Quantification of global DNA methylation with infrared fluorescence in liver and muscle tissues of differentially fed boars

Methylation of cytosine residues at CpG sites is involved in various biological processes to control gene regulation and gene expression. Global DNA methylation is changed in different tumors and in cloned animals. Global DNA methylation can be accurately quantified by dot blot analysis with infrared (IR) fluorophores. Methylated lambda DNA was used as model DNA to develop and validate an immunochemical assay with IR fluorescence detection. Two different IR fluorophores were used, one to detect 5-methylcytosine and another to account for DNA loading. A sensitive infrared detection method was established which is suitable for accurate and reproducible quantification of global DNA methylation across a wide dynamic range. This method was subsequently employed to quantify global DNA methylation in liver and in muscle tissues of boars which have received either a control diet or a methyl supplemented diet in an ongoing study. A significant difference in global DNA methylation is indicated in muscle but not in liver tissue between the two groups of boars.

Characteristics and functional relevance of apolipoprotein-A1 and cholesterol binding in mammary gland tissues and epithelial cells

Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of (125)I-apoA-I and (3)H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular (3)H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell(®) plates. The amounts of isolated EPM and the maximal binding capacity of (125)I-apoA-I to EPM differed depending on the MG's physiological state, while the kinetics of (3)H-cholesterol and (125)I-apoA-I binding were similar. (3)H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of (125)I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of (125)I-apoA-I ranged between 40-74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and (125)I-apoA-I binding. The ABCA1 inhibitor Probucol displaced (125)I-apoA-I binding to EPM and reduced (3)H-cholesterol efflux in MeBo. Time-dependent (3)H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell(®) plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of (3)H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the importance of the apoA-I/ABCA1 pathway in MG cholesterol transport and suggest its role in influencing milk composition and directing cholesterol back into the bloodstream.

Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling.

Priapism, abnormally prolonged penile erection in the absence of sexual excitation, is associated with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. It is common among males with sickle cell disease (SCD), and SCD transgenic mice are an accepted model of the disorder. Current strategies to manage priapism suffer from a poor fundamental understanding of the molecular mechanisms underlying the disorder. Here we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected priapic activity. ADA enzyme therapy successfully corrected the priapic activity both in vivo and in vitro, suggesting that it was dependent on elevated adenosine levels. Further genetic and pharmacologic evidence demonstrated that A2B adenosine receptor-mediated (A2BR-mediated) cAMP and cGMP induction was required for elevated adenosine-induced prolonged penile erection. Finally, priapic activity in SCD transgenic mice was also caused by elevated adenosine levels and A2BR activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased A2BR signaling in both Ada -/- and SCD transgenic mice. These findings provide insight regarding the molecular basis of priapism and suggest that strategies to either reduce adenosine or block A2BR activation may prove beneficial in the treatment of this disorder.

Increased FOXP3 expression in tumour-associated tissues of horses affected with equine sarcoid disease.

Recent studies suggest that regulatory T cells (Tregs) are associated with disease severity and progression in papilloma virus induced neoplasia. Bovine papilloma virus (BPV) is recognised as the most important aetiological factor in equine sarcoid (ES) disease. The aim of this study was to compare expression levels of Treg markers and associated cytokines in tissue samples of ES-affected equids with skin samples of healthy control horses. Eleven ES-affected, and 12 healthy horses were included in the study. Expression levels of forkhead box protein 3 (FOXP3), interleukin 10 (IL10), interleukin 4 (IL4) and interferon gamma (IFNG) mRNA in lesional and tumour-distant samples from ES-affected horses, as well as in dermal samples of healthy control horses were measured using quantitative reverse transcription polymerase chain reaction (PCR). Expression levels were compared between lesional and tumour-distant as well as between tumour-distant and control samples. Furthermore, BPV-1 E5 DNA in samples of ES-affected horses was quantified using quantitative PCR, and possible associations of viral load, disease severity and gene expression levels were evaluated. Expression levels of FOXP3, IL10 and IFNG mRNA and BPV-1 E5 copy numbers were significantly increased in lesional compared to tumour-distant samples. There was no difference in FOXP3 and cytokine expression in tumour-distant samples from ES- compared with control horses. In tumour-distant samples viral load was positively correlated with IL10 expression and severity score. The increased expression of Treg markers in tumour-associated tissues of ES-affected equids indicates a local, Treg-induced immune suppression.

