Proteomic changes in the rat brain induced by homogenous irradiation and by the bystander effect resulting from high energy synchrotron X-ray microbeams

Abstract Purpose: To further evaluate the use of microbeam irradiation (MBI) as a potential means of non-invasive brain tumor treatment by investigating the induction of a bystander effect in non-irradiated tissue. Methods: Adult rats were irradiated with 35 or 350 Gy at the European Synchotron Research Facility (ESRF), using homogenous (broad beam) irradiation (HI) or a high energy microbeam delivered to the right brain hemisphere only. The proteome of the frontal lobes were then analyzed using two-dimensional electrophoresis (2-DE) and mass spectrometry. Results: HI resulted in proteomic responses indicative of tumourigenesis; increased albumin, aconitase and triosphosphate isomerase (TPI), and decreased dihydrolipoyldehydrogenase (DLD). The MBI bystander effect proteomic changes were indicative of reactive oxygen species mediated apoptosis; reduced TPI, prohibitin and tubulin and increased glial fibrillary acidic protein (GFAP). These potentially anti-tumourigenic apoptotic proteomic changes are also associated with neurodegeneration. However the bystander effect also increased heat shock protein (HSP) 71 turnover. HSP 71 is known to protect against all of the neurological disorders characterized by the bystander effect proteome changes. Conclusions: These results indicate that the collective interaction of these MBI-induced bystander effect proteins and their mediation by HSP 71, may confer a protective effect which now warrants additional experimental attention.

Preventive effect of ozone on the development of white spot lesions during multibracket appliance therapy

OBJECTIVE: To test the null hypotheses: (1) there is no difference in the caries protective effect of ozone and Cervitec/Fluor Protector during multibracket (MB) appliance therapy, and (2) DIAGNOdent and quantitative light-induced fluorescence (QLF) are not superior to a visual evaluation of initial caries lesions. MATERIALS AND METHODS: Twenty right-handed patients with a very poor oral hygiene who required full MB appliance therapy were analyzed during 26 months. In a split-mouth-design, the four quadrants of each patient were either treated with ozone, a combination of Cervitec and Fluor Protector, or served as untreated controls. The visible plaque index (VPI) and white spot formation were analyzed clinically. DIAGNOdent and QLF were used for a quantitative assessment of white spot formation. RESULTS: The average VPI in all four dental arch quadrants amounted to 55.6% and was independent of the preventive measure undertaken. In the quadrants treated with Cervitec/Fluor Protector, only 0.7% of the areas developed new, clinically visible white spots. This was significantly (P < .05) less than in the quadrants treated with ozone (3.2%). The lesions detected with QLF only partially corresponded to the clinically detected white spots, while DIAGNOdent proved to be unable to detect any changes at all. CONCLUSIONS: The caries protective effect of Cervitec/Fluor Protector during MB therapy was superior to ozone, and a visual evaluation of initial caries lesions was superior to both DIAGNOdent and QLF.

Regulatory mechanism of the light-activable allosteric switch LOV-TAP for the control of DNA binding: a computer simulation study

The spatio-temporal control of gene expression is fundamental to elucidate cell proliferation and deregulation phenomena in living systems. Novel approaches based on light-sensitive multiprotein complexes have recently been devised, showing promising perspectives for the noninvasive and reversible modulation of the DNA-transcriptional activity in vivo. This has lately been demonstrated in a striking way through the generation of the artificial protein construct light-oxygen-voltage (LOV)-tryptophan-activated protein (TAP), in which the LOV-2-Jα photoswitch of phototropin1 from Avena sativa (AsLOV2-Jα) has been ligated to the tryptophan-repressor (TrpR) protein from Escherichia coli. Although tremendous progress has been achieved on the generation of such protein constructs, a detailed understanding of their functioning as opto-genetical tools is still in its infancy. Here, we elucidate the early stages of the light-induced regulatory mechanism of LOV-TAP at the molecular level, using the noninvasive molecular dynamics simulation technique. More specifically, we find that Cys450-FMN-adduct formation in the AsLOV2-Jα-binding pocket after photoexcitation induces the cleavage of the peripheral Jα-helix from the LOV core, causing a change of its polarity and electrostatic attraction of the photoswitch onto the DNA surface. This goes along with the flexibilization through unfolding of a hairpin-like helix-loop-helix region interlinking the AsLOV2-Jα- and TrpR-domains, ultimately enabling the condensation of LOV-TAP onto the DNA surface. By contrast, in the dark state the AsLOV2-Jα photoswitch remains inactive and exerts a repulsive electrostatic force on the DNA surface. This leads to a distortion of the hairpin region, which finally relieves its tension by causing the disruption of LOV-TAP from the DNA.

