Histone deacetylase (HDAC) 1 and 2 complexes regulate both histone acetylation and crotonylation in vivo.

Proteomic analysis of histones has shown that they are subject to a superabundance of acylations, which extend far beyond acetylation, to include: crotonylation, propionylation, butyrylation, malonylation, succinylation, ß-hydroxybutyrylation and 2-hydroxyisobutyrylation. To date, much of the functional data has focussed on histone crotonylation which, similar to acetylation, has been associated with positive gene regulation and is added by the acyltransferase, p300. Although Sirtuins 1-3, along with HDAC3, have been shown to possess decrotonylase activity in vitro, there is relatively little known about the regulation of histone crotonylation in vivo. Here we show that Histone Deacetylase 1 and 2 (HDAC1/2), the catalytic core of numerous co-repressor complexes, are important histone decrotonylase enzymes. A ternary complex of HDAC1/CoREST1/LSD1 is able to hydrolyse both histone H3 Lys18-acetyl (H3K18ac) and H3 Lys18-crotonyl (H3K18cr) peptide substrates. Genetic deletion of HDAC1/2 in ES cells increases global levels of histone crotonylation and causes an 85% reduction in total decrotonylase activity. Furthermore, we mapped H3K18cr in cells using ChIP-seq, with and without HDAC1/2, and observed increased levels of crotonylation, which largely overlaps with H3K18ac in the vicinity of transcriptional start sites. Collectively, our data indicate that HDAC1/2 containing complexes are critical regulators of histone crotonylation in vivo.

Raman spectroscopy of stored red blood cell concentrate within sealed transfusion blood bags.

The standard practice in blood banks worldwide involves storage of red blood cells (RBCs) in plastic bags until they are needed for transfusion. During storage, the cells gradually degrade in functionality, a condition described as RBC storage lesion. Standard analytical techniques cannot assess the blood quality without breaching the sterility of the transfusion bag. In this study, we employed a commercially available spatially offset Raman spectroscopy (SORS) system using a custom designed protocol to non-invasively explore the biochemical changes in RBC concentrate of healthy donors over a storage period of approximately 42 days in standard transfusion bags, under standard storage conditions. The results reveal an increase in the oxygenation state of haemoglobin over the storage period for all donors, but different profiles for each donor. This study demonstrates the feasibility of acquiring consistent biochemical information relevant to the quality of stored blood, in situ through sealed blood transfusion bags using a commercially available instrument.

Non-invasive spectroscopy of transfusable red blood cells stored inside sealed plastic blood-bags.

After being separated from (donated) whole blood, red blood cells are suspended in specially formulated additive solutions and stored (at 4 °C) in polyvinyl chloride (PVC) blood-bags until they are needed for transfusion. With time, the prepared red cell concentrate (RCC) is known to undergo biochemical changes that lower effectiveness of the transfusion, and thus regulations are in place that limit the storage period to 42 days. At present, RCC is not subjected to analytical testing prior to transfusion. In this study, we use Spatially Offset Raman Spectroscopy (SORS) to probe, non-invasively, the biochemistry of RCC inside sealed blood-bags. The retrieved spectra compare well with conventional Raman spectra (of sampled aliquots) and are dominated by features associated with hemoglobin. In addition to the analytical demonstration that SORS can be used to retrieve RCC spectra from standard clinical blood-bags without breaking the sterility of the system, the data reveal interesting detail about the oxygenation-state of the stored cells themselves, namely that some blood-bags unexpectedly contain measurable amounts of deoxygenated hemoglobin after weeks of storage. The demonstration that chemical information can be obtained non-invasively using spectroscopy will enable new studies of RCC degeneration, and points the way to a Raman-based instrument for quality-control in a blood-bank or hospital setting.

Do individuals with intellectual disability select appropriate objects as landmarks when learning a new route?

