Frontal Cryosectioning: An Improved Protocol for Sectioning Large Areas of Fibrous Scaffolds

Fibrous tissue engineering scaffolds, such as those produced by electrospinning, cannot achieve their clinical potential until deep cell-scaffold interactions are understood. Even the most advanced imaging techniques are limited to capturing data at depths of 100 µm due to light scatter associated with the fibers that compose these scaffolds. Conventional cross-sectional analysis provides information on relatively small volumes of space and frontal sections are difficult to generate. Current understanding of cellular penetration into fibrous scaffolds is limited predominantly to the scaffold surface. Although some information is available from cross-sections, sections vary in quality, can distort spatial scaffold properties, and offer virtually no spatial cues as to what scaffold properties instigate specific cellular responses. Without the definitive ability to understand how cells interact with the architecture of an entire scaffold it is difficult to justify scaffold modifications or in-depth cell penetration analyses until appropriate techniques are developed. To address this limitation we have developed a cryosectioning protocol that makes it possible to obtain serial frontal sections from electrospun scaffolds. Microscopic images assembled into montage images from serial sections were then used to create three-dimensional (3D) models of cellular infiltration throughout the entire scaffold

Rebuttal to Irons

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Intelligent Design Will Survive: Kitzmiller v. Dover

Intelligent Design Will Surviv

Intelligent Design Will Survive: Kitzmiller v. Dover

Intelligent Design Will Surviv

Primary Beam Shape Calibration from Mosaicked, Interferometric Observations

Image quality in mosaicked observations from interferometric radio telescopes is strongly dependent on the accuracy with which the antenna primary beam is calibrated. The next generation of radio telescope arrays such as the Allen Telescope Array (ATA) and the Square Kilometer Array (SKA) have key science goals that involve making large mosaicked observations filled with bright point sources. We present a new method for calibrating the shape of the telescope's mean primary beam that uses the multiple redundant observations of these bright sources in the mosaic. The method has an analytical solution for simple Gaussian beam shapes but can also be applied to more complex beam shapes through $\chi^2$ minimization. One major benefit of this simple, conceptually clean method is that it makes use of the science data for calibration purposes, thus saving telescope time and improving accuracy through simultaneous calibration and observation. We apply the method both to 1.43 GHz data taken during the ATA Twenty Centimeter Survey (ATATS) and to 3.14 GHz data taken during the ATA's Pi Gigahertz Sky Survey (PiGSS). We find that the beam's calculated full width at half maximum (FWHM) values are consistent with the theoretical values, the values measured by several independent methods, and the values from the simulation we use to demonstrate the effectiveness of our method on data from future telescopes such as the expanded ATA and the SKA. These results are preliminary, and can be expanded upon by fitting more complex beam shapes. We also investigate, by way of a simulation, the dependence of the accuracy of the telescope's FWHM on antenna number. We find that the uncertainty returned by our fitting method is inversely proportional to the number of antennas in the array.Comment: Accepted by PASP. 8 pages, 8 figure

Transdiagnostic treatment of bipolar disorder and comorbid anxiety using the Unified Protocol for Emotional Disorders: A pilot feasibility and acceptability trial

BACKGROUND Comorbid anxiety in bipolar disorder (BD) is associated with greater illness severity, reduced treatment response, and greater impairment. Treating anxiety in the context of BD is crucial for improving illness course and outcomes. The current study examined the feasibility, acceptability and preliminary efficacy of the Unified Protocol (UP), a transdiagnostic cognitive behavioral therapy, as an adjunctive treatment to pharmacotherapy for BD and comorbid anxiety disorders. METHODS Twenty-nine patients with BD and at least one comorbid anxiety disorder were randomized to pharmacotherapy treatment-as-usual (TAU) or TAU with 18 sessions of the UP (UP+TAU). All patients completed assessments every four weeks to track symptoms, functioning, emotion regulation and temperament. Linear mixed-model regressions were conducted to track symptom changes over time and to examine the relationship between emotion-related variables and treatment response. RESULTS Satisfaction ratings were equivalent for both treatment groups. Patients in the UP+TAU group evidenced significantly greater reductions over time in anxiety and depression symptoms (Cohen's d's>0.80). Baseline levels of neuroticism, perceived affective control, and emotion regulation ability predicted magnitude of symptom change for the UP+TAU group only. Greater change in perceived control of emotions and emotion regulation skills predicted greater change in anxiety related symptoms. LIMITATIONS This was a pilot feasibility and acceptability trial; results should be interpreted with caution. CONCLUSIONS Treatment with the UP+TAU was rated high in patient satisfaction, and resulted in significantly greater improvement on indices of anxiety and depression relative to TAU. This suggests that the UP may be a feasible treatment approach for BD with comorbid anxiety.This work was supported by a Postdoctoral National Research Service Award from the National Institutes of Health [F32 MH098490] to K. Ellard. (F32 MH098490 - Postdoctoral National Research Service Award from the National Institutes of Health)Accepted manuscrip

