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Chromatin consists of nucleosomes as well as nonnucleosomal histone-containing particles. Here we describe the prenucleosome, which is a stable conformational isomer of the nucleosome that associates with ∼80 bp DNA. Prenucleosomes are formed rapidly upon the deposition of histones onto DNA and can be converted into canonical nucleosomes by an ATP-driven chromatin assembly factor such as ACF. Different lines of evidence reveal that there are prenucleosome-sized DNA-containing particles with histones in the upstream region of active promoters. Moreover, p300 acetylates histone H3K56 in prenucleosomes but not in nucleosomes, and H3K56 acetylation is found at active promoters and enhancers. These findings therefore suggest that there may be prenucleosomes or prenucleosome-like particles in the upstream region of active promoters. More generally, we postulate that prenucleosomes or prenucleosome-like particles are present at dynamic chromatin, whereas canonical nucleosomes are at static chromatin.

The significant biological and functional differences between small and large platelets suggested by recent studies could have profound implications for transfusion medicine. However, investigating the relationship between platelet size and function is challenging because separating platelets by size without affecting their properties is difficult. A standard approach is centrifugation, but it inevitably leads to premature activation and aggregation of separated platelets. This paper describes the development and validation of a microfluidic device based on controlled incremental filtration (CIF) for separating platelets by size without the cell damage and usability limitations associated with centrifugation. Platelet samples derived from whole blood were used to evaluate the dependence of the CIF device separation performance on design parameters and flow rate, and to compare the properties of PLT fractions generated by the CIF device with those produced using a centrifugation protocol in a split-sample study. This was accomplished by quantifying the platelet size distribution, mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet activation before and after processing for all input and output samples. The 'large platelet' fractions produced by the CIF device and the centrifugation protocol were essentially equivalent (no significant difference in MPV and P-LCR). Platelets in the 'small platelet' fraction produced by the CIF device were significantly smaller than those produced by centrifugation (lower MPV and P-LCR). This was because the CIF 'small platelet' fraction was contaminated by much fewer large platelets (∼2-times lower recovery of >12 fL platelets) and retained the smallest platelets that were discarded by the centrifugation protocol. There was no significant difference in platelet activation between the two methods. However, centrifugation required a substantial amount of additional anticoagulant to prevent platelet aggregation during pelleting. Unlike centrifugation, the CIF device offered continuous, flow-through, single-step processing that did not cause platelet aggregation. Such a capability has the potential to accelerate the basic studies of the relationship between platelet size and function, and ultimately improve transfusion practice, particularly in the pediatric setting, where the need for low-volume, high-quality platelet transfusions is most urgent. [ABSTRACT FROM AUTHOR]

The majority of adoptive cellular therapies are produced from peripheral mononuclear cells obtained via leukapheresis and further enriched for the cells of interest (e.g., T cells). Here, we present a first‐of‐its‐kind closed system, which effectively removes ~85% of monocytes and ~88% of platelets, while recovering ~88% of concentrated T cells in a separate output stream, as the leukapheresis sample flows through a microfluidic device at 5 mL/min. The system is driven by a common peristaltic pump, enabled by a novel pressure wave dampener, and operates in a closed bag‐to‐bag configuration, without requiring any specialized, dedicated equipment. When compared to standard density gradient centrifugation on paired samples, the new system demonstrated a 1.5‐fold increase in T cell recovery and a 2‐fold reduction in inter‐sample variability for this separation outcome. The T cell‐to‐monocyte ratio of the leukapheresis sample was increased to 20:1, whereas with density gradient processing it decreased to 2:1. As a result of superior purity and/or gentler processing, T cells enriched by the system showed a 2.7‐times higher fold expansion during subsequent culture, and an overall 3.5‐times higher cumulative yield. This centrifugation‐free and label‐free closed system for enriching lymphocytes could significantly simplify and standardize the manufacturing of life‐saving cellular therapies. [ABSTRACT FROM AUTHOR]

Tetrodotoxin analogues (TTXs) used in research and development were originated from biological sources for the last decades. Among TTXs‐bearing aquatic animals, horseshoe crab Carcinoscorpius rotundicauda from Vietnam's Coast was reported to contain a high level of TTXs, indicating a promising source for extraction and purification of TTXs. In this work, horseshoe crab C. rotundicauda collected from Can Gio Coast, Vietnam was used for TTX extraction using homogenization‐assisted method with acetic acid solvent. The extraction process was analysed and optimized by single‐factor investigation and response surface methodology (RSM). Single‐factor investigation pointed out that the optimal conditions for TTX recovery yield from C. rotundicauda's soft tissues were homogenization time of 20 min, acetic acid concentration of 1.0%, solvent/matrix ratio of 4 mL/g, extraction temperature of 70°C, extraction time of 30 min and number of extraction of two times. The most significant factors including acetic acid concentration, solvent/matrix ratio and extraction temperature were utilized for constructing the RSM model to predict TTX recovery yield. The maximal TTX recovery yield predicted from RSM model was 97.3% under solvent/matrix ratio of 4 mL/g, extraction temperature of 70°C and acetic acid concentration of 1% (v/v). The RSM model displayed a high stability and precision for prediction of TTX recovery yield from experiments of 1–10 kg C. rotundicauda's soft tissues/batch extractions producing low errors (−1.95% to 1.85%). TTXs in C. rotundicauda's soft tissues were elucidated by HPLC‐MS/MS, revealing the presence of three analogues TTX, 4epi‐TTX and Anh‐TTX with their contents of 43.02, 22.54 and 45.07 μg/g, respectively. [ABSTRACT FROM AUTHOR]