BACKGROUND: The present study was aimed at investigating the selection of landmarks by individuals with intellectual disability (ID). The hypothesis was that they would be less efficient than individuals without IDs in the selection of landmarks when learning a new route. METHODS: The experiment took place in a natural setting with a group of participants with ID and a group of control participants matched by chronological age. The participants were first guided along a route situated in an unfamiliar district. Then, they had to guide the experimenter along the route while pointing to all the objects and features they found useful for wayfinding. RESULTS: The designated objects were categorised as a function of their landmarks properties. There were significant differences between the two groups for non-permanent landmarks, distant landmarks and non-unique landmarks. The two groups selected landmarks near intersections in the same proportions. However, the individuals with ID selected more non-unique landmarks and less textual signage than the control group at these decision points. CONCLUSION: Individuals with ID seem to be less efficient than individuals without disability in landmark selection. This may limit their wayfinding abilities in their day-to-day travelling. This may also account for their difficulties in obtaining the kind of spatial knowledge which relates to the configural structure of their environment.

A qualitative investigation of school-aged children's answers to items from a generic quality of life measure.

BACKGROUND: The development of instruments to measure child self-reported quality of life (QOL) is dependent on whether children can understand the concepts behind items. Researchers need more information on how children are interpreting and answering items. This paper aims to investigate the strategies school-aged children use to answer QOL items. METHODS: A generic 30-item QOL measure (the TedQL) was administered to 266 healthy children (5-6, 7-9 years old). Children were asked to 'think aloud' while answering a selection of 10 TedQL items (n = 4 ability, n = 4 social, n = 2 mood items), and their responses were recorded verbatim. RESULTS: The strategies children reported using when answering items were coded into five categories: (1) social comparisons; (2) stable character references; (3) concrete examples; (4) other reasons; or (5) no reason given. Concrete examples were used most frequently by children. Strategy type was dependent on age, with 7-9-year-olds reporting social comparisons and concrete examples more frequently than 5-6-year-olds. Five-to-six-year-olds gave no reasons for their response choices more frequently than 7-9-year-olds. Strategy type also differed by item type, with social comparisons used more frequently for ability items, and stable character references for social items. However, concrete examples were used consistently highly across ability and social items. CONCLUSIONS: Children aged 5-9 years most commonly report using concrete examples of specific instances when answering QOL items. However, strategy use varies as a function of age and types of items. Our results highlight the importance of keeping in mind children's developmental age when interpreting responses from child QOL instruments.

Pulse optimization for Raman spectroscopy with cross-correlation frequency resolved optical gating.

We propose to employ the technique of femtosecond pulse shaping for improving the performance of the recently suggested method of complete characterization of molecular vibrations, in which both the amplitude and phase of the laser induced vibrational coherence are detected with high resolution. The amplitude-phase information is retrieved from the cross-correlation frequency resolved optical gating of Raman modes. By creating rich interference pattern in the measured two-dimensional spectrogram of coherent anti-Stokes Raman scattering we enhance the accuracy of the retrieved spectral and temporal response and increase the robustness of the method against noise.

Investigation of a 2D two-point maximum entropy regularization method for signal-to-noise ratio enhancement: application to CT polymer gel dosimetry.

This study presents a new method of image signal-to-noise ratio (SNR) enhancement by utilizing a newly developed 2D two-point maximum entropy regularization method (TPMEM). When utilized as an image filter, it is shown that 2D TPMEM offers unsurpassed flexibility in its ability to balance the complementary requirements of image smoothness and fidelity. The technique is evaluated for use in the enhancement of x-ray computed tomography (CT) images of irradiated polymer gels used in radiation dosimetry. We utilize a range of statistical parameters (e.g. root-mean square error, correlation coefficient, error histograms, Fourier data) to characterize the performance of TPMEM applied to a series of synthetic images of varying initial SNR. These images are designed to mimic a range of dose intensity patterns that would occur in x-ray CT polymer gel radiation dosimetry. Analysis is extended to a CT image of a polymer gel dosimeter irradiated with a stereotactic radiation therapy dose distribution. Results indicate that TPMEM performs strikingly well on radiation dosimetry data, significantly enhancing the SNR of noise-corrupted images (SNR enhancement factors >15 are possible) while minimally distorting the original image detail (as shown by the error histograms and Fourier data). It is also noted that application of this new TPMEM filter is not restricted exclusively to x-ray CT polymer gel dosimetry image data but can in future be extended to a wide range of radiation dosimetry data.

