, there was at least one anatomic location where the skin mark from mammography was posterior to that from breast CT (prone, upright) positioning. The maximum amount by which the skin mark from mammography was posterior to breast CT (prone and upright) over all six locations was quantified for each study participant and pair-wise comparison did not exhibit statistically significant difference between prone and upright breast CT (paired t- test, p = 0.4). Quantitatively, for 95% of the study participants the skin mark from mammography was posterior to breast CT (prone or upright) by at the most 9 mm over all six locations. Based on the study observations, geometric design considerations targeting chest-wall coverage with breast CT equivalent to mammography, wherein part of the x-ray beam images through the swale during breast CT are provided. Assuming subjects can extend their chest in to a swale, the optimal swale-depth required to achieve equivalent coverage with breast CT images as mammograms for 95% of the subjects varies in the range of approximately 30-50 mm for clinical prototypes and was dependent on the system geometry.
Nicotinic acetylcholine receptors (nAChRs) containing either the alpha4 and/or alpha6 subunit are robustly expressed in dopaminergic nerve terminals in dorsal striatum where they are hypothesized to modulate dopamine (DA) release via acetylcholine (ACh) stimulation from cholinergic interneurons. However, pharmacological blockade of nAChRs or genetic deletion of individual nAChR subunits, including alpha4 and alpha6, in mice, yields little effect on motor behavior. Based on the putative role of nAChRs containing the alpha4 subunit in modulation of DA in dorsal striatum, we hypothesized that mice expressing a single point mutation in the alpha4 nAChR subunit, Leu9'Ala, that renders nAChRs hypersensitive to agonist, would exhibit exaggerated differences in motor behavior compared to WT mice. To gain insight into these differences, we challenged WT and Leu9'Ala mice with the alpha4beta2 nAChR antagonist dihydro-beta-erythroidine (DHbetaE). Interestingly, in Leu9'Ala mice, DHbetaE elicited a robust, reversible motor impairment characterized by hypolocomotion, akinesia, catalepsy, clasping, and tremor; whereas the antagonist had little effect in WT mice at all doses tested. Pre-injection of nicotine (0.1 mg/kg) blocked DHbetaE-induced motor impairment in Leu9'Ala mice confirming that the phenotype was mediated by antagonism of nAChRs. In addition, SKF82958 (1 mg/kg) and amphetamine (5 mg/kg) prevented the motor phenotype. DHbetaE significantly activated more neurons within striatum and substantia nigra pars reticulata in Leu9'Ala mice compared to WT animals, suggesting activation of the indirect motor pathway as the circuit underlying motor dysfunction. ACh evoked DA release from Leu9'Ala striatal synaptosomes revealed agonist hypersensitivity only at alpha4(non-alpha6)* nAChRs. Similarly, alpha6 nAChR subunit deletion in an alpha4 hypersensitive nAChR (Leu9'Ala/alpha6 KO) background had little effect on the DHbetaE-induced phenotype, suggesting an alpha4(non-alpha6)* nAChR-dependent mechanism. Together, these data indicate that alpha4(non-alpha6)* nAChR have an impact on motor output and may be potential molecular targets for treatment of disorders associated with motor impairment.
Structural basis of the relaxed state of a Ca2+-regulated myosin filament and its evolutionary implications
Myosin filaments of muscle are regulated either by phosphorylation of their regulatory light chains or Ca(2+) binding to the essential light chains, contributing to on-off switching or modulation of contraction. Phosphorylation-regulated filaments in the relaxed state are characterized by an asymmetric interaction between the two myosin heads, inhibiting their actin binding or ATPase activity. Here, we have tested whether a similar interaction switches off activity in myosin filaments regulated by Ca(2+) binding. Cryo-electron microscopy and single-particle image reconstruction of Ca(2+)-regulated (scallop) filaments reveals a helical array of myosin head-pair motifs above the filament surface. Docking of atomic models of scallop myosin head domains into the motifs reveals that the heads interact in a similar way to those in phosphorylation-regulated filaments. The results imply that the two major evolutionary branches of myosin regulation-involving phosphorylation or Ca(2+) binding-share a common structural mechanism for switching off thick-filament activity in relaxed muscle. We suggest that the Ca(2+)-binding mechanism evolved from the more ancient phosphorylation-based system to enable rapid response of myosin-regulated muscles to activation. Although the motifs are similar in both systems, the scallop structure is more tilted and higher above the filament backbone, leading to different intermolecular interactions. The reconstruction reveals how the myosin tail emerges from the motif, connecting the heads to the filament backbone, and shows that the backbone is built from supramolecular assemblies of myosin tails. The reconstruction provides a native structural context for understanding past biochemical and biophysical studies of this model Ca(2+)-regulated myosin.
