A re-examination of the Ghrelin and Ghrelin receptor genes

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

Establishment of a mouse model of colitis and its use to evaluate the anti-inflammatory effects of two ghrelin peptides

Ghrelin is a gut-brain peptide hormone that induces appetite, stimulates the release of growth hormone, and has recently been shown to ameliorate inflammation. Recent studies have suggested that ghrelin may play a potential role in inflammation-related diseases such as inflammatory bowel diseases (IBD). A previous study with ghrelin in the TNBS mouse model of colitis demonstrated that ghrelin treatment decreased the clinical severity of colitis and inflammation and prevented the recurrence of disease. Ghrelin may be acting at the immunological and epithelial level as the ghrelin receptor (GHSR) is expressed by immune cells and intestinal epithelial cells. The current project investigated the effect of ghrelin in a different mouse model of colitis using dextran sodium sulphate (DSS) – a luminal toxin. Two molecular weight forms of DSS were used as they give differing effects (5kDa and 40kDa). Ghrelin treatment significantly improved clinical colitis scores (p=0.012) in the C57BL/6 mouse strain with colitis induced by 2% DSS (5kDa). Treatment with ghrelin suppressed colitis in the proximal colon as indicated by reduced accumulative histopathology scores (p=0.03). Whilst there was a trend toward reduced scores in the mid and distal colon in these mice this did not reach significance. Ghrelin did not affect histopathology scores in the 40kDa model. There was no significant effect on the number of regulatory T cells or TNF-α secretion from cultured lymph node cells from these mice. The discovery of C-terminal ghrelin peptides, for example, obestatin and the peptide derived from exon 4 deleted proghrelin (Δ4 preproghrelin peptide) have raised questions regarding their potential role in biological functions. The current project investigated the effect of Δ4 peptide in the DSS model of colitis however no significant suppression of colitis was observed. In vitro epithelial wound healing assays were also undertaken to determine the effect of ghrelin on intestinal epithelial cell migration. Ghrelin did not significantly improve wound healing in these assays. In conclusion, ghrelin treatment displays a mild anti-inflammatory effect in the 5kDa DSS model. The potential mechanisms behind this effect and the disparity between these results and those published previously will be discussed.

The relationship between subjective appetite sensations, markers of inflammation and appetite in dialysis patients

Background: Poor appetite is a marker of morbidity and mortality in hemodialysis patients, making it an important area for research. Visual analog scales (VAS) can capture a range of subjective sensations related to appetite (such as hunger, desire to eat or fullness), but have not been commonly used to measure appetite in dialysis patients. The aim of this study was to explore the association between retrospective ratings of appetite using VAS and a range of clinical variables as well as biomarkers of appetite in hemodialysis patients.----- Methods: 28 hemodialysis patients (mean age 61±17y, 50% male, median dialysis vintage 19.5(4-101) months) rated their appetite using VAS for hunger, fullness and desire to eat and a 5-point categorical scale measuring general appetite. Blood levels of the appetite peptides leptin, ghrelin and peptide YY were also measured.----- Results: Hunger ratings measured by VAS were significantly (p<0.05) correlated with a range of clinical, nutritional and inflammatory markers: age (r=-0.376), co-morbidities, (r=-0.380) PG-SGA score (r=-0.451), weight (r=-0.375), fat-free mass (r=-0.435), C-Reactive Protein (CRP) (r=-0.383) and Intercellular adhesion molecule (sICAM-1) (r=-0.387). There was a consistent relationship between VAS and appetite on a 5-point categorical scale for questions of hunger, and a similar trend for desire to eat, but not for fullness. Neither method of measuring subjective appetite correlated with appetite peptides.----- Conclusions: Retrospective ratings of hunger on a VAS are associated with a range of clinical variables and further studies are warranted to support their use as a method of measuring appetite in dialysis patients.

New insights into appetite, inflammation and the use of fish oil in hemodialysis patients