The effect of interviewing techniques on young children's responses to questions.

BACKGROUND: Research into the effect of interviewing techniques has been predominantly within the paradigm of eyewitness testimony. This review focuses on the issues of questioning and examines whether children's responses are affected by questioning techniques, and whether these effects are generic to all interviewing contexts. METHODS: Systematic literature searches were used to identify areas of concern and current findings in research on interviewing young children (aged 4-12). RESULTS: The style and wording of questioning can affect children's responses and accuracy positively and negatively. These effects were especially apparent in interviews with the youngest children. CONCLUSIONS: The implications of these findings are relevant in all contexts where an adult questions a child. It has been demonstrated that interviewing techniques can affect responses from children and that it is therefore imperative that interviewers are aware of, understand and control their influence in order to elicit complete, accurate and reliable information from the child.

Association of CXCL13 and CCL21 expression with the progressive organization of lymphoid-like structures in Sjögren's syndrome.

OBJECTIVE: Ectopic lymphoneogenesis can occur in the salivary glands of Sjögren's syndrome (SS) patients and is associated with local antigen-driven B cell responses, autoantibody formation, and potential lymphomatous transformation. CXCL13 and CCL21 have been identified in salivary glands, but their role in ectopic lymphoneogenesis in SS remains unclear. This study aimed to evaluate the microanatomic association between CXCL13 and CCL21 expression and the acquisition of lymphoid features in periductal foci. METHODS: Salivary glands from 37 SS patients and 9 chronic sialadenitis patients were analyzed by immunohistochemistry for T cell/B cell segregation, CD21+ follicular dendritic cell networks, and peripheral lymph node addressin (PNAd)-positive high endothelial venules (HEVs) in relationship to the size of the aggregates and the expression of CXCL13 and CCL21 within infiltrating cells, epithelium, and endothelium. RESULTS: Grade 1 aggregates (10-50 lymphocytes) demonstrated predominance of nonorganized CD3+ cells, while grade 2 (>50 lymphocytes) and grade 3 (>50 with germinal centers) showed a progressive increase in CD20+ B cells and T cell/B cell segregation. This higher degree of lymphoid organization was significantly related to an increased expression of CXCL13 within infiltrating cells and PNAd+ HEV-associated CCL21-producing cells. Conversely, no association between lymphoid organization and lymphoid chemokine expression by epithelial cells was observed. CONCLUSION: The acquisition of lymphoid features by inflammatory foci in SS is critically associated with the enlargement of the inflammatory foci and with the expression of CXCL13 and CCL21 within the infiltrate, but is not associated with their expression by epithelial cells. These data strongly support an active participation of CXCL13 and CCL21 in regulating the progressive organization and maintenance of periductal foci.

Accuracy and precision of manual baseline determination.

Vibrational spectra often require baseline removal before further data analysis can be performed. Manual (i.e., user) baseline determination and removal is a common technique used to perform this operation. Currently, little data exists that details the accuracy and precision that can be expected with manual baseline removal techniques. This study addresses this current lack of data. One hundred spectra of varying signal-to-noise ratio (SNR), signal-to-baseline ratio (SBR), baseline slope, and spectral congestion were constructed and baselines were subtracted by 16 volunteers who were categorized as being either experienced or inexperienced in baseline determination. In total, 285 baseline determinations were performed. The general level of accuracy and precision that can be expected for manually determined baselines from spectra of varying SNR, SBR, baseline slope, and spectral congestion is established. Furthermore, the effects of user experience on the accuracy and precision of baseline determination is estimated. The interactions between the above factors in affecting the accuracy and precision of baseline determination is highlighted. Where possible, the functional relationships between accuracy, precision, and the given spectral characteristic are detailed. The results provide users of manual baseline determination useful guidelines in establishing limits of accuracy and precision when performing manual baseline determination, as well as highlighting conditions that confound the accuracy and precision of manual baseline determination.

