Moreover maximum increase in GR and GP activities, 2.1 and 2.5 times, respectively, was observed at 96 h after exposure to CCl4, what coincided with the maximum of free radical oxidation processes. Using a combination of reverse transcription and real-time polymerase chain reaction, expression of the glutathione peroxidase and glutathione reductase genes (Gpx1 and Gsr) was analyzed by the determination of their respective mRNAs in the rat liver tissue under toxic hepatitis conditions. The analyses of Gpx1 and Gsr expression revealed that the transcript levels increased in 2.5- and 3.0-folds, respectively. Western blot analysis revealed that the amounts of hepatic Gpx1 and Gsr proteins increased considerably after CCl4 administration. It can be proposed that the overexpression of these enzymes could be a mechanism of enhancement of hepatocytes tolerance to oxidative stress.Correlation between intensity of free radical processes estimated by biochemiluminesce parameters, content of lipoperoxidation products, and changes of glutathione peroxidase  and glutathione reductase  activities at rats liver injury, after 12, 36, 70, 96, 110, and 125 hours & tetrachloromethane administration have been investigated. The histological examination of the liver sections of rats showed that prominent hepatocytes with marked vacuolisation and inflammatory cells which were arranged around the necrotic tissue are more at 96 h after exposure to CCl After that, the tube was placed in ice and the reagents were added in the following order: 4 μL 5x-RT buffer, 1 μL RiboLock (20 μL/μL), and 2 μL 10 mM dNTP mix. The mixture was incubated at 37°C for 5 min. Then 2 μL of M-MuLV reverse transcriptase (20 units/μL) to a final volume of 20 μL was added. The mixture was subsequently incubated at 37°C for 60 min. The reaction was stopped by heating at 70°C for 10 min. cDNA obtained during reverse transcription was used to analyze gene expression of Gpx1 and Gsr by RT-PCR.To assess the expression of Gpx1 and Gsr, the total RNA from liver tissue of control and experimental animals was isolated using a set of YellowSolve . The degree of RNA degradation was evaluated by electrophoresis in denaturing agarose gel (1%). An amount of RNA was measured using a Hitachi-U1900 spectrophotometer at a wavelength of 260 nm. For reverse transcription, 5 micrograms of total RNA was annealed with 1 β-actin (β-Act), and ribosomal protein L3. Primers for all five investigated genes were selected using Genomics Expression software ; and 30 sec at 72°C. Each tissue sample was analyzed three times for each gene. The obtained threshold cycle reactions values were normalized with respect to Ct of three “housekeeping” genes and statistically processed using the “Relative Expression Software Tool (REST)” [To normalize the expression level of the studied genes, three out group genes were used: albumin (Alb), software . RT-PCR  (REST)” . The chaΔCt GOI; REF Conc1, 2…= ΔCt efficiencyREF 1, 2, where ΔCt GOI is a difference of threshold cycles of control and experimental groups obtained during the study of gene amplification; ΔCt REF Conc1, 2…—a difference of threshold cycles of control and experimental groups obtained during the reference genes amplification.Concentration of the studied genes and reference genes was calculated as GOI Conc = efficiencyE = 10A) according to the protocol of manufacturer. The PCR products were sequenced on an automatic analyzer ABI Prizm 3130 Genetic Analyzer. The obtained sequences were analyzed for specificity using the software BLAST.μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blot analysis was performed using an anti-rat GR antibody, and GP antibody (1 : 1000) purchased from Almabion Co. Ltd. (Russia). The blots were probed with a goat polyclonal actin antibody (1 : 500) from Almabion Co. Ltd (Russia) to ensure that the amount of protein loaded into each lane of the gel was constant. Blots were incubated with the appropriate peroxidase-conjugated secondary antibodies. After removal of the secondary antibodies, the blots were washed and developed using the ECL-Western blotting system.After homogenization of the liver from all experimental rats using a T10 homogenizer, the homogenates  and the maximum flash intensity (Imax), reflecting the intensity of FRO, significantly increased and reached a peak 96 h after induction of pathology  was observed. Such change indicated that the total antioxidant activity increased 2.25 times compared to the norm. This suggests that under pathological conditions an effective compensatory mechanisms reducing FRO level in the cell are necessary. The changes in GP activity during the development of experimental toxic hepatitis showed that the maximum activity was observed at 96 h after administration of tetrachloromethane. In this case, the activity per gram of fresh weight and specific activity increased 4.60 and 2.50 times, respectively.The light sum of chemiluminescence , such as O2•−, H2O2, and OH•, which damage the liver [κB) and interleukin-6 at the time of G1/S transition, induction of cyclin D1 expression associated with an increase of mitosis, and a time-dependent increase of TNF consistent with the appearance of necrosis [κB might regulate Gpx and Gsr [κB by inactivation of IkBa [κB also regulates genes involved in inflammation, apoptosis, proliferation, and regeneration in liver [κB induced expression of Gpx1 and Gsr  and glutathione reductase (GR) system plays key role in organism defense under toxic hepatitis.The Gpx1 and Gsr enzyme's genes expression was detected at the action of tetrachloromethane.The enzyme's expression can be a mechanism of tissue tolerance to oxidative stress. This might be the first sign of the role of xenobiotics in the overexpression of the GP/GR system enzymes.