Protein-energy wasting (PEW) is commonly seen in patients with chronic kidney disease (CKD). The condition is characterised by chronic, systemic low-grade inflammation which affects nutritional status by a variety of mechanisms including reducing appetite and food intake and increasing muscle catabolism. PEW is linked with co-morbidities such as cardiovascular disease, and is associated with lower quality of life, increased hospitalisations and a 6-fold increase in risk of death1. Significant gender differences have been found in the severity and effects of several markers of PEW. There have been limited studies testing the ability of anti-inflammatory agents or nutritional interventions to reduce the effects of PEW in dialysis patients. This thesis makes a significant contribution to the understanding of PEW in dialysis patients. It advances understanding of measurement techniques for two of the key components, appetite and inflammation, and explores the effect of fish oil, an anti-inflammatory agent, on markers of PEW in dialysis patients. The first part of the thesis consists of two methodological studies conducted using baseline data. The first study aims to validate retrospective ratings of hunger, desire to eat and fullness on visual analog scales (VAS) (paper and pen and electronic) as a new method of measuring appetite in dialysis patients. The second methodological study aims to assess the ability of a variety of methods available in routine practice to detect the presence of inflammation. The second part of the thesis aims to explore the effect of 12 weeks supplementation with 2g per day of Eicosapentaenoic Acid (EPA), a longchain fatty acid found in fish oil, on markers of PEW. A combination of biomarkers and psychomarkers of appetite and inflammation are the main outcomes being explored, with nutritional status, dietary intake and quality of life included as secondary outcomes. A lead in phase of 3 months prior to baseline was used so that each person acts as their own historical control. The study also examines whether there are gender differences in response to the treatment. Being an exploratory study, an important part of the work is to test the feasibility of the intervention, thus the level of adherence and factors associated with adherence are also presented. The studies were conducted at the hemodialysis unit of the Wesley Hospital. Participants met the following criteria: adult, stage 5 CKD on hemodialysis for at least 3 months, not expected to receive a transplant or switch to another dialysis modality during the study, absence of intellectual impairment or mental illness impairing ability to follow instructions or complete the intervention. A range of intermediate, clinical and patient-centred outcome measures were collected at baseline and 12 weeks. Inflammation was measured using five biomarkers: c-reactive protein (CRP), interleukin-6 (IL6), intercellular adhesion molecule (sICAM-1), vascular cell adhesion molecule (sVCAM-1) and white cell count (WCC). Subjective appetite was measured using the first question from the Appetite and Dietary Assessment (ADAT) tool and VAS for measurements of hunger, desire to eat and fullness. A novel feature of the study was the assessment of the appetite peptides leptin, ghrelin and peptide YY as biomarkers of appetite. Nutritional status/inflammation was assessed using the Malnutrition-Inflammation Score (MIS) and the Patient-Generated Subjective Global Assessment (PG-SGA). Dietary intake was measured using 3-day records. Quality of life was measured using the Kidney Disease Quality of Life Short Form version 1.3 (KDQOL-SF™ v1.3 © RAND University), which combines the Short-Form 36 (SF36) with a kidney-disease specific module2. A smaller range of these variables was available for analysis during the control phase (CRP, ADAT, dietary intake and nutritional status). Statistical analysis was carried out using SPSS version 14 (SPSS Inc, Chicago IL, USA). Analysis of the first part of the thesis involved descriptive and bivariate statistics, as well as Bland-Altman plots to assess agreement between methods, and sensitivity analysis/ROC curves to test the ability of methods to predict the presence of inflammation. The unadjusted (paired ttests) and adjusted (linear mixed model) change over time is presented for the main outcome variables of inflammation and appetite. Results are shown for the whole group followed by analyses according to gender and adherence to treatment. Due to the exploratory nature of the study, trends and clinical significance were considered as important as statistical significance. Twenty-eight patients (mean age 61±17y, 50% male, dialysis vintage 19.5 (4- 101) months) underwent baseline assessment. Seven out of 28 patients (25%) reported sub-optimal appetite (self-reported as fair, poor or very poor) despite all being well nourished (100% SGA A). Using the VAS, ratings of hunger, but not desire to eat or fullness, were significantly (p<0.05) associated with a range of relevant clinical variables including age (r=-0.376), comorbidities (r=-0.380) nutritional status (PG-SGA score, r=-0.451), inflammatory markers (CRP r=-0.383; sICAM-1 r=-0.387) and seven domains of quality of life. Patients expressed a preference for the paper and pen method of administering VAS. None of the tools (appetite, MIS, PG-SGA, albumin or iron) showed an acceptable ability to detect patients who are inflamed. It is recommended that CRP should be tested more frequently as a matter of course rather than seeking alternative methods of measuring inflammation. 27 patients completed the 12 week intervention. 20 patients were considered adherent based on changes in % plasma EPA, which rose from 1.3 (0.94)% to 5.2 (1.1)%, p<0.001, in this group. The major barriers to adherence were forgetting to take the tablets as well as their size. At 12 weeks, inflammatory markers remained steady apart from the white cell count which decreased (7.6(2.5) vs 7.0(2.2) x109/L, p=0.058) and sVCAM-1 which increased (1685(654) vs 2249(925) ng/mL, p=0.001). Subjective appetite using VAS increased (51mm to 57mm, +12%) and there was a trend towards reduction in peptide YY (660(31) vs 600(30) pg/mL, p=0.078). There were some gender differences apparent, with the following adjusted change between baseline and week 12: CRP (males -3% vs females +17%, p=0.19), IL6 (males +17% vs females +48%, p=0.77), sICAM-1 (males -5% vs females +11%, p=0.07), sVCAM-1 (males +54% vs females +19%, p=0.08) and hunger ratings (males 20% vs females -5%, p=0.18). On balance, males experienced a maintainence or reduction in three inflammatory markers and an improvement in hunger ratings, and therefore appeared to have responded better to the intervention. Compared to those who didn’t adhere, adherent patients maintained weight (mean(SE) change: +0.5(1.6) vs - 0.8(1.2) kg, p=0.052) and fat-free mass (-0.1 (1.6) vs -1.8 (1.8) kg, p=0.045). There was no difference in change between the intervention and control phase for CRP, appetite, nutritional status or dietary intake. The thesis makes a significant contribution to the evidence base for understanding of PEW in dialysis patients. It has advanced knowledge of methods of assessing inflammation and appetite. Retrospective ratings of hunger on a VAS appear to be a valid method of assessing appetite although samples which include patients with very poor appetite are required to confirm this. Supplementation with fish oil appeared to improve subjective appetite and dampen the inflammatory response. The effectiveness of the intervention is influenced by gender and adherence. Males appear to be more responsive to the primary outcome variables than females, and the quality of response is improved with better adherence. These results provide evidence to support future interventions aimed at reducing the effects of PEW in dialysis patients.