Revealing system dynamics through decomposition of the perturbation domain in two-dimensional correlation spectroscopy.

A technique is presented to simply and effectively decompose the perturbation domain in two-dimensional (2D) correlation maps calculated on a given set of vibrational spectra. Decomposition of the perturbation domain exposes a wealth of kinetic information complementary to the information extracted from conventional 2D correlation spectroscopy. It is shown that the technique produces "perturbation profile maps" that can be utilized in both the interpretation of the conventional 2D correlation maps and the independent kinetic analysis of the given system. Discrimination between spectral features exhibiting similar, but not identical, dynamics is facilitated by the decomposition, and spectral features exhibiting identical dynamics over the perturbation interval are quickly identified. Spectral features exhibiting similar dynamics over only a sub-range of the full perturbation are also identifiable. Interpretation of phase information illuminated in synchronous and asynchronous maps is simplified. Comparison between similar spectral features present in different samples is facilitated with the technique. The simplicity and ease of implementation of the technique make decomposition of the perturbation domain a valuable addition to the tools available in 2D correlation analysis.

Identification and interpretation of generalized two-dimensional correlation spectroscopy features through decomposition of the perturbation domain.

Generalized two-dimensional correlation spectroscopy offers great scope for revealing the behavior of relationships between components of a system under empirical study. We have developed methods that aid in the interpretation of two-dimensional correlation spectroscopy. These methods include reference patterns for two-dimensional correlation and correlation coefficient maps, their superposition and joint interpretation, and the use of delta functions to decompose them in the perturbation domain. We show how their joint use permits discrimination between similar two-dimensional correlation map features on the basis of different correlation coefficients. We also show how the decomposition of maps into the perturbation domain reflects the dynamic behavior of spectral features over the course of the perturbation and permits discrimination between otherwise highly similar two-dimensional correlation cross-peaks. These approaches simplify the interpretation of two-dimensional correlation spectroscopy maps and facilitate access to their rich information content.

Direct determination of polycyclic aromatic hydrocarbons in solid matrices using laser desorption/laser photoionization ion trap mass spectrometry.

The development and characterization of a new instrument for solid sampling which couples IR laser desorption followed by UV laser photo-ionization and analysis using an ion trap mass spectrometer has been investigated. For calibration, a new type of solid sample preparation involving activated charcoal as the solid substrate was used. This solid sample provided a steady signal for several thousand laser shots, which allowed optimization of the experimental procedure. It was found that both the IR and UV intensity and the delay between them play an important role in both the magnitude and type of signals observed. A method of gas phase accumulation with multiple laser shots was examined. Finally, this technique was demonstrated to be effective in providing direct qualitative information for N.I.S.T. SRM 1944 river sediment sample with no sample pre-treatment.

[Role of cell migration in the pathogenesis of rheumatoid arthritis: in vivo studies in SCID mice transplanted with human synovial membrane]. / Ruolo della migrazione cellulare nella patogenesi dell'artrite reumatoide: studi in vivo in topi SCID trapiantati con membrana sinoviale umana.

OBJECTIVE: Adhesion mechanisms play a central role in the recruitment of leukocytes which characteristically infiltrate rheumatoid synovium. Therefore, we adapted an animal model, in which human rheumatoid synovium was transplanted into severe combined immunodeficient (SCID) mice, to study the effects of Tumor Necrosis Factor-alpha (TNF-alpha) in modulatine leukocyte migration and to investigate the chemotactic potential of Stromal Derived Factor-1 alpha (SDF-1 alpha). MATERIALS AND METHODS: Human synovium samples, obtained from patients undergoing joint replacement, were divided into two parts. One was analysed by immunohistology and the other was implanted subcutaneously into SCID mice under general anaesthesia. Four weeks post-transplantation, grafts were injected with optimal dose of SDF-1, TNF-alpha or saline (negative control). At the same time, animals were injected iv with fluorescently labelled cells. 48 hours later mice were sacrificed and grafts removed for cryo-hystology. The number of cells migrating to the grafts was determined by UV-microscopy and the results expressed as cells per high power field. RESULTS AND CONCLUSIONS: In these studies we provide the evidence that: 1) the animal model, in which human tissues are grafted into SCID mice, can be used to study cell migration under controlled experimental conditions; 2) direct intragraft injection of TNF-alpha increases lymphocytes migration and up-regulates the expression of human adhesion molecules (CAMs) and 3) SDF-1 alphainjected intragraft increases the migration of the pro-myelo-monocytic U937 cells to synovial transplants, even more efficiently than TNF-alpha, but without modifications of CAMs' expression.