Small, noncoding microRNAs (miRNAs) regulate diverse biological functions in the liver and increasing evidence suggests that they have a role in liver pathology. This Review summarizes advances in the field of miRNAs in liver diseases, inflammation and cirrhosis. MicroRNA-122, the most abundant miRNA in hepatocytes, has well-defined roles in HCV replication, and data indicate that it also serves as a viable therapeutic target. The role of miR-122 is also emerging in other liver diseases. Ample evidence exists for the important regulatory potential of other miRNAs in conditions associated with liver inflammation related to alcohol use, the metabolic syndrome or autoimmune processes. In addition, a broad array of miRNAs have been associated with the development of liver fibrosis both in animal models and human studies. The significance of the function and cellular distribution of miRNAs in the liver and the potential of miRNAs as a means of communication between cells and organs is discussed as well as the emerging utility of circulating miRNAs as biomarkers of different forms of liver damage and as early markers of disease and progression in hepatocellular carcinoma. Importantly, miRNA modulation in the liver represents a new therapeutic approach in the treatment armamentarium of hepatologists in the future.
Notch signaling regulates a broad spectrum of cell fate decisions and differentiation. Both oncogenic and tumor suppressor functions have been shown for Notch signaling. However, little is known about the underlying mechanisms of its tumor suppressor function. Here, we report that expression of Notch3, a member of Notch family transmembrane receptors, was elevated in human cells during senescence activated by various senescence-inducing stimuli. This upregulation of Notch3 was required for the induction of p21 expression in senescent cells. Downregulation of Notch3 led to a delayed onset of senescence and extended replicative lifespan, whereas adventitious expression of Notch3 was sufficient to activate senescence and p21 expression. The ability of Notch3 to induce senescence and p21 expression was dependent on the canonical Notch singling. Deletion of p21 in cells significantly attenuated Notch3-induced senescence.
Furthermore, a significant decrease in Notch3 expression was observed in human tumor cell lines as well as primary human breast cancer and melanoma samples compared with normal tissues. Restoration of Notch3 expression in human tumor cells resulted in inhibition of cell proliferation and activation of senescence. Collectively, our results reveal a novel function of Notch3 in senescence regulation and tumor suppression. Cancer Res; 73(11); 1-9. (c)2013 AACR.
Tunable Near Infrared to Ultraviolet Upconversion Luminescence Enhancement in (alpha-NaYF :Yb,Tm)/CaF Core/Shell Nanoparticles for In situ Real-time Recorded Biocompatible Photoactivation
A family of upconverting nanoparticles (UCNPs) with a tunable UV enhancement is developed via a facile approach. The design leads to a maximum 9-fold enhancement in comparison with known optimal beta-phase core/shell UCNPs in water. A highly effective and rapid in situ real-time live-cell photoactivation is recorded for the first time with such nanoparticles.
BACKGROUND: Despite the availability of effective therapies, heart failure (HF) remains a highly prevalent disease and the leading cause of hospitalizations in the U.S. Few data are available, however, describing changing trends in the use of various cardiac medications to treat patients with HF and factors associated with treatment. The objectives of this population-based study were to examine decade-long trends (1995 - 2004) in the use of several cardiac medications in patients hospitalized with acute decompensated heart failure (ADHF) and factors associated with evidence-based treatment.
METHODS: We reviewed the medical records of 9,748 residents of the Worcester, MA, metropolitan area who were hospitalized with ADHF at all 11 central Massachusetts medical centers in 1995, 2000, 2002, and 2004.
RESULTS: Between 1995 and 2004, respectively, the prescription upon hospital discharge of beta-blockers (23%; 67%), angiotensin pathway inhibitors (47%; 55%), statins (5%; 43%), and aspirin (35%; 51%) increased markedly, while the use of digoxin (51%; 29%), nitrates (46%; 24%), and calcium channel blockers (33%; 22%) declined significantly; nearly all patients received diuretics. Patients in the earliest study year, those with a history of obstructive pulmonary disease or anemia, incident HF, non-specific symptoms, and women were less likely to receive beta blockers and angiotensin pathway inhibitors than respective comparison groups. In 2004, 82% of patients were discharged on at least one of these recommended agents; however, only 41% were discharged on medications from both recommended classes.
CONCLUSIONS: Our data suggest that opportunities exist to further improve the use of HF therapeutics.
Haemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder resulting from immune dysfunction reflecting either primary immune deficiency or acquired failure of normal immune homeostasis. Familial HLH includes autosomal recessive and X-linked disorders characterized by uncontrolled activation of T cells and macrophages and overproduction of inflammatory cytokines, secondary to defects in genes encoding proteins involved in granule-dependent cytolytic pathways. In older children and adults, HLH is associated more often with infections, malignancies, autoimmune diseases, and acquired immune deficiencies. HLH, macrophage activation syndrome, sepsis, and systemic inflammatory response syndrome are different clinical entities that probably represent a common immunopathological state, termed cytokine storm. These conditions may be clinically indistinguishable; all include massive inflammatory response, elevated serum cytokine levels, multi-organ involvement, haemophagocytic macrophages, and often death. Tissues of haematopoietic and lymphoid function are directly involved; other organs are secondarily damaged by circulating cytokines and chemokines. Haemophagocytic disorders are now increasingly diagnosed in the context of severe inflammatory reactions to viruses, malignancies and systemic connective tissue diseases. Many of these cases may reflect underlying genetic predispositions to HLH. The detection of gene defects has contributed considerably to our understanding of HLH, but the mechanisms leading to acquired HLH have yet to be fully determined.