Having controlled for the Suess effect and for increase CO2 in seawater, δ13C values of Antarctic penguins and of king penguins did not change over time, while δ13C of other subantarctic and subtropical species were lower in the 1970s. The data therefore suggest a decrease in ecosystem carrying capacity of the southern Indian Ocean during the temperature regime-shift in subtropical and subantarctic waters but not in the vicinity of the Polar Front and in southward high-Antarctic waters. The resulting lower secondary productivity could be the main driving force explaining the decline of subtropical and subantarctic (but not Antarctic) penguins that occurred in the 1970s. Feather δ15N values did not show a consistent temporal trend among species, suggesting no major change in penguins’ diet. This study highlights the usefulness of developing long-term tissue sampling and data bases on isotopic signature of key marine organisms to track potential changes in their isotopic niches and in the carrying capacity of the environment.A temperature-defined regime shift occurred in the 1970s in the southern Indian Ocean, with simultaneous severe decreases in many predator populations. We tested a possible biological link between the regime shift and predator declines by measuring historic and contemporary feather isotopic signatures of seven penguin species with contrasted foraging strategies and inhabiting a large latitudinal range. We first showed that contemporary penguin isotopic variations and chlorophyll a concentration were positively correlated, suggesting the usefulness of predator δ The warming of Earth’s climate since the 1970s has been at a rate greater that at any other time in the last thousand years, producing sharp biological and ecological consequences 13C and δ15N values reflecting the consumers’ foraging habitat and trophic level, respectively 13C values of predators were also used as a proxy for ecosystem primary productivity  concentrations  that approved all our fieldwork (program n° 109).Aptenodytes forsteri) and Adélie  penguins breeding in Adélie Land ; the king , gentoo , macaroni  and southern rockhopper  penguins breeding at Possession Island ; and the northern rockhopper penguin  breeding at Amsterdam island . Amsterdan Island is located north of the Subtropical Front (STF) in the Subtropical Zone (STZ), Crozet Islands lay between the STF and Polar Front (PF) within the Subantarctic Zone sensus lato (SAZ) and Adélie Land is positioned further south, in the high-Antarctic waters of the Antarctic Zone (AZ)  .Contemporary feathers (old and new) were collected on live moulting adult penguins . Old and15N values sample/Rstandard) −1] x 1000) where X is 13C or 15N and R is the corresponding ratio 13C/12C or 15N/14N. Rstandard are PeeDee Belemnite and atmospheric N2 for δ13C and δ15N, respectively. Replicate measurements of internal laboratory standards (acetanilide) indicated measurement errors <0.15‰ and <0.20‰ for δ13C and δ15N, respectively.Prior to isotopic analysis, the tip (oldest part) of each feather was cut and discarded, because penguin feather synthesis begins at sea  were assessed using paired t-tests, because data followed Gaussian distributions  during both 2006 and 2007. For each year, Chl a concentration data were extracted and averaged from a square centred on the islands (10° longitude per 10° latitude) and for a period of two months before the initiation of the moulting fast on land (13C and Chl a was subsequently assessed using a Pearson’s correlation test (Differences in the δibutions . A possi on land . The corion test .p>0.05), except for feather NRP δ13C values that decreased in the 1970s (p = 0.004). Long-term changes were then investigated by non-parametric comparisons of groups .Historic samples were not regularly collected over time, with most of the penguins sampled in the 1950s and 1970s, a few in the 1900s and none between those years. Samples were therefore pooled in 4 groups of years: 1900s, 1950s, 1970s and 2000s  and 3. T13C values of penguin feathers were adjusted following 2 in response to human fossil fuel burning. Firstly, the resulting higher [CO2]aq increases in turn phytoplankton fractionation, thus reducing its δ13C isotopic values 13C than background carbon, thus inducing an accelerating decrease in δ13C in the biosphere  than adjusted δ13C values in the comparison between old (1900s) and more recent values (1970s and 2000s). Conversely, the δ13C differences between 1970s and 2000s samples might be exacerbated by the addition of Suess correction factors. We cannot rule out that the true effects are precisely evaluated, but there are increasing evidences of a strong effect of human fossil fuel burning on marine carbon isotopic ratios 13C change between 2006 and 2007 due to both Suess and aqueous CO2 effects was negligible (∼0.01‰).In our long-term analyses using historic and contemporary samples, the raw δ13C values were 0.1–1.3‰ higher in 2007 than in 2006. Surface Chl a concentrations were also 5–19% higher in 2007 than in 2006 in the different water masses of the southern Indian Ocean (from high-Antarctica to the subtropics), as indicated by the positive differences in Chl a concentration between 2007 and 2006 .All penguin species, but one (SRP) showed distinct inter-annual moulting isotopic niches . Dependiand 2006 . Interes15N values of three penguin species  were significantly ∼1‰ higher in 2007 than in 2006, while the three remaining species  did not