Three-dimensional imaging methods for quantitative analysis of facial soft tissues and skeletal morphology in patients with orofacial clefts: a systematic review.

BACKGROUND Current guidelines for evaluating cleft palate treatments are mostly based on two-dimensional (2D) evaluation, but three-dimensional (3D) imaging methods to assess treatment outcome are steadily rising. OBJECTIVE To identify 3D imaging methods for quantitative assessment of soft tissue and skeletal morphology in patients with cleft lip and palate. DATA SOURCES Literature was searched using PubMed (1948-2012), EMBASE (1980-2012), Scopus (2004-2012), Web of Science (1945-2012), and the Cochrane Library. The last search was performed September 30, 2012. Reference lists were hand searched for potentially eligible studies. There was no language restriction. STUDY SELECTION We included publications using 3D imaging techniques to assess facial soft tissue or skeletal morphology in patients older than 5 years with a cleft lip with/or without cleft palate. We reviewed studies involving the facial region when at least 10 subjects in the sample size had at least one cleft type. Only primary publications were included. DATA EXTRACTION Independent extraction of data and quality assessments were performed by two observers. RESULTS Five hundred full text publications were retrieved, 144 met the inclusion criteria, with 63 high quality studies. There were differences in study designs, topics studied, patient characteristics, and success measurements; therefore, only a systematic review could be conducted. Main 3D-techniques that are used in cleft lip and palate patients are CT, CBCT, MRI, stereophotogrammetry, and laser surface scanning. These techniques are mainly used for soft tissue analysis, evaluation of bone grafting, and changes in the craniofacial skeleton. Digital dental casts are used to evaluate treatment and changes over time. CONCLUSION Available evidence implies that 3D imaging methods can be used for documentation of CLP patients. No data are available yet showing that 3D methods are more informative than conventional 2D methods. Further research is warranted to elucidate it.

The histological features and physical properties of eroded dental hard tissues.

Erosive demineralisation causes characteristic histological features. In enamel, mineral is dissolved from the surface, resulting in a roughened structure similar to an etching pattern. If the acid impact continues, the initial surface mineral loss turns into bulk tissue loss and with time a visible defect can develop. The microhardness of the remaining surface is reduced, increasing the susceptibility to physical wear. The histology of eroded dentine is much more complex because the mineral component of the tissue is dissolved by acids whereas the organic part is remaining. At least in experimental erosion, a distinct zone of demineralised organic material develops, the thickness of which depends on the acid impact. This structure is of importance for many aspects, e.g. the progression rate or the interaction with active agents and physical impacts, and needs to be considered when quantifying mineral loss. The histology of experimental erosion is increasingly well understood, but there is lack of knowledge about the histology of in vivo lesions. For enamel erosion, it is reasonable to assume that the principal features may be similar, but the fate of the demineralised dentine matrix in the oral cavity is unclear. As dentine lesions normally appear hard clinically, it can be assumed that it is degraded by the variety of enzymes present in the oral cavity. Erosive tooth wear may lead to the formation of reactionary or reparative dentine.

Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues.

PURPOSE Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the (125)iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer (125)I-GLP-1(7-36)amide. METHODS Receptor autoradiography studies with (125)I-GLP-1(7-36)amide agonist or (125)I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. RESULTS The antagonist (125)I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer (125)I-GLP-1(7-36)amide. For comparison, (125)I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. CONCLUSION The GLP-1 receptor antagonist exendin(9-39) labelled with (125)I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients.

Segmented filamentous bacterium uses secondary and tertiary lymphoid tissues to induce gut IgA and specific T helper 17 cell responses.