We present version 3.0 of our publicly available protein-protein docking benchmark. This update includes 40 new test cases, representing a 48% increase from Benchmark 2.0. For all of the new cases, the crystal structures of both binding partners are available. As with Benchmark 2.0, Structural Classification of Proteins (Murzin et al., J Mol Biol 1995;247:536-540) was used to remove redundant test cases. The 124 unbound-unbound test cases in Benchmark 3.0 are classified into 88 rigid-body cases, 19 medium-difficulty cases, and 17 difficult cases, based on the degree of conformational change at the interface upon complex formation. In addition to providing the community with more test cases for evaluating docking methods, the expansion of Benchmark 3.0 will facilitate the development of new algorithms that require a large number of training examples. Benchmark 3.0 is available to the public at http://zlab.bu.edu/benchmark.
The insulator binding protein CTCF positions 20 nucleosomes around its binding sites across the human genome
Chromatin structure plays an important role in modulating the accessibility of genomic DNA to regulatory proteins in eukaryotic cells. We performed an integrative analysis on dozens of recent datasets generated by deep-sequencing and high-density tiling arrays, and we discovered an array of well-positioned nucleosomes flanking sites occupied by the insulator binding protein CTCF across the human genome. These nucleosomes are highly enriched for the histone variant H2A.Z and 11 histone modifications. The distances between the center positions of the neighboring nucleosomes are largely invariant, and we estimate them to be 185 bp on average. Surprisingly, subsets of nucleosomes that are enriched in different histone modifications vary greatly in the lengths of DNA protected from micrococcal nuclease cleavage (106-164 bp). The nucleosomes enriched in those histone modifications previously implicated to be correlated with active transcription tend to contain less protected DNA, indicating that these modifications are correlated with greater DNA accessibility. Another striking result obtained from our analysis is that nucleosomes flanking CTCF sites are much better positioned than those downstream of transcription start sites, the only genomic feature previously known to position nucleosomes genome-wide. This nucleosome-positioning phenomenon is not observed for other transcriptional factors for which we had genome-wide binding data. We suggest that binding of CTCF provides an anchor point for positioning nucleosomes, and chromatin remodeling is an important component of CTCF function.
Structure-based design of a T-cell receptor leads to nearly 100-fold improvement in binding affinity for pepMHC
T-cell receptors (TCRs) are proteins that recognize peptides from foreign proteins bound to the major histocompatibility complex (MHC) on the surface of an antigen-presenting cell. This interaction enables the T cells to initiate a cell-mediated immune response to terminate cells displaying the foreign peptide on their MHC. Naturally occurring TCRs have high specificity but low affinity toward the peptide-MHC (pepMHC) complex. This prevents the usage of solubilized TCRs for diagnosis and treatment of viral infections or cancers. Efforts to enhance the binding affinity of several TCRs have been reported in recent years, through randomized libraries and in vitro selection. However, there have been no reported efforts to enhance the affinity via structure-based design, which allows more control and understanding of the mechanism of improvement.
Here, we have applied structure-based design to a human TCR to improve its pepMHC binding. Our design method evolved based on iterative steps of prediction, testing, and generating more predictions based on the new data. The final design function, named ZAFFI, has a correlation of 0.77 and average error of 0.35 kcal/mol with the binding free energies of 26 point mutations for this system that we measured by surface plasmon resonance (SPR). Applying the filter that we developed to remove nonbinding predictions, this correlation increases to 0.85, and the average error decreases to 0.3 kcal/mol. Using this algorithm, we predicted and tested several point mutations that improved binding, with one giving over sixfold binding improvement. Four of the point mutations that improved binding were then combined to give a mutant TCR that binds the pepMHC 99 times more strongly than the wild-type TCR.
BACKGROUND: Single nucleotide polymorphisms (SNPs) have been used extensively in genetics and epidemiology studies. Traditionally, SNPs that did not pass the Hardy-Weinberg equilibrium (HWE) test were excluded from these analyses. Many investigators have addressed possible causes for departure from HWE, including genotyping errors, population admixture and segmental duplication. Recent large-scale surveys have revealed abundant structural variations in the human genome, including copy number variations (CNVs). This suggests that a significant number of SNPs must be within these regions, which may cause deviation from HWE.
RESULTS: We performed a Bayesian analysis on the potential effect of copy number variation, segmental duplication and genotyping errors on the behavior of SNPs. Our results suggest that copy number variation is a major factor of HWE violation for SNPs with a small minor allele frequency, when the sample size is large and the genotyping error rate is 0~1%.
CONCLUSIONS: Our study provides the posterior probability that a SNP falls in a CNV or a segmental duplication, given the observed allele frequency of the SNP, sample size and the significance level of HWE testing.