Stromal cell-derived factor 1 (CXCL12) induces monocyte migration into human synovium transplanted onto SCID Mice.

OBJECTIVE: The mechanisms by which monocyte/macrophage cells migrate to the joint involve a series of integrated adhesion and signaling events in which chemokines and their receptors are strongly implicated. This study was undertaken to investigate the hypothesis that stromal cell-derived factor 1 (SDF-1), a CXC chemokine (CXCL12), plays a critical role in monocyte/macrophage localization to synovium. METHODS: SDF-1 and CXC receptor 4 (CXCR4) expression in rheumatoid arthritis (RA) and osteoarthritis synovium and graft SDF-1, tumor necrosis factor alpha (TNF alpha), and human and murine vascular markers were examined by immunohistochemistry and double-immunofluorescence. The functional capacity of SDF-1 to modulate monocyte migration into joints was investigated by examining the localization of pro-myelomonocytic U937 cells into synovial tissue transplanted into SCID mice. SDF-1, TNF alpha, or saline was injected into graft sites and response determined by the number of fluorescently labeled U937 cells (injected intravenously) detected in grafts by ultraviolet microscopy. RESULTS: SDF-1 and CXCR4 were highly expressed in CD68+ cells in the RA synovium. SDF-1 induced U937 cell migration in vitro and in vivo in a dose-dependent manner and, in vivo, SDF-1 was more effective than TNF alpha. In contrast to TNF alpha, SDF-1 did not induce intracellular adhesion molecule 1 in transplant microvasculature. Furthermore, intragraft injection of SDF-1 did not up-regulate TNF alpha, or vice versa. CONCLUSION: This study demonstrates, for the first time, that SDF-1 is functional in vivo when injected into synovial grafts. In addition, SDF-1 is more potent than TNF alpha, and its mechanisms of action appear to be autonomous. Therefore, SDF-1 may be an important TNF-independent molecule involved in the migration to and retention of inflammatory effector cells in the joint.

Tumour necrosis factor-alpha (TNF-alpha) enhances lymphocyte migration into rheumatoid synovial tissue transplanted into severe combined immunodeficient (SCID) mice.

Adhesion mechanisms play a major role in the recruitment of peripheral blood lymphocytes (PBL) which characteristically infiltrate rheumatoid arthritis (RA) synovium and other chronically inflamed tissues. Through a sequential series of complex integrated adhesion and signalling events, 'multistep model of migration', specific subsets of PBL are recruited into inflamed tissues. In this process both leucocyte receptors and microvascular endothelial (MVE) counter-receptors play a critical role. The MVE in particular, during an inflammatory state, is the target of various inflammatory mediators that cause the up-regulation of several cell adhesion molecules (CAM). One of the most important factors known to be a powerful inducer of MVE CAM is TNF-alpha. Conversely, blocking TNF-alpha causes a down-modulation of CAM expression. To test directly the capacity of TNF-alpha to induce cell migration into RA synovium we adapted a model in which synovial grafts were implanted into SCID mice subcutaneously. Using this model we demonstrate that: (i) transplants remain viable and become vascularized and fed by mouse subdermal vessels; (ii) the mouse vasculature connects to the transplant vasculature which maintains the ability to express human CAM; (iii) intragraft injections of TNF-alpha up-regulate the expression of human CAM, following the down-regulation which occurred 4 weeks post-transplantation; and (iv) the up-regulation of graft CAM is associated with increased human PBL migration into the transplants. This study provides direct evidence in vivo of the capacity of TNF-alpha to induce cell migration. In addition, it provides the experimental background for the optimal use of this model.