Bothnia dystrophy is caused by domino-like rearrangements in cellular retinaldehyde-binding protein mutant R234W

Cellular retinaldehyde-binding protein (CRALBP) is essential for mammalian vision by routing 11-cis-retinoids for the conversion of photobleached opsin molecules into photosensitive visual pigments. The arginine-to-tryptophan missense mutation in position 234 (R234W) in the human gene RLBP1 encoding CRALBP compromises visual pigment regeneration and is associated with Bothnia dystrophy. Here we report the crystal structures of both wild-type human CRALBP and of its mutant R234W as binary complexes complemented with the endogenous ligand 11-cis-retinal, at 3.0 and 1.7 A resolution, respectively. Our structural model of wild-type CRALBP locates R234 to a positively charged cleft at a distance of 15 A from the hydrophobic core sequestering 11-cis-retinal. The R234W structural model reveals burial of W234 and loss of dianion-binding interactions within the cleft with physiological implications for membrane docking. The burial of W234 is accompanied by a cascade of side-chain flips that effect the intrusion of the side-chain of I238 into the ligand-binding cavity. As consequence of the intrusion, R234W displays 5-fold increased resistance to light-induced photoisomerization relative to wild-type CRALBP, indicating tighter binding to 11-cis-retinal. Overall, our results reveal an unanticipated domino-like structural transition causing Bothnia-type retinal dystrophy by the impaired release of 11-cis-retinal from R234W.

Action of food preservatives on 14-days dental biofilm formation, biofilm vitality and biofilm-derived enamel demineralisation in situ

AIMS The aims of this double-blind, controlled, crossover study were to assess the influence of food preservatives on in situ dental biofilm growth and vitality, and to evaluate their influence on the ability of dental biofilm to demineralize underlying enamel over a period of 14 days. MATERIALS AND METHODS Twenty volunteers wore appliances with six specimens each of bovine enamel to build up intra-oral biofilms. During four test cycles of 14 days, the subjects had to place the appliance in one of the assigned controls or active solutions twice a day for a minute: negative control 0.9 % saline, 0.1 % benzoate (BA), 0.1 % sorbate (SA) and 0.2 % chlorhexidine (CHX positive control). After 14 days, the biofilms on two of the slabs were stained to visualize vital and dead bacteria to assess biofilm thickness (BT) and bacterial vitality (BV). Further, slabs were taken to determine mineral loss (ML), by quantitative light-induced laser fluorescence (QLF) and transversal microradiography (TMR), moreover the lesion depths (LD). RESULTS Nineteen subjects completed all test cycles. Use of SA, BA and CHX resulted in a significantly reduced BV compared to NaCl (p < 0.001). Only CHX exerted a statistically significant retardation in BT as compared to saline. Differences between SA and BA were not significant (p > 0.05) for both parameters. TMR analysis revealed the highest LD values in the NaCl group (43.6 ± 44.2 μm) and the lowest with CHX (11.7 ± 39.4 μm), while SA (22.9 ± 45.2 μm) and BA (21.4 ± 38.5 μm) lay in between. Similarly for ML, the highest mean values of 128.1 ± 207.3 vol% μm were assessed for NaCl, the lowest for CHX (-16.8 ± 284.2 vol% μm), while SA and BA led to values of 83.2 ± 150.9 and 98.4 ± 191.2 vol% μm, respectively. With QLF for both controls, NaCl (-33.8 ± 101.3 mm(2) %) and CHX (-16.9 ± 69.9 mm(2) %), negative values were recorded reflecting a diminution of fluorescence, while positive values were found with SA (33.9 ± 158.2 mm(2) %) and BA (24.8 ± 118.0 mm(2) %) depicting a fluorescence gain. These differences were non-significant (p > 0.05). CONCLUSION The biofilm model permited the assessment of undisturbed oral biofilm formation influenced by antibacterial components under clinical conditions for a period of 14 days. An effect of BA and SA on the demineralization of enamel could be demonstrated by TMR and QLF, but these new findings have to be seen as a trend. As part of our daily diet, these preservatives exert an impact on the metabolism of the dental biofilm, and therefore may even influence demineralization processes of the underlying dental enamel in situ.

Preparing Offspring for a Dangerous World: Potential Costs of Being Wrong

Adaptive maternal responses to stressful environments before young are born can follow two non-exclusive pathways: either the mother reduces current investment in favor of future investment, or influences offspring growth and development in order to fit offspring phenotype to the stressful environment. Inducing such developmental cues, however, may be risky if the environment changes meanwhile, resulting in maladapted offspring. Here we test the effects of a predator-induced maternal effect in a predator-free postnatal environment. We manipulated perceived predation-risk for breeding female great tits by exposing them to stuffed models of either a predatory bird or a non-predatory control. Offspring were raised either in an environment matching the maternal one by exchanging whole broods within a maternal treatment group, or in a mismatching environment by exchanging broods among the maternal treatments. Offspring growth depended on the matching of the two environments. While for offspring originating from control treated mothers environmental mismatch did not significantly change growth, offspring of mothers under increased perceived predation risk grew faster and larger in matching conditions. Offspring of predator treated mothers fledged about one day later when growing under mismatching conditions. This suggests costs paid by the offspring if mothers predict environmental conditions wrongly.