Coating of biomaterial scaffolds with the collagen-mimetic peptide GFOGER for bone defect repair

Healing large bone defects and non-unions remains a significant clinical problem. Current treatments, consisting of auto and allografts, are limited by donor supply and morbidity, insufficient bioactivity and risk of infection. Biotherapeutics, including cells, genes and proteins, represent promising alternative therapies, but these strategies are limited by technical roadblocks to biotherapeutic delivery, cell sourcing, high cost, and regulatory hurdles. In the present study, the collagen-mimetic peptide, GFOGER, was used to coat synthetic PCL scaffolds to promote bone formation in critically-sized segmental defects in rats. GFOGER is a synthetic triple helical peptide that binds to the [alpha]2[beta]1 integrin receptor involved in osteogenesis. GFOGER coatings passively adsorbed onto polymeric scaffolds, in the absence of exogenous cells or growth factors, significantly accelerated and increased bone formation in non-healing femoral defects compared to uncoated scaffolds and empty defects. Despite differences in bone volume, no differences in torsional strength were detected after 12 weeks, indicating that bone mass but not bone quality was improved in this model. This work demonstrates a simple, cell/growth factor-free strategy to promote bone formation in challenging, non-healing bone defects. This biomaterial coating strategy represents a cost-effective and facile approach, translatable into a robust clinical therapy for musculoskeletal applications.

The effects of exercise-induced weight loss on appetite-related peptides and motivation to eat

Context: The magnitude of exercise-induced weight loss depends on the extent of compensatory responses. An increase in energy intake is likely to result from changes in the appetite control system toward an orexigenic environment; however, few studies have measured how exercise impacts on both orexigenic and anorexigenic peptides. ---------- Objective: The aim of the study was to investigate the effects of medium-term exercise on fasting/postprandial levels of appetite-related hormones and subjective appetite sensations in overweight/obese individuals. ---------- Design and Setting: We conducted a longitudinal study in a university research center. ---------- Participants and Intervention: Twenty-two sedentary overweight/obese individuals (age, 36.9 ± 8.3 yr; body mass index, 31.3 ± 3.3 kg/m2) took part in a 12-wk supervised exercise programme (five times per week, 75% maximal heart rate) and were requested not to change their food intake during the study. ---------- Main Outcome Measures: We measured changes in body weight and fasting/postprandial plasma levels of glucose, insulin, total ghrelin, acylated ghrelin (AG), peptide YY, and glucagon-like peptide-1 and feelings of appetite. ---------- Results: Exercise resulted in a significant reduction in body weight and fasting insulin and an increase in AG plasma levels and fasting hunger sensations. A significant reduction in postprandial insulin plasma levels and a tendency toward an increase in the delayed release of glucagon-like peptide-1 (90–180 min) were also observed after exercise, as well as a significant increase (127%) in the suppression of AG postprandially. ---------- Conclusions: Exercise-induced weight loss is associated with physiological and biopsychological changes toward an increased drive to eat in the fasting state. However, this seems to be balanced by an improved satiety response to a meal and improved sensitivity of the appetite control system.