show any significant inter-annual differences in their feather nitrogen signatures  and three out of four subantarctic species  had lower feather δ13C values in the 1970s than in the 2000s . Older historic NRP samples (1900s) showed higher δ13C values than in 1970s  and no significant difference with contemporary (2000s) samples . For SRP, only two samples were available in 1900s but they confirmed latest analyses. In contrast, the two Antarctic species (EP and AP) and KP did not show significant temporal change between 1950s and 2000s .Corrected feather δ13C anomalies from δ13C values, i.e. the mean feather δ13C value was subtracted from each δ13C value for a given penguin species. Latitudinal effect on δ13C value p<0.0001 and p<0.001, respectively)  and subtropical species, we calculated δctively) .15N values were significant higher than contemporary samples for EP (in the 1950s and 1970s) and MP (in the 1970s) penguins . The five other penguin species did not present significant change in their feather δ15N values over time  isotopic variations of penguin feathers and showed for the first time concurrent variations in consumer δ13C values reflect the oceanic δ13C latitudinal gradient at the base of the food web. The low δ13C values of EP and AP, intermediate values of KP, MP and SRP, and high value of NRP are representative of Antarctic, subantarctic and subtropical waters, respectively 13C values 15N values reflect mainly their dietary habits, with EP, KP and GP preying primarily upon fish, and thus higher in the food web than AP, MP and SRP that feed on both fish and crustaceans 15N value characterizing the baseline level in warm subtropical waters Little is known about the penguin biology during the pre-moult period, because birds spend 2–3 weeks away at sea to build up energy reserves, hence, the use of indirect methods to investigate their feeding ecology at this time i.e. changes in foraging locations) can be rejected for penguin δ13C values for two reasons. Firstly, penguins are spatio-temporally constrained in the vicinity of their colonies during the pre-moulting foraging period, because they have to moult ashore at their breeding sites afterwards. Secondly, the same trend in feather δ13C values was found in 5 penguin species , thus strongly suggesting a broad-scale explanation rather than species-specific changes in foraging habitats. Indeed, surface Chl a concentration was higher in 2007 than in 2006 over a broad latitudinal gradient that includes high-Antarctic, subantarctic and subtropical waters, therefore indicating that the carrying capacity of the southern Indian Ocean was higher in 2007 than in 2006. A major finding of the present work is that the inter-annual differences in penguin feather δ13C values and surface Chl a concentrations were positively correlated.Carbon and nitrogen isotopic signatures of penguin feathers presented substantial inter-annual variations. Such short-term variations were rarely investigated and they were generally interpreted in terms of changes in the birds’ feeding ecology 13C values increase 13C values of top predators have been used as bio-indicators of ecosystem productivity 13C values of top predators and productivity of the pelagic ecosystem. Our analysis was performed on a significant number of penguin species, but during a relatively short temporal window (two consecutive years only). Clearly, the relationship between penguin δ13C value and surface Chl a concentration needs to be investigated during a longer period. Also, the higher inter-annual change of king penguin δ13C values  or broad geographical areas (δ13C).The southern Indian Ocean was marked by a temperature regime shift in the 1970s associated with population changes of predators, including penguins 15N values. Feather δ15N changes were species-specific, suggesting temporal variations in penguin diet rather than in ecosystem baseline δ15N levels. Short-term δ15N changes were previously observed in AP. They were interpreted as varying proportions of fish and krill in their diet, with the higher δ15N value of fish over krill inducing a corresponding increase in predator δ15N signature Two penguin species (EP and MP) showed long-term temporal changes in their feather δ13C values of Antarctic penguins did not change over time, while the carbon isotopic signature of subantarctic and subtropical species presented lower values in the 1970s during the temperature regime shift. Similarly to short-term variations, long-term variations in δ13C values can be explained in two ways, a change in penguin foraging location and/or in primary productivity of the environment. The consistent δ13C changes over species and localities strongly support the latter hypothesis. The ecosystemic explanation includes no major changes in the carrying capacity of high-Antarctic waters in the vicinity of Adélie Land. By contrast, historic and contemporary penguin δ13C values suggest a lower carrying capacity of the pelagic ecosystem in both subantarctic (Crozet) and subtropical (Amsterdam) waters in the 1970s when compared to older and present times. Such a trend was also observed from feather δ13C values of SRP from Kerguelen Islands, i.e. another subantarctic locality from the southern Indian Ocean Click here for additional data file.Table S213C values.Suess effect correction factors (‰) added to feather δ They are presented for each penguin species and each group of sampling years.(DOC)Click here for additional data file.


