Contributions to our understanding of T cell physiology through unveiling the T cell proteome

Since the sequencing of the human genome was completed, attention has turned to examining the functionality of the molecular machinery, in particular of protein expression. Differential proteome analysis by two-dimensional electrophoresis has been adopted to study changes in T cell proteomes during T cell activation, and this work is increasing our understanding of the complexity of signals elicited across multiple pathways. The purpose of this review is to summarize the available evidence in the application of proteomic techniques and methodologies to understand T cell receptor activation from lipid raft and cytoskeletal rearrangements, through to signalling cascades, transcription factor modulation and changes in protein expression patterns. These include post-translational modifications, which are not encoded by the genome. © 2007 British Society for Immunology.

The development of methods of steroid analysis in reproductive physiology

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Parental diet, pregnancy outcomes and offspring health:metabolic determinants in developing oocytes and embryos

The periconceptional period, embracing the terminal stages of oocyte growth and post-fertilisation development up to implantation, is sensitive to parental nutrition. Deficiencies or excesses in a range of macro- and micronutrients during this period can lead to impairments in fertility, fetal development and long-term offspring health. Obesity and genotype-related differences in regional adiposity are associated with impaired liver function and insulin resistance, and contribute to fatty acid-mediated impairments in sperm viability and oocyte and embryo quality, all of which are associated with endoplasmic reticulum stress and compromised fertility. Disturbances to maternal protein metabolism can elevate ammonium concentrations in reproductive tissues and disturb embryo and fetal development. Associated with this are disturbances to one-carbon metabolism, which can lead to epigenetic modifications to DNA and associated proteins in offspring that are both insulin resistant and hypertensive. Many enzymes involved in epigenetic gene regulation use metabolic cosubstrates (e.g. acetyl CoA and S-adenosyl methionine) to modify DNA and associated proteins, and so act as 'metabolic sensors' providing a link between parental nutritional status and gene regulation. Separate to their genomic contribution, spermatozoa can also influence embryo development via direct interactions with the egg and by seminal plasma components that act on oviductal and uterine tissues. © IETS 2014.

Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring

Early embryonic development is known to be susceptible to maternal undernutrition, leading to a disease-related postnatal phenotype. To determine whether this sensitivity extended into oocyte development, we examined the effect of maternal normal protein diet (18% casein; NPD) or isocaloric low protein diet (9% casein; LPD) restricted to one ovulatory cycle (3.5 days) prior to natural mating in female MF-1 mice. After mating, all females received NPD for the remainder of gestation and all offspring were litter size adjusted and fed standard chow. No difference in gestation length, litter size, sex ratio or postnatal growth was observed between treatments. Maternal LPD did, however, induce abnormal anxiety-related behaviour in open field activities in male and female offspring (P <0.05). Maternal LPD offspring also exhibited elevated systolic blood pressure (SBP) in males at 9 and 15 weeks and in both sexes at 21 weeks (P <0.05). Male LPD offspring hypertension was accompanied by attenuated arterial responsiveness in vitro to vasodilators acetylcholine and isoprenaline (P <0.05). LPD female offspring adult kidneys were also smaller, but had increased nephron numbers (P <0.05). Moreover, the relationship between SBP and kidney or heart size or nephron number was altered by diet treatment (P <0.05). These data demonstrate the sensitivity of mouse maturing oocytes in vivo to maternal protein undernutrition and identify both behavioural and cardiovascular postnatal outcomes, indicative of adult disease. These outcomes probably derive from a direct effect of protein restriction, although indirect stress mechanisms may also be contributory. Similar and distinct postnatal outcomes were observed here compared with maternal LPD treatment during post-fertilization preimplantation development which may reflect the relative contribution of the paternal genome. © Journal compilation © 2008 The Physiological Society.

Changes in ocular physiology, tear film characteristics, and symptomatology with 18 months silicone hydrogel contact lens wear

Purpose. The purpose of this study was to evaluate the longitudinal changes in ocular physiology, tear film characteristics, and symptomatology experienced by neophyte silicone hydrogel (SiH) contact lens wearers in a daily-wear compared with a continuous-wear modality and with the different commercially available lenses over an 18-month period. Methods. Forty-five neophyte subjects were enrolled in the study and randomly assigned to wear one of two SiH materials: lotrafilcon A or balafilcon A lenses on either a daily- (LDW; BDW) or continuous-wear (LCW; BCW) basis. Additionally, a group of noncontact lens-wearing subjects (control group) was also recruited and followed over the same study period. Objective and subjective grading of ocular physiology were carried out together with tear meniscus height (TMH) and noninvasive tear breakup time (NITBUT). Subjects also subjectively rated symptoms and judgments with lens wear. After initial screening, subsequent measurements were taken after 1, 3, 6, 12, and 18 months. Results. Subjective and objective grading of ocular physiology revealed a small increase in bulbar, limbal, and palpebral hyperemia as well as corneal staining over time with both lens materials and regimes of wear (p < 0.05). No significant changes in NITBUT or TMH were found (p > 0.05). Subjective symptoms and judgment were not material- or modality-specific. Conclusions. Daily and continuous wear of SiH contact lenses induced small but statistically significant changes in ocular physiology and symptomatology. Clinical measures of tear film characteristics were unaffected by lens wear. Both materials and regimes of wear showed similar clinical performance. Long-term SiH contact lens wear is shown to be a successful option for patients. Copyright © 2006 American Academy of Optometry.