Segmented filamentous bacterium (SFB) is a symbiont that drives postnatal maturation of gut adaptive immune responses. In contrast to nonpathogenic E. coli, SFB stimulated vigorous development of Peyer's patches germinal centers but paradoxically induced only a low frequency of specific immunoglobulin A (IgA)-secreting cells with delayed accumulation of somatic mutations. Moreover, blocking Peyer's patch development abolished IgA responses to E. coli, but not to SFB. Indeed, SFB stimulated the postnatal development of isolated lymphoid follicles and tertiary lymphoid tissue, which substituted for Peyer's patches as inductive sites for intestinal IgA and SFB-specific T helper 17 (Th17) cell responses. Strikingly, in mice depleted of gut organized lymphoid tissue, SFB still induced a substantial but nonspecific intestinal Th17 cell response. These results demonstrate that SFB has the remarkable capacity to induce and stimulate multiple types of intestinal lymphoid tissues that cooperate to generate potent IgA and Th17 cell responses displaying only limited target specificity.

Assessment of STR Typing Success Rate in Soft Tissues from Putrefied Bodies Based on a Quantitative Grading System for Putrefaction.

To date, there is no systematic investigation of the association of short tandem repeat (STR) typing success rate in soft tissues with different signs of putrefaction. Herein, putrefaction was rated using a newly developed 19-parameter system in soft tissues from a collective of 68 decaying bodies, and DNA yield was determined in 408 samples. DNA integrity was rated using a self-devised pentaplex PCR generating an "integrity score" (Si ). STR typing success rate was then assessed for selected cases. DNA yield and Si differed significantly between tissues with kidney on average exhibiting the highest Si values. Statistical analysis revealed that nine parameters were significantly and positively correlated with Si . The observed values for each of these nine parameters were summed up to generate a putrefaction score (Sp ) for each sample. Our results show that STR typing success rate can be predicted based on Sp before expensive multiplex STR profiling is performed.

A specialist root herbivore exploits defensive metabolites to locate nutritious tissues

he most valuable organs of plants are often particularly rich in essential elements, but also very well defended. This creates a dilemma for herbivores that need to maximise energy intake while minimising intoxication. We investigated how the specialist root herbivore Diabrotica virgifera solves this conundrum when feeding on wild and cultivated maize plants. We found that crown roots of maize seedlings were vital for plant development and, in accordance, were rich in nutritious primary metabolites and contained higher amounts of the insecticidal 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and the phenolic compound chlorogenic acid. The generalist herbivores Diabrotica balteata and Spodoptera littoralis were deterred from feeding on crown roots, whereas the specialist D. virgifera preferred and grew best on these tissues. Using a 1,4-benzoxazin-3-one-deficient maize mutant, we found that D. virgifera is resistant to DIMBOA and other 1,4-benzoxazin-3-ones and that it even hijacks these compounds to optimally forage for nutritious roots

Search for Low-Molecular-Weight Biomarkers in Plant Tissues and Seeds Using Metabolomics: Tools, Strategies, and Applications

This chapter summarises the metabolomic strategies currently in force used in plant science and describes the methods used. The metabolite profiling and fingerprinting of plant tissues through MS- and/or NMR-based approaches and the subsequent identification of biomarkers is detailed. Strategies for the microisolation and de novo identification of unknown biomarkers are also discussed. The various approaches are illustrated by a metabolomic study of the maize response to herbivory. A review of recent metabolomic studies performed on seed and crop plant tissues involving various analytical strategies is provided.