Immunoblots from buffered solutions or suction blister fluid reveal that propagation of photooxidative damage to other residues such as Tyr or disulfide bonds produces intra- and intermolecular bonds in apolipoproteins A-I, A-II, and B100. Partial repair of phenoxyl tyrosyl radicals (TyrO•) by α-tocopherol is observed with LDL and HDL on millisecond or second time scales, whereas limited repair of α-tocopherol by carotenoids occurs in only HDL. More effective repair of Tyr and α-tocopherol is observed with the flavonoid, quercetin, bound to serum albumin, but quercetin is less potent than new synthetic polyphenols in inhibiting LDL lipid peroxidation or restoring α-tocopherol. The systemic consequences of HDL and LDL oxidation and the activation and/or inhibition of signalling pathways by oxidized LDL and their ability to enhance transcription factor DNA binding activity are also reviewed.There are numerous studies concerning the effect of UVB light on skin cells but fewer on other skin components such as the interstitial fluid. This review highlights high-density lipoprotein (HDL) and low-density lipoprotein (LDL) as important targets of UVB in interstitial fluid. Tryptophan residues are the sole apolipoprotein residues absorbing solar UVB. The UVB-induced one-electron oxidation of Trp produces  Human skin is chronically attacked by deleterious environmental agents such as ultraviolet (UV) light, ionizing radiation, and air pollutants, for example, ozone. These may generate free radicals and other reactive oxygen species (ROS), which—through processes of oxidative stress –3—can agDuring the last three decades, the incidence of cutaneous cancers due to exaggerated exposure to solar UV radiation has markedly increased as a result of outdoor occupations and sun-bathing habits. The spectral limit for the solar UV radiation reaching the Earth is ~295 nm. The biological effects of UV light have led photobiologists to separate the solar UV spectrum into two domains, namely, UVB 295–315 nm) and UVA (315–390 nm). While UVA mainly produces oxidative stress 15 nm and, UVB is Our group—which involves collaboration among dermatologists, biochemists, chemists, and biophysicists from several countries—has spent more than 20 years investigating the molecular and cellular aspects of UV-induced photooxidative stress related to skin pathophysiology. This review deals with several aspects of our contribution to dermatological science and to the photobiology of skin.A full comprehension of all mediators involved in the response of human skin to UV light requires that the molecular bases of the multifocal biological effects of the UV radiation be understood. Not only epidermal cells but also extracellular skin components have been considered in our studies. For example, we have been interested in the effect of UV light on some components of the interstitial fluid which feeds the dermis and epidermis, as this fluid plays an essential role in mediating the transport of nutrients, hormones, essential proteins, and lipids required for cell growth and differentiation. In addition to studies concerning the deleterious effects on cells and other components of the skin, preventive molecular strategies against oxidative stress have also been developed by searching for new families of antioxidants.α-tocopherol (αTocOH) and carotenoids (Car) [Early on, it appeared to us that high-density lipoprotein (HDL) and low-density lipoprotein (LDL) are important UVB targets. First, it has been shown that oxidation of some amino acid residues of lipoproteins such as tyrosine (Tyr), tryptophan (Trp), and lysine (Lys) leads to apolipoprotein alterations and lipid peroxidation . Secondlds (Car) , which cds (Car) . Lastly,ds (Car) .μM, whereas that of the LDL apolipoprotein B100 (apoB100) is on the order of 0.4 μM. Each of apoA-I and apoB100 contains 4 and 37 Trp residues, respectively. As the indole ring of Trp has appreciable molar extinction coefficients of 1500 M−1 cm−1 at 295 nm and even 510 M−1 cm−1 at 300 nm, one may conclude that the most significant fraction of UVB light absorbance by lipoproteins occurs in the 120 μM of HDL3 Trp (HDL3 has 2 apoA-I) compared to the 15 μM of LDL Trp. For comparison, the concentration of the single Trp of albumin in the interstitial fluid is about 160 μM. Consequently, in the ~50 μm thick layer of the epidermis reached by the UVB radiation, the Trp residues of lipoproteins absorb more than half the quantity of light absorbed by albumin. Thus, despite its large concentration, albumin cannot be considered as an effective sunscreen for the lipoproteins. All these data support the contention that lipoproteins must be considered as mediators in the overall effects of UVB on human skin through the specific or unspecific interaction of photochemically oxidized HDL and LDL with human skin cells.HDL and LDL like most other macromolecular components of blood can cross vessel walls by a process resembling ultrafiltration. Thus, the interstitial fluid feeding epidermal cells can be considered as a serum ultrafiltrate. Therefore, the concentration of serum proteins in this ultrafiltrate is determined by their molecular size , 12. Tha3 with UVB light, Trp residues of apoA-I and apoB100 are readily destroyed. The quantum yields of Trp photolysis are 5 × 10−4 and 2 × 10−3 for LDL and HDL3, respectively. This Trp destruction is accompanied by formation of lipid peroxidation decomposition products as measured by the thiobarbituric acid assay (TBARS) and by the consumption of αTocOH and Car carried by the lipoproteins. On the other hand, neither Trp destruction nor antioxidant consumption is observed when solutions are irradiated with the UVA radiation [Upon irradiation of aerated solutions of LDL and HDLadiation . From UVadiation , demonstadiation . As a coadiation .