Clinical monitoring of ocular physiology using digital image analysis

Aim: To examine the use of image analysis to quantify changes in ocular physiology. Method: A purpose designed computer program was written to objectively quantify bulbar hyperaemia, tarsal redness, corneal staining and tarsal staining. Thresholding, colour extraction and edge detection paradigms were investigated. The repeatability (stability) of each technique to changes in image luminance was assessed. A clinical pictorial grading scale was analysed to examine the repeatability and validity of the chosen image analysis technique. Results: Edge detection using a 3 × 3 kernel was found to be the most stable to changes in image luminance (2.6% over a +60 to -90% luminance range) and correlated well with the CCLRU scale images of bulbar hyperaemia (r = 0.96), corneal staining (r = 0.85) and the staining of palpebral roughness (r = 0.96). Extraction of the red colour plane demonstrated the best correlation-sensitivity combination for palpebral hyperaemia (r = 0.96). Repeatability variability was <0.5%. Conclusions: Digital imaging, in conjunction with computerised image analysis, allows objective, clinically valid and repeatable quantification of ocular features. It offers the possibility of improved diagnosis and monitoring of changes in ocular physiology in clinical practice. © 2003 British Contact Lens Association. Published by Elsevier Science Ltd. All rights reserved.

Oxidiative lipidomics coming of age:advances in analysis of oxidized phospholipids in physiology and pathology

Significance: Oxidized phospholipids are now well-recognized as markers of biological oxidative stress and bioactive molecules with both pro-inflammatory and anti-inflammatory effects. While analytical methods continue to be developed for studies of generic lipid oxidation, mass spectrometry (MS) has underpinned the advances in knowledge of specific oxidized phospholipids by allowing their identification and characterization, and is responsible for the expansion of oxidative lipidomics. Recent Advances: Studies of oxidized phospholipids in biological samples, both from animal models and clinical samples, have been facilitated by the recent improvements in MS, especially targeted routines that depend on the fragmentation pattern of the parent molecular ion and improved resolution and mass accuracy. MS can be used to identify selectively individual compounds or groups of compounds with common features, which greatly improves the sensitivity and specificity of detection. Application of these methods have enabled important advances in understanding the mechanisms of inflammatory diseases such as atherosclerosis, steatohepatitis, leprosy and cystic fibrosis, and offer potential for developing biomarkers of molecular aspects of the diseases. Critical Issues and Future Directions: The future in this field will depend on development of improved MS technologies, such as ion mobility, novel enrichment methods and databases and software for data analysis, owing to the very large amount of data generated in these experiments. Imaging of oxidized phospholipids in tissue MS is an additional exciting direction emerging that can be expected to advance understanding of physiology and disease.

The influence of end of day silicone hydrogel daily disposable contact lens fit on ocular comfort, physiology and lens wettability

Purpose: To quantify the end-of-day silicone-hydrogel daily disposable contact lens fit and its influence of on ocular comfort, physiology and lens wettability. Methods: Thirty-nine subjects (22.1. ±. 3.5 years) were randomised to wear each of 3 silicone-hydrogel daily-disposable contact lenses (narafilcon A, delefilcon A and filcon II 3), bilaterally, for one week. Lens fit was assessed objectively using a digital video slit-lamp at 8, 12 and 16. h after lens insertion. Hyperaemia, non-invasive tear break-up time, tear meniscus height and comfort were also evaluated at these timepoints, while corneal and conjunctival staining were assessed on lens removal. Results: Lens fit assessments were not different between brands (P > 0.05), with the exception of the movement at blink where narafilcon A was more mobile. Overall, lag reduced but push-up speed increased from 8 to 12. h (P <. 0.05), but remained stable from 12 to 16. h (P > 0.05). Movement-on-blink was unaffected by wear-time (F = 0.403, P = 0.670). A more mobile lens fit with one brand did not indicate that person would have a more mobile fit with another brand (r = -0.06 to 0.63). Lens fit was not correlated with comfort, ocular physiology or lens wettability (P > 0.01). Conclusions: Among the lenses tested, objective lens fit changed between 8. h and 12. h of lens wear. The weak correlation in individual lens fit between brands indicates that fit is dependent on more than ocular shape. Consequently, substitution of a different lens brand with similar parameters will not necessarily provide comparable lens fit.