Chromatin assembly involves the combined action of ATP-dependent motor proteins and histone chaperones. Because motor proteins in chromatin assembly also function as chromatin remodeling factors, we investigated the relationship between ATP-driven chromatin assembly and chromatin remodeling in the generation of periodic nucleosome arrays. We found that chromatin remodeling-defective Chd1 motor proteins are able to catalyze ATP-dependent chromatin assembly. The resulting nucleosomes are not, however, spaced in periodic arrays. Wild-type Chd1, but not chromatin remodeling-defective Chd1, can catalyze the conversion of randomly-distributed nucleosomes into periodic arrays. These results reveal a functional distinction between ATP-dependent nucleosome assembly and chromatin remodeling, and suggest a model for chromatin assembly in which randomly-distributed nucleosomes are formed by the nucleosome assembly function of Chd1, and then regularly-spaced nucleosome arrays are generated by the chromatin remodeling activity of Chd1. These findings uncover an unforeseen level of specificity in the role of motor proteins in chromatin assembly. DOI:http://dx.doi.org/10.7554/eLife.00863.001.

Background and Objective: Spatial Frequency Domain Imaging (SFDI) is a non-contact wide-field optical imaging technology currently being developed to investigate the feasibility of quantitative non-invasive evaluation of burn wound severity in a rat model. Our objective is to determine the potential of SFDI for mapping quantitative changes in spatially resolved tissue oxygen saturation and water concentration may be indicative of burn wound severity, healing, and further complications. In this portion of the investigation, we focus on the development of a rat burn model and the acute response of tissue to burn wounds. Study Design/Materials and Methods: A controlled burn protocol involving a heated brass comb was applied to 6 rats. Imaging was acquired at 17 evenly spaced wavelengths in the near-infrared from 650 to 970 nm. Over the course of the 3 hour post-burn period, we were able to map quantitative changes in spatially resolved chromophores. Burn severities were verified post-experiment using standard H and E histology and optical microscopy. Results/Conclusion: In total, we were able to induce 12 superficial-partial thickness burns, 8 deep-partial thickness burns, and 4 full thickness burns in our rat models. While several tissue chromophores were tracked, we found that changes in oxygen saturation and water concentration to be sensitive indicators of burn severity. Future work will include additional longitudinal studies over a period of days in order to investigate which parameters are correlated to tissue healing. © 2013 SPIE.

A photoemission electron microscope (PEEM) was recently commissioned at the NIST. To benchmark its capabilities, epitaxial graphene on 4H-SiC (0001) was imaged and analyzed in the PEEM and compared to other complementary imaging techniques. We determine our routine spatial resolution to be about 50 nm. Using the well-known electronic structure of graphene as a reference, we outline a procedure to calibrate our instrument in energy and momenta in the micrometer-angle-resolved photoemission spectroscopy (μ-ARPES). We also determine the energy and momenta resolution to be about 300 meV, 0.08 Å−1 (ky), and 0.2 Å−1 (kx), respectively. We identify distinct regions of the graphene surface based on intensity contrast rising from topographic and electronic contrasts as well as μ-ARPES. These regions are one layer graphene, one SiC buffer layer, and ≥2 layers of graphene (or graphite). These assignments are confirmed using confocal laser scanning microscopy and Raman spectroscopy. Finally, the PEEM instrument had enough sensitivity to observe the flatband in monolayer epitaxial graphene, which we attribute to the presence of compressive strain, −1.2%, in the graphene sample. [ABSTRACT FROM AUTHOR]

BackgroundObesity is one of the most important modifiable risk factors for the prevention of type 2 diabetes. The aim of this study was to examine the prevalence of diabetes with increasing severity of obesity and the distribution of HbA1c levels in diabetics participating in the latest National Health and Nutrition Examination Survey (NHANES).MethodsData from a representative sample of adults with diabetes participating in the NHANES between 1999 and 2006 were reviewed. The prevalence of diabetes and levels of fasting glucose, insulin, c-peptide, and HbA1c were examined across different weight classes with normal weight, overweight, and obesity classes 1, 2, and 3 were defined as body mass index (BMI) of 8.0%) in 23%. The mean fasting glucose and HbA1c levels were highest for diabetics with BMI

Obesity has been linked with a chronic state of inflammation which may be involved in the development of metabolic syndrome, cardiovascular disease, non-alcoholic steatohepatitis, and even cancer. The objective of this study was to examine the association between obesity class and levels of inflammatory biomarkers from men and women who participated in the 1999–2004 National Health and Nutrition Examination Survey (NHANES).Serum concentrations of C-reactive protein (CRP) and fibrinogen were measured among US participants of the 1999–2004 NHANES. We examined biomarker levels across different weight classes with normal weight, overweight, and obesity classes 1, 2, and 3 were defined as BMI of