•Trp) as well as a hydrated electron (eaq) [eaq is scavenged by O2 at diffusion-controlled rate in competition with reactions involving endogenous electrophilic residues  to produce the superoxide anion whose dismutation produces H2O2, making it a primary product of Trp photolysis [eaq production in one-electron oxidation of Trp residues is the inhibition of 30% of the TBARS upon saturation of the lipoprotein solutions with a mixture of N2O/O2 (80/20 v/v). This transforms approximately 80% of the eaq (hence of •O2−) into strongly oxidizing •OH radicals. The •OH radicals react nonspecifically with all constituents of HDL or LDL, not only unsaturated lipids but also most residues of apolipoproteins including Trp itself, and a 50% increase of the rate of Trp destruction is observed upon N2O/O2 saturation [•Trp radical has been shown to be involved in the induction of lipid peroxidation, which occurs during the well-established process of Cu2+-induced LDL autooxidation. The binding of the redox Cu2+ ions in the vicinity of 7 of the 37 apoB100 Trp residues in LDL probably catalyzes the Trp autooxidation [The primary process of Trp residue photolysis by UVB radiation in HDL and LDL is undoubtedly the one-electron oxidation  of the indole ring with formation of a neutral Trp radical (n (eaq) , 15. In otolysis , 16. An turation . Interesxidation .3 composed of two main irregularly distributed proteins: apoA-I (MW: 28 kDa) and apolipoprotein A-II . The remaining proteins comprise less than 10% of the total protein content. On average, 75% of HDL3 particles contain 2 apoA-I and 2 apoA-II, while the rest are devoid of apoA-II. The apoA-II is a dimer of 77 residues connected by a disulphide bond with no Trp, free Cys, His, or Arg.The major HDL fraction in human serum is that of HDL2+-induced oxidation of dialyzed plasma as obtained with isolated HDL3 [•Trp can oxidize intact Tyr residues in LDL [•) through long range electron transfer reactions [eaq and disulphide bond are split by eaq, leading to additional radical formation. All these apolipoprotein radical species  can, in turn, contribute to cross-linking and/or polymer formation during Trp photolysis.It has been long known that free radical formation in proteins can induce their cleavage or cross-linking . This ruted HDL3 . Furtherted HDL3 , 14 suggs in LDL  to produeactions . In HDL,2 at 300 nm, 50 mJ/cm2 at 304 nm, and 1000 mJ/cm2 at 313 nm [stratum corneum transmits about 45% and 30% at 313 nm and 304 nm, respectively, the UVB absorbed in the ~50 μm of the epidermal layer [3. Thus, considering the light doses used in [3, there is more than enough light at 1 MED to induce lipoprotein alterations in skin similar to those reported in this reference.As oxidized lipoproteins cannot properly perform their biological functions , these rt 313 nm . Since tal layer  at 1 MED used in , for exa3 fraction and the 37 Trp residues of apoB100 absorb practically 80% as much light as absorbed by the single Trp residue of human serum albumin (HSA). Hence, the HDL and LDL of the suction blister fluid or of a “reconstituted fluid” based on protein concentrations reported in [in vitro [in vivo [A convenient stratagem for gathering the interstitial fluid feeding the dermis and epidermis is to collect blister fluid (0.7 mL) from 2 cm diameter blisters formed by mild suction (−175 mm Hg). In our studies, the methodology followed to demonstrate the oxidative modifications of apolipoprotein by a UVB stress in neutral buffered aqueous solutions has been applied to the suction blister fluid gathered before irradiation . The 8 Torted in , 12 may orted in . Althougin vitro  and in v[in vivo , it is nThe TBARS formation as well as the structural modifications of apolipoproteins and HSA in the suction blister fluid are induced by UVB irradiation of skin at doses well below 1 MED see  legend. It is of note that, in contrast to buffered solutions of purified lipoproteins , irradiaαTocOH and Car at low absorbed doses of UVB irradiation cannot be attributed solely to their photobleaching. Although Car strongly absorb UVA  than those obtained under steady-state irradiation (≪pM) with incident light doses comparable to those used in [•Trp concentrations can be produced rather selectively by pulse radiolysis [•OH and eaq as major radical species with H• atoms as a minor component. Subsequently, •Trp radicals are formed within 50 μs by reaction of Trp residues with •Br2− radical-anions. These radical-anions are selective, mild oxidants formed by scavenging the •OH radicals with Br− anions. Most importantly, •Br2− is the sole radical formed in N2O-saturated solutions, while both •Br2− and •O2− radical-anions are simultaneously produced at almost equal yields in air- or O2-saturated solutions.The investigation of kinetics in such reactions requires the production of  used in , 15, 25.diolysis , a fast λmax) as well as molar extinction coefficient (εmax) at λmax, of the •Trp, TyrO•, and α-tocopheroxyl (αTocO•) radicals have been measured in the UV-visible spectral regions. With these parameters kinetics of their formation and/or disappearance have been determined in lipoprotein aqueous solutions on time scales extending from microseconds to seconds. The λmax characteristics for •Trp, TyrO•, and αTocO• radicals are 520 nm, 410 nm, and 430 nm, respectively, while the corresponding εmax are 1750 M−1 cm−1, 2700 M−1 cm−1, and 7100 M−1 cm−1, respectively. The αTocO• radical absorbance also presents a shoulder at 410 nm with εmax = 4500 M−1 cm−1 [2O-saturated LDL and HDL solutions following oxidation with •Br2− radical-anions. While the repair of •Trp by Tyr residues with formation of TyrO• radicals takes place on a time scale of a few hundreds of μs [• radicals are repaired by αTocOH on longer time scales with formation of the αTocO•. An unrepaired population of •Trp species remains stable for more than 2.5 s. For the repairable damage, the repair reaction can be schematized asαTocOH. In oxidized LDL, αTocO• radicals formation is already completed after 2.5 ms with no indication of remaining TyrO• transient absorbance  not repaired by superoxide radical-anions can be repaired by HSA-bound quercetin with formation of •Q but to a much lesser extent in LDL than in HDL [The transient absorbance spectra observed in Oradicals , 4b), a, a2-saturadicals  observedn in HDL .The extensive repair of oxidative damage to lipoproteins by QH is particularly interesting as QH exists in plasma as conjugated derivatives with antioxidant activities comparable to or exceeding that of unconjugated QH .2+-induced LDL oxidation model [αTocOH [2+-induced LDL oxidation model [Several recent studies suggest that newly synthesized flavones such as 3-alkyl-3′,4,5,7-tetrahydroxyflavones  or hydroon model  or in re [αTocOH . Becauseon model . It is kon model . Thus, ton model .The present review illustrates that photooxidation and autooxidation of serum lipoproteins share common mechanisms although they occur on quite different time scales. These involve the direct oxidation of Trp residues with propagation of damage to other residues such as Tyr and induction of lipid peroxidation leading to formation of multiple TBARS. These, in turn, can react with free amino groups of the apolipoproteins. For example, aldehydic end products of lipid peroxidation react with Lys residues responsible for LDL binding to their cell receptors. Furthermore, both oxidation modes lead to consumption of vitamin E and Car, the natural antioxidants that lipoproteins carry in human serum. A major difference between photo- and autooxidation is that HDL is strongly altered by light, owing to its large excess in the interstitial fluid as compared to LDL. As a result, this oxidized HDL cannot properly fulfil one of its most important functions, the protection of LDL from autooxidation . FurtherBeside systemic effects, lipoprotein oxidation induces or impairs numerous crucial cell physiological responses. For instance, we have shown that oxidation of HDL by various sources of oxidative stress decreases cholesterol efflux from human cultured fibroblasts. The reduced ability of HDL to remove intracellular cholesterol pools and to bind to their receptors has been attributed to apoA-I and apoA-II alterations involving Lys and Trp residues .κB, AP, and STAT1/3  signalling by oxidized LDL in cultured human fibroblasts is an excellent system to illustrate these properties as these cells have a wide range of biological responses to this hormone . As show factors . Furtherormation .αTocOH and Car consumption after propagation of initial radical damage from Trp residues to other apolipoprotein residues as well as to the lipid core. This review illustrates that, in addition to systemic effects, UVB-induced alterations of proteins of the interstitial fluid may make consequential contributions to inflammation and degenerative processes of skin exposed to UVB attack. Moreover, the local and systemic perturbations reported here concern normal skin. They may also contribute to the mechanism of action and long-term adverse effects observed with the UVB phototherapy of chronic inflammatory skin diseases such as psoriasis and atopic dermatitis [The main reasons for the paucity of studies addressing the effects of UVB on the interstitial fluid—as compared to the innumerable ones on skin cells—are probably due to the difficulties in obtaining samples large enough through suction blister fluid collection compared to ready accessibility of skin cells. The analysis of the effects of UVB radiation on major lipoproteins  of the interstitial fluid, which bathes the epidermis, demonstrates that to achieve a full understanding of the skin aging process, this medium should not be ignored. Further, the need for such understanding is supported by data which have established the primary processes leading to rmatitis , 50.


A hallmark of Alzheimer's disease (AD) is the rearrangement of the β-amyloid (Aβ) peptide to a non-native conformation that promotes the formation of toxic, nanoscale aggregates. Recent studies have pointed to the role of sample preparation in creating polymorphic fibrillar species. One of many potential pathways for Aβ toxicity may be modulation of lipid membrane function on cellular surfaces. There are several mutations clustered around the central hydrophobic core of Aβ near the α-secretase cleavage site . These point mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimer's disease pathology with plaques and tangles. We investigated how these point mutations alter Aβ aggregation in the presence of supported lipid membranes comprised of total brain lipid extract. Brain lipid extract bilayers were used as a physiologically relevant model of a neuronal cell surface. Intact lipid bilayers were exposed to predominantly monomeric preparations of Wild Type or different mutant forms of Aβ, and atomic force microscopy was used to monitor aggregate formation and morphology as well as bilayer integrity over a 12 hour period. The goal of this study was to determine how point mutations in Aβ, which alter peptide charge and hydrophobic character, influence interactions between Aβ and the lipid surface. While fibril morphology did not appear to be significantly altered when mutants were prepped similarly and incubated under free solution conditions, aggregation in the lipid membranes resulted in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also had a variable ability to disrupt bilayer integrity. The neuropathological and neurochemical hallmarks of Alzheimer's disease (AD) include: synaptic loss and selective neuronal cell death; decreases in markers for certain neurotransmitters; and abnormalities in neurons and their processes (neurofibrillary tangles comprised of Tau and dystrophic neurites) as well as in the extracellular space  in vitroIt has been well established that many amyloid forming peptides have the ability to aggregate into a variety of morphologically distinct and stable fibril structures A potential environmental factor influencing Aβ aggregate formation is the presence of lipid surfaces. In particular, it has been hypothesized that a potential pathway for Aβ toxicity may lie in its ability to modulate lipid membrane function. Thus, elucidating the interaction between Aβ and membrane lipids could be critical in understanding potential pathways of Aβ toxicity, especially given the results of studies demonstrating that changes in membrane composition occur in AD in vitroThere are several point mutations associated with familial forms of AD. We were particularly interested in point mutations clustered around the central hydrophobic core of Aβ  . With onoC and sampled at various times to check for the presence of fibrils. To control for the role of sample preparation and solution conditions, which have been shown to result in distinct polymorphic