Gender differences in the effect of fish oil on appetite, inflammation and nutritional status in haemodialysis patients

Background: Haemodialysis patients show signs of chronic inflammation and reduced appetite, which is associated with a worse clinical status and an increased mortality risk. Fish oil has anti-inflammatory properties and may be useful as a therapeutic treatment. There is limited evidence to indicate the feasibility and efficacy of this intervention in dialysis patients. The present study aimed to compare the effect of 12 weeks of supplementation with fish oil on markers of appetite and inflammation in male and female haemodialysis patients. Methods: The study was conducted in 28 haemodialysis patients. All patients were prescribed 3 g of fish oil per day for 12 weeks. Changes in appetite, plasma fatty acid profiles and inflammatory markers were measured at baseline and at 12 weeks. Results: The mean (SD) increase in percent plasma eicosapentaenoic acid was statistically significant [1.1 (0.8) to 4.1 (2.2), P < 0.001], which was a strong indicator of good adherence. There were trends towards reductions in peptide YY (−9%; P = 0.078) and an increase in subjective sensations of hunger (+12%; P = 0.406), which reflects an increase in motivation to eat. Males (n = 13) experienced a more marked increase in hunger compared to females (+23% versus −6%), which was associated with maintenance in C-reactive protein and interleukin-6, and a reduction in soluble intercellular adhesion molecule-1. Conclusions: The results obtained demonstrate meaningful trends towards improvements in subjective appetite and certain inflammatory markers (although no change in dietary intake) and this effect was more pronounced in males. However, the levels of some inflammatory markers increased in females and this requires further study. The high level of adherence achieved indicates that an intervention requiring patients to consume four fish oil capsules per day is achievable. This was a short-term study and the effects need to be confirmed in a randomised controlled trial.

Prandial subcutaneous injections of glucagon-like peptide-1 cause weight loss in obese human subjects

Recombinant glucagon-like peptide-1 (7–36)amide (rGLP-1) was recently shown to cause significant weight loss in type 2 diabetics when administered for 6 weeks as a continuous subcutaneous infusion. The mechanisms responsible for the weight loss are not clarified. In the present study, rGLP-1 was given for 5d by prandial subcutaneous injections (PSI) (76nmol 30min before meals, four times daily; a total of 302·4nmol/24h) or by continuous subcutaneous infusion (CSI) (12·7nmol/h; a total of 304·8nmol/24h). This was performed in nineteen healthy obese subjects (mean age 44·2 (sem 2·5) years; BMI 39·0 (sem 1·2)kg/m2) in a prospective randomised, double-blind, placebo-controlled, cross-over study. Compared with the placebo, rGLP-1 administered as PSI and by CSI generated a 15% reduction in mean food intake per meal (P=0·02) after 5d treatment. A weight loss of 0·55 (sem 0·2) kg (P<0·05) was registered after 5d with PSI of rGLP-1. Gastric emptying rate was reduced during both PSI (P<0·001) and CSI (P<0·05) treatment, but more rapidly and to a greater extent with PSI of rGLP-1. To conclude, a 5d treatment of rGLP-1 at high doses by PSI, but not CSI, promptly slowed gastric emptying as a probable mechanism of action of increased satiety, decreased hunger and, hence, reduced food intake with an ensuing weight loss.

Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates.