Listeriolysin S, a Novel Peptide Haemolysin Associated with a Subset of Lineage I Listeria monocytogenes

peer-reviewedStreptolysin S (SLS) is a bacteriocin-like haemolytic and cytotoxic virulence factor that plays a key role in the virulence of Group A Streptococcus (GAS), the causative agent of pharyngitis, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. Although it has long been thought that SLS and related peptides are produced by GAS and related streptococci only, there is evidence to suggest that a number of the most notorious Gram-positive pathogenic bacteria, including Listeria monocytogenes, Clostridium botulinum and Staphylococcus aureus, produce related peptides. The distribution of the L. monocytogenes cluster is particularly noteworthy in that it is found exclusively among a subset of lineage I strains; i.e., those responsible for the majority of outbreaks of listeriosis. Expression of these genes results in the production of a haemolytic and cytotoxic factor, designated Listeriolysin S, which contributes to virulence of the pathogen as assessed by murine- and human polymorphonuclear neutrophil–based studies. Thus, in the process of establishing the existence of an extended family of SLS-like modified virulence peptides (MVPs), the genetic basis for the enhanced virulence of a proportion of lineage I L. monocytogenes may have been revealed.Work is funded by the Irish Government under the National Development Plan, through a Science Foundation Ireland Investigator award to CH, PR and PC (06/IN.1/B98)

Efficient delivery of small interfering RNA for inhibition of IL-12p40 expression in vivo

Background: RNA interference is an evolutionary conserved immune response mechanism that can be used as a tool to provide novel insights into gene function and structure. The ability to efficiently deliver small interfering RNA to modulate gene expression in vivo may provide new therapeutic approaches to currently intractable diseases. Methods: In vitro, siRNA targeting IL-12p40 was delivered to the murine macrophage cell line (J774A.1) encapsulated in a liposome with an IL-12 inducing agent (LPS/IFN-γ) over a number of time points. Controls included a variety of non-target specific siRNA reagents. Supernatants were analyzed for cytokine production while the cells were removed for mRNA profiling. In vivo, siRNA-targeting IL-12p40 was delivered to the murine peritoneal cavity in a therapeutic fashion, after endotoxin (LPS) challenge. Cells from the peritoneal cavity were removed by lavage and analyzed by flow cytometry. Levels of IL-12 present in lavage and in serum were also examined by ELISA. Results: In this report, we show that IL-12p40 siRNA can specifically silence macrophage expression of IL-12p40 mRNA and IL-12p70 protein in vitro. We extend this finding to demonstrate that delivery of liposome encapsulated siRNA targeting IL-12p40 to the murine peritoneal cavity can modulate an inflammatory stimulus in vivo. Furthermore, specific siRNA can be used therapeutically after endotoxin challenge to reduce both the local and systemic inflammatory response. Thus, the delivery of siRNA can be used to elicit specific non-permanent inhibition of endogenous protein expression. Conclusion: In vitro silencing of IL-12p40 using siRNA at selected doses leads to specific knockdown of IL-12p70 protein production without inducing type I interferons. Furthermore, siRNA targeting murine IL-12p40 can be used therapeutically to counter an inflammatory response in vivo

Efficient delivery of small interfering RNA for inhibition of IL-12p40 expression in vivo

Background: RNA interference is an evolutionary conserved immune response mechanism that can be used as a tool to provide novel insights into gene function and structure. The ability to efficiently deliver small interfering RNA to modulate gene expression in vivo may provide new therapeutic approaches to currently intractable diseases. Methods: In vitro, siRNA targeting IL-12p40 was delivered to the murine macrophage cell line (J774A.1) encapsulated in a liposome with an IL-12 inducing agent (LPS/IFN-γ) over a number of time points. Controls included a variety of non-target specific siRNA reagents. Supernatants were analyzed for cytokine production while the cells were removed for mRNA profiling. In vivo, siRNA-targeting IL-12p40 was delivered to the murine peritoneal cavity in a therapeutic fashion, after endotoxin (LPS) challenge. Cells from the peritoneal cavity were removed by lavage and analyzed by flow cytometry. Levels of IL-12 present in lavage and in serum were also examined by ELISA. Results: In this report, we show that IL-12p40 siRNA can specifically silence macrophage expression of IL-12p40 mRNA and IL-12p70 protein in vitro. We extend this finding to demonstrate that delivery of liposome encapsulated siRNA targeting IL-12p40 to the murine peritoneal cavity can modulate an inflammatory stimulus in vivo. Furthermore, specific siRNA can be used therapeutically after endotoxin challenge to reduce both the local and systemic inflammatory response. Thus, the delivery of siRNA can be used to elicit specific non-permanent inhibition of endogenous protein expression. Conclusion: In vitro silencing of IL-12p40 using siRNA at selected doses leads to specific knockdown of IL-12p70 protein production without inducing type I interferons. Furthermore, siRNA targeting murine IL-12p40 can be used therapeutically to counter an inflammatory response in vivo