A prospective, randomized, controlled study using OsseoSpeed™ implants placed in maxillary fresh extraction socket: soft tissues response

PURPOSE The aim of this work was to study the peri-implant soft tissues response, by evaluating both the recession and the papilla indexes, of patients treated with implants with two different configurations. In addition, data were stratified by tooth category, smoking habit and thickness of buccal bone wall. MATERIALS AND METHODS The clinical trial was designed as a prospective, randomized-controlled multicenter study. Adults in need of one or more implants replacing teeth to be removed in the maxilla within the region 15-25 were recruited. Following tooth extraction, the site was randomly allocated to receive either a cylindrical or conical/cylindrical implant. The following parameters were studied: (i) Soft tissue recession (REC) measured by comparing the gingival zenith (GZ) score at baseline (permanent restoration) with that of the yearly follow-up visits over a period of 3 years (V1, V2 and V3). (ii) Interdental Papilla Index (PI): PI measurements were performed at baseline and compared with that of the follow-up visits. In addition, data were stratified by different variables: tooth category: anterior (incisors and canine) and posterior (first and second premolar); smoking habit: patient smoker (habitual or occasional smoker at inclusion) or non-smoker (non-smoker or ex-smoker at inclusion) and thickness of buccal bone wall (TB): TB ≤ 1 mm (thin buccal wall) or TB > 1 mm (thick buccal wall). RESULTS A total of 93 patients were treated with 93 implants. At the surgical re-entry one implant was mobile and then removed; moreover, one patient was lost to follow-up. Ninety-one patients were restored with 91 implant-supported permanent single crowns. After the 3-year follow-up, a mean gain of 0.23 mm of GZ was measured; moreover, 79% and 72% of mesial and distal papillae were classified as >50%/ complete, respectively. From the stratification analysis, not significant differences were found between the mean GZ scores of implants with TB ≤ 1 mm (thin buccal wall) and TB > 1 mm (thick buccal wall), respectively (P < 0.05, Mann-Whitney U-test) at baseline, at V1, V2 and V3 follow-up visits. Also, the other variables did not seem to influence GZ changes over the follow-up period. Moreover, a re-growth of the interproximal mesial and distal papillae was the general trend observed independently from the variables studied. CONCLUSIONS Immediate single implant treatment may be considered a predictable option regarding soft tissue stability over a period of 3 years of follow-up. An overall buccal soft tissue stability was observed during the GZ changes from the baseline to the 3 years of follow-up with a mean GZ reduction of 0.23 mm. A nearly full papillary re-growth can be detectable over a minimum period of 2 years of follow-up for both cylindrical and conical/cylindrical implants. Both the interproximal papilla filling and the midfacial mucosa stability were not influenced by variables such as type of fixture configuration, tooth category, smoke habit, and thickness of buccal bone wall of ≤ 1 mm (thin buccal wall).

The physiological roles of ICAM-1 and ICAM-2 in neutrophil migration into tissues

PURPOSE OF REVIEW Neutrophil extravasation from the blood into tissues is initiated by tethering and rolling of neutrophils on endothelial cells, followed by neutrophil integrin activation and shear resistant arrest, crawling, diapedesis and breaching the endothelial basement membrane harbouring pericytes. Endothelial intercellular cell adhesion molecule (ICAM)-1 and ICAM-2, in conjunction with ICAM-1 on pericytes, critically contribute to each step. In addition, epithelial ICAM-1 is involved in neutrophil migration to peri-epithelial sites. The most recent findings on the role of ICAM-1 and ICAM-2 for neutrophil migration into tissues will be reviewed here. RECENT FINDINGS Signalling via endothelial ICAM-1 and ICAM-2 contributes to stiffness of the endothelial cells at sites of chronic inflammation and junctional maturation, respectively. Endothelial ICAM-2 contributes to neutrophil crawling and initiation of paracellular diapedesis, which then proceeds independent of ICAM-2. Substantial transcellular neutrophil diapedesis across the blood-brain barrier is strictly dependent on endothelial ICAM-1 and ICAM-2. Endothelial ICAM-1 or ICAM-2 is involved in neutrophil-mediated plasma leakage. ICAM-1 on pericytes assists the final step of neutrophil extravasation. Epithelial ICAM-1 rather indirectly promotes neutrophil migration to peri-epithelial sites. SUMMARY ICAM-1 and ICAM-2 are involved in each step of neutrophil extravasation, and have redundant but also distinct functions. Analysis of the role of endothelial ICAM-1 requires simultaneous consideration of ICAM-2.