Vibrational spectroscopic studies of wool

Fourier transfonn (FT) Raman, Raman microspectroscopy and Fourier transform infrared (FTIR) spectroscopy have been used for the structural analysis and characterisation of untreated and chemically treated wool fibres. For FT -Raman spectroscopy novel methods of sample presentation have been developed and optimised for the analysis of wool. No significant fluorescence was observed and the spectra could be obtained routinely. The stability of wool keratin to the laser source was investigated and the visual and spectroscopic signs of sample damage were established. Wool keratin was found to be extremely robust with no signs of sample degradation observed for laser powers of up to 600 m W and for exposure times of up to seven and half hours. Due to improvements in band resolution and signal-to-noise ratio, several previously unobserved spectral features have become apparent. The assignment of the Raman active vibrational modes of wool have been reviewed and updated to include these features. The infrared spectroscopic techniques of attenuated total reflectance (ATR) and photoacoustic (P A) have been used to examine shrinkproofed and mothproofed wool samples. Shrinkproofing is an oxidative chemical treatment used to selectively modifY the surface of a wool fibre. Mothproofing is a chemical treatment applied to wool for the prevention of insect attack. The ability of PAS and A TR to vary the penetration depth by varying certain instrumental parameters was used to obtain spectra of the near surface regions of these chemically treated samples. These spectra were compared with those taken with a greater penetration depth, which therefore represent more of the bulk wool sample. The PA and ATR spectra demonstrated that oxidation was restricted to the near-surface layer of wool. Extensive curve fitting of ATR spectra of untreated wool indicated that cuticle was composed of a mixed protein conformation, but was predominately that of an a.-helix. The cortex was proposed to be a mixture of both a.helical and ~-pleated sheet protein conformations. These findings were supported by PAS depth profiling results. Raman microspectroscopy was used in an extensive investigation of the molecular structure of the wool fibre. This included determining the orientation of certain functional groups within the wool fibre and the symmetry of particular vibrations. The orientation ofbonds within the wool fibre was investigated by orientating the wool fibre axis parallel and then perpendicular to the plane of polarisation of the electric vector of the incident radiation. It was experimentally determined that the majority of C=O and N-H bonds of the peptide bond of wool lie parallel to the fibre axis. Additionally, a number of the important vibrations associated with the a-helix were also found to lie parallel to the fibre axis. Further investigation into the molecular structure of wool involved determining what effect stretching the wool fibre had on bond orientation. Raman spectra of stretched and unstretched wool fibres indicated that extension altered the orientation ofthe aromatic rings, the CH2 and CH3 groups of the amino acids. Curve fitting results revealed that extension resulted in significant destruction of the a-helix structure a substantial increase in the P-pleated sheet structure. Finally, depolarisation ratios were calculated for Raman spectra. The vibrations associated with the aromatic rings of amino acids had very low ratios which indicated that the vibrations were highly symmetrical.

Impacts of periodic tillage on soil C stocks : a synthesis

Long-term loss of soil C stocks under conventional tillage and accrual of soil C following adoption of no-tillage have been well documented. No-tillage use is spreading, but it is common to occasionally till within a no-till regime or to regularly alternate between till and no-till practices within a rotation of different crops. Short-term studies indicate that substantial amounts of C can be lost from the soil immediately following a tillage event, but there are few field studies that have investigated the impact of infrequent tillage on soil C stocks. How much of the C sequestered under no-tillage is likely to be lost if the soil is tilled? What are the longer-term impacts of continued infrequent no-tillage? If producers are to be compensated for sequestering C in soil following adoption of conservation tillage practices, the impacts of infrequent tillage need to be quantified. A few studies have examined the short-term impacts of tillage on soil C and several have investigated the impacts of adoption of continuous no-tillage. We present: (1) results from a modeling study carried out to address these questions more broadly than the published literature allows, (2) a review of the literature examining the short-term impacts of tillage on soil C, (3) a review of published studies on the physical impacts of tillage and (4) a synthesis of these components to assess how infrequent tillage impacts soil C stocks and how changes in tillage frequency could impact soil C stocks and C sequestration. Results indicate that soil C declines significantly following even one tillage event (1-11 % of soil C lost). Longer-term losses increase as frequency of tillage increases. Model analyses indicate that cultivating and ripping are less disruptive than moldboard plowing, and soil C for those treatments average just 6% less than continuous NT compared to 27% less for CT. Most (80%) of the soil C gains of NT can be realized with NT coupled with biannual cultivating or ripping. (C) 2007 Elsevier B.V. All rights reserved.

Stabilization mechanisms of soil organic matter: Implications for C-saturation of soils

The relationship between soil structure and the ability of soil to stabilize soil organic matter (SOM) is a key element in soil C dynamics that has either been overlooked or treated in a cursory fashion when developing SOM models. The purpose of this paper is to review current knowledge of SOM dynamics within the framework of a newly proposed soil C saturation concept. Initially, we distinguish SOM that is protected against decomposition by various mechanisms from that which is not protected from decomposition. Methods of quantification and characteristics of three SOM pools defined as protected are discussed. Soil organic matter can be: (1) physically stabilized, or protected from decomposition, through microaggregation, or (2) intimate association with silt and clay particles, and (3) can be biochemically stabilized through the formation of recalcitrant SOM compounds. In addition to behavior of each SOM pool, we discuss implications of changes in land management on processes by which SOM compounds undergo protection and release. The characteristics and responses to changes in land use or land management are described for the light fraction (LF) and particulate organic matter (POM). We defined the LF and POM not occluded within microaggregates (53-250 mum sized aggregates as unprotected. Our conclusions are illustrated in a new conceptual SOM model that differs from most SOM models in that the model state variables are measurable SOM pools. We suggest that physicochemical characteristics inherent to soils define the maximum protective capacity of these pools, which limits increases in SOM (i.e. C sequestration) with increased organic residue inputs.