Performance of a new laser fluorescence device for the detection of occlusal caries in vitro

The new device DIAGNOdent pen based on red laser light induced fluorescence was introduced for the detection of approximal and occlusal caries. The aim of this study was to test its performance on occlusal surfaces. The new device comes with two different sapphire fibre tips: a cylindrical tip and a conical tip. The two new sapphire fibre tips were used and compared with the tip currently available with DIAGNOdent (DD). METHODS: The teeth were selected from a pool of extracted permanent human molars, which were stored frozen at -20 degrees C, until use. Prior to being measured the teeth were defrosted and cleaned. One hundred and nineteen teeth were selected and measured with the old tip and with the two new tips of the new device by two independent assessments. The teeth were histologically prepared and assessed for caries extension. RESULTS: Specificity values for D(1), D(2) and D(3) ranged between 0.69 and 0.89, sensitivity between 0.78 and 0.96. There were no statistically significant differences obtained between the two tips of the new and the one tip of the old device (p>0.05). Intra-examiner reliability with kappa values of >0.83 was high. CONCLUSIONS: In this study, the new laser fluorescence device performed on occlusal surfaces as well as the available device.

Novel lesion detection aids

Several non-invasive and novel aids for the detection of (and in some cases monitoring of) caries lesions have been introduced in the field of 'caries diagnostics' over the last 15 years. This chapter focusses on those available to dentists at the time of writing; continuing research is bound to lead to further developments in the coming years. Laser fluorescence is based on measurements of back-scattered fluorescence of a 655-nm light source. It enhances occlusal and (potentially) approximal lesion detection and enables semi-quantitative caries monitoring. Systematic reviews have identified false-positive results as a limitation. Quantitative light-induced fluorescence is another sensitive method to quantitatively detect and measure mineral loss both in enamel and some dentine lesions; again, the trade-offs with lower specificity when compared with clinical visual detection must be considered. Subtraction radiography is based on the principle of digitally superimposing two radiographs with exactly the same projection geometry. This method is applicable for approximal surfaces and occlusal caries involving dentine but is not yet widely available. Electrical caries measurements gather either site-specific or surface-specific information of teeth and tooth structure. Fixed-frequency devices perform best for occlusal dentine caries but the method has also shown promise for lesions in enamel and other tooth surfaces with multi-frequency approaches. All methods require further research and further validation in well-designed clinical trials. In the future, they could have useful applications in clinical practice as part of a personalized, comprehensive caries management system.

Tamoxifen inhibits TRPV6 activity via estrogen receptor-independent pathways in TRPV6-expressing MCF-7 breast cancer cells

The epithelial calcium channel TRPV6 is upregulated in breast carcinoma compared with normal mammary gland tissue. The selective estrogen receptor modulator tamoxifen is widely used in breast cancer therapy. Previously, we showed that tamoxifen inhibits calcium uptake in TRPV6-transfected Xenopus oocytes. In this study, we examined the effect of tamoxifen on TRPV6 function and intracellular calcium homeostasis in MCF-7 breast cancer cells transiently transfected with EYFP-C1-TRPV6. TRPV6 activity was measured with fluorescence microscopy using Fura-2. The basal calcium level was higher in transfected cells compared with nontransfected cells in calcium-containing solution but not in nominally calcium-free buffer. Basal influxes of calcium and barium were also increased. In transfected cells, 10 mumol/L tamoxifen reduced the basal intracellular calcium concentration to the basal calcium level of nontransfected cells. Tamoxifen decreased the transport rates of calcium and barium in transfected cells by 50%. This inhibitory effect was not blocked by the estrogen receptor antagonist, ICI 182,720. Similarly, a tamoxifen-induced inhibitory effect was also observed in MDA-MB-231 estrogen receptor-negative cells. The effect of tamoxifen was completely blocked by activation of protein kinase C. Inhibiting protein kinase C with calphostin C decreased TRPV6 activity but did not alter the effect of tamoxifen. These findings illustrate how tamoxifen might be effective in estrogen receptor-negative breast carcinomas and suggest that the therapeutic effect of tamoxifen and protein kinase C inhibitors used in breast cancer therapy might involve TRPV6-mediated calcium entry. This study highlights a possible role of TRPV6 as therapeutic target in breast cancer therapy.