The effect of a nonverbal aid on preschoolers' recall for color.

The purpose of the present study was to investigate whether the provision of a nonverbal memory aid would improve preschoolers' recall of color. Forty 4-year-old children carried out 2 tasks with the same set of colored objects. Colors were not referred to, nor were children told that their recall would later be tested. One day later, the children were split into 2 groups. One group was given a chart containing both the colors of the objects and distractor colors. The other group was not given a chart. Recall for object color was tested. There was an effect of chart provision; children who used the chart recalled more colors correctly than did those who did not use a chart. This result indicates (a) that even very young children can make use of props to facilitate their recall and (b) that such memory aids need not be exact copies of previously seen objects. Implications of these findings for eyewitness recall are discussed.

Key residues approach to the definition of protein families and analysis of sparse family signatures.

We extend the concept of the motif as a tool for characterizing protein families and explore the feasibility of a sparse "motif" that is the length of the protein sequence itself. The type of motif discussed is a sparse family signature consisting of a set of N key residue positions (A1, A2...AN) preceded by gaps (G) thus G1A1G2A2. ...GNAN. Both a residue and gap can be variable. A signature is matched to a protein sequence and scored using a dynamic programming algorithm which permits variability in gap distance and residue type. Generating a signature involves identifying residues associated with points of contact in interactions between secondary structure elements. A raw signature consists of a set of positions with potential key structural roles sampled from a sequence alignment constructed with reference to this contact data. Raw signatures are refined by sampling different gap-residue pairs until the specificity of a signature for the family cannot be further improved. We summarize signatures for nine families of protein of diverse fold and function and present results of scans against the OWL protein sequence database. The implications of such signatures are discussed.

An interface with a linear quadrupole ion guide for an electrospray-ion trap mass spectrometer system.

A new ion sampling interface for an electrospray ionization 3D ion trap mass spectrometer system is described. The interface uses linear rf quadrupoles as ion guides and ion traps to enhance the performance of the 3D trap. Trapping ions in the linear quadrupoles is demonstrated to improve the duty cycle of the system. Dipolar excitation of ions trapped in a linear quadrupole is used to eject unwanted ions. A resolution of ejection of up to 254 is demonstrated for protonated reserpine ions (m/z 609.3). A composite waveform with a notch in frequency space is used to eject a wide range of matrix ions and to isolate trace analyte ions in a linear quadrupole before ions are injected into the 3D trap. This is useful to overcome space charge problems in the 3D trap caused by excess matrix ions. For trace reserpine in a 500-fold molar excess of poly(propylene glycol) (PPG), it is demonstrated that the resolution and sensitivity of the 3D trap can be increased dramatically with ejection of the excess PPG matrix ions. In comparison to ejection of matrix ions in the 3D trap with a similar broad-band waveform, a 5-fold increase in sensitivity with a 7 times shorter acquisition time was achieved.

Measurement of some small-molecule and peptide neurotransmitters in-vitro using a fiber-optic probe with pulsed ultraviolet resonance Raman spectroscopy.

Many techniques have been developed to investigate the chemistry associated with brain activity. These techniques generally fall into two categories: fast techniques with species-limited sensitivity; and generally slower techniques with broader species sensitivity. Therefore, a need exists for a fast, minimally invasive technique that is sensitive to a wide array of biologically relevant compounds in order to measure chemical brain events in real time. The work presented here describes the development of a novel spectroscopic neurotransmitter probe for the rapid and simultaneous detection of a variety of neurotransmitters. A fiber-optic-linked Raman and tunable ultraviolet resonance Raman system was assembled with custom designed optical fiber probes. Using this system, the ultraviolet resonance Raman spectra of some small-molecule and peptide neurotransmitters were measured in-vitro with a fiber-optic probe and are reported here for the first time. The probe has furthermore been used to measure neurotransmitter secretions obtained from depolarized rat pheochromocytoma (PC12) cells. These results demonstrate the general utility of this approach which, due to the fiber-optic implementation, could potentially also be applied to in-vivo neurotransmitter determinations.