fibrillar structures of Wild Type Aβ Ex situ AFM images and height profiles of fibrils formed by Wild Type, Arctic, Italian, Iowa, or Flemish Aβ (1–40) are shown in To compare the resulting morphological features of fibrils formed by Wild Type and the mutant forms of Aβ under free solution conditions (without the presence of a lipid surface), 20 µM solutions of Wild Type, Arctic, Italian, Iowa, or Flemish Aβ (1–40) were incubated at 37Supported bilayers on mica used in this study were produced through the fusion of total brain lipid extract (TBLE) vesicles . The fusExperiments exposing defect-free TBLE bilayers to freshly prepared Wild Type Aβ (1–40) verified previously reported observations When TBLE bilayers were exposed to a freshly prepared aliquots of 20 µM solution of Arctic Aβ (1–40), small oligomeric aggregates similar in morphology to those associated with Wild Type Aβ (1–40)  appearedWe next investigated the interaction of Italian Aβ (1–40) with supported TBLE bilayers . Upon exUpon exposure of a supported TBLE bilayer to a 20 µM solution of Iowa Aβ (1–40), a large number of discrete oligomeric aggregates appeared on the lipid surface within 2–3 hours . Like olWhen TBLE bilayers were exposed to freshly prepared aliquots of 20 µM solutions of Flemish Aβ (1–40) via injection into the fluid cell, oligomeric aggregates were not typically observed for 3–5 hours . Once olWe next compared measurable morphological features of oligomer and fibrillar aggregates of Wild Type and mutant forms of Aβ (1–40) formed in the presence of supported TBLE bilayers . To obtaThe average height above the surface along the contour of elongated, fibrillar aggregates was also determined . Unlike 2 . Despite different aggregate types (or lack thereof) being associated with regions of bilayer disruption for Wild Type or mutant forms of Aβ (1–40), there was no significant differences in the measured RMS roughness of these regions when comparing each mutant peptide to Wild Type.We next used image processing software to analyze the extent of bilayer disruption. A freshly formed bilayer had a root mean square (RMS) surface roughness of 0.24±0.09 nm measured over a total of 148 µm2 . To prev2 (2 . LikewisAlthough the resulting surface roughness associated with bilayer disruption by Wild Type or mutant forms of Aβ (1–40) was similar in magnitude after 10 hours of exposure, the fraction of the bilayer surface area exhibiting some amount of increased roughness varied   peptides with supported TBLE lipid bilayers with a focus on aggregate morphology and bilayer disruption. Point mutations have been shown to promote protein aggregation by destabilizing the native, globular state of a protein Results presented here highlight the potential role electrostatic and hydrophobic properties of Aβ play in its ability to bind, insert, and potentially disrupt lipid membranes. The Arctic mutation replaces a polar, negatively charged glutamic acid with a nonpolar, neutral glycine. The Arctic mutation also represents a change in the hydropathy index The central peptide region, where the mutations studied here are located, has been implicated as being important in Aβ aggregation and fibril structure. EPR studies of Aβ fibril structure using site-directed spin-labeling demonstrated that this central region is sandwiched between two more highly ordered regions in the fibril structure nd residue of Aβ. In both cases, a negative charge is removed, indicating that this residue may experience specific electrostatic interactions with lipids that influence its ability to form distinct fibrillar polymorphs. However, it appears that the exact composition of the bilayer and the peptide preparation protocol could also play a role in this interaction, as the more highly curved fibril structures have been observed for Wild Type Aβ (1–40) on TBLE bilayers by other investigators A striking feature of many amyloid forming peptides is their ability to form more than one stable fibril structure Synthetic Wild Type, Arctic, Italian, Iowa, and Flemish Aβ (1–40) peptides were obtained from AnaSpec Inc. . All peptides were prepared in the same manner Total brain lipid extract (porcine) was purchased from Avanti Polar Lipids  in a lypholized state and was resuspended in PBS (pH 7.3) at a concentration of 1 mg/ml. Using an acetone/dry ice bath, bilayers and multilayer lipid sheets were formed by five cycles of freeze-thaw treatment in situ. Once a 40×40 µm patch of defect-free bilayer was formed, the cell was flushed to remove vesicles remaining in solution. Only defect-free bilayers that were 40×40 µm were used for studies with Aβ. Next, 40 µL of 20 µM of freshly prepared Aβ (1–40) solution in PBS was added via the channels in the fluid cell.In situ AFM experiments were performed with a Nanoscope V MultiMode scanning probe microscope  equipped with a sealable fluid cell and a closed-loop vertical engage J-scanner. Images were taken with V-shaped oxide-sharpened silicon nitride cantilever with a nominal spring constant of 0.5 N/m. Scan rates were set at 1 to 2 Hz with cantilever drive frequencies ranging from ∼8–10 kHz. Concentrated TBLE vesicle solution was added to the cell in 40 µL aliquots by the hanging drop method and allowed to fuse Image analysis of all AFM images was performed using Matlab equipped with the image processing toolbox . Physical dimensions of most aggregates were measured automatically in this way: 1) Images were imported into Matlab. 2) Images were flattened to correct for curvature due to the imaging process. 3) Flattened images were converted into binary maps of aggregate locations by using a height threshold (set at ∼1 nm). This was accomplished by assigning values of 1 to any pixel of the image that represented a height above the threshold and assigning a value of 0 to any pixel corresponding to a height below the threshold. 4) The binary map was used to locate aggregates within the original AFM image using pattern recognition algorithms. 5) Once a discreet aggregate was located, dimensions  were automatically measured. Each aggregate was also assigned an individual number so that aggregates chosen based on specific measured properties could be located, allowing us to verify that chosen dimensions corresponded to specific aggregate types. In this way large data sets were automatically constructed that could be used to keep track of thousands of individual aggregates and sorted based on specific dimensional characteristics. Based on inspection of individual representative aggregate types and verified using our tracking system, we assigned specific dimensional characteristics to specific aggregate types. Oligomers were defined as 2–10 nm in height with an aspect ratio (longest distance across to shortest distance across) less than 2, indicating a globular structure. Fibrils were defined as aggregates greater than 3 nm in height that had an aspect ratio greater than 2.5. For determination of fibril contour length and end to end distance, individual objects in an AFM image were identified by height thresholding and filtering based on aspect ratio. The software then incorporated a fast parallel thinning algorithm Volume measurements were partially corrected for error associated with the finite size of the AFM probe based on geometric models


The systematic investigation of susceptibility-induced contrast in MRI is important to better interpret the influence of microvascular and microcellular morphology on DSC-MRI derived perfusion data. Recently, a novel computational approach called the Finite Perturber Method (FPM), which enables the study of susceptibility-induced contrast in MRI arising from arbitrary microvascular morphologies in 3D has been developed. However, the FPM has lower efficiency in simulating water diffusion especially for complex tissues. In this work, an improved computational approach that combines the FPM with a matrix-based finite difference method (FDM), which we call the Finite Perturber the Finite Difference Method (FPFDM), has been developed in order to efficiently investigate the influence of vascular and extravascular morphological features on susceptibility-induced transverse relaxation. The current work provides a framework for better interpreting how DSC-MRI data depend on various phenomena, including contrast agent leakage in cancerous tissues and water diffusion rates. In addition, we illustrate using simulated and micro-CT extracted tissue structures the improved FPFDM along with its potential applications and limitations. Furthermore, leakage of contrast agent in tumors with BBB disruption causes additional T1 and T2* shortening with subsequent distortion of DSC-MRI signal profiles The passage of paramagnetic or superparamagnetic contrast agents (CA) through brain tissue induces a measurable drop in Tpk for normal brain and tumor In order to better understand susceptibility-based image contrast, several theoretical For realistic complex tissues, the MC method needs to track the diffusion of a large number of spins to capture complex structural features, which in turn can increase the computation time. As an alternative, the Bloch-Torrey partial differential equation describing the transverse magnetization can be directly solved using finite difference methods (FDM). This approach has been previously shown to improve the computational efficiency of such simulations In this study, we propose to evaluate the combination of the finite pertuber and finite difference methods, termed the FPFDM, as a tool for modeling susceptibility based contrast mechanisms. Such an approach leverages the strengths of the FPM, for computing magnetic field perturbations for arbitrarily shaped structures, and the FDM, for efficiently computing the resulting MRI signal evolution. The accuracy of the FPFDM is validated by comparison to traditional Monte Carlo methods. The potential of the FPFDM to compute DSC-MRI signals arising from realistic three-dimensional cellular and vascular models as well as micro-CT based renal angiograms is demonstrated. Going forward, the FPFDM provides a useful tool with which to investigate the influence of vascular morphology, contrast agent kinetics and extravasation on DSC-MRI signals.In this section, we first describe a new approach for generating more realistic, three-dimensional tissue structures that can be used for the systematic investigation of DSC-MRI signals arising from heterogeneous tissues. We then describe the computation of the appropriate magnetic field perturbations and the associated MR signal evolution, including the influence of water diffusion, using the FPFDM.3 voxels. The motivation for exploring whether cellular properties influence DSC-MRI data originates from previous reports demonstrating that contrast agent extravasation and compartmentalization around cells can induce measurable and dynamic changes in gradient echo acquired signals in vivo cellular distribution and shape. In particular, packed spheres intrinsically provide no means for modeling orientation heterogeneity and are unable to achieve cellular densities that approximate those found in vivo. To overcome these limitations we explore here the use of randomly packed ellipsoids Tissue structures consisting of cells, vessels or both were simulated in a 3D volume sampled with NTypically, randomly oriented cylinders are used to explore susceptibility contrast mechanisms V indicating whether a given point within the simulation volume lies inside or outside the simulated compartments, i.e.:All simulated tissue structures were represented by a binary function To further illustrate the versatility of the FPFDM, in addition to the simulated structures, micro-CT was used to create a three-dimensional rendering of a murine kidney vasculature perfused with Microfil . Following perfusion and fixation in 10% neutral buffered formalin, the kidney was scanned in a microCT50 . Cross-sectional images of the entire kidney were acquired with an isotropic voxel size of 5.0 µm using an energy of 55 kVp, 200 µA intensity, 700 msec sample time, and 1000 projections per rotation using the manufacturers 1200 mg HA/ccm beam hardening correction algorithm in a 10.24 mm field of view. Using the manufacturer’s software, we assembled individual slices into a z-stack and contrast-filled vessels were segmented from soft tissue by applying a threshold of 260 mg HA/ccm  and a three-dimensional Gaussian noise filter with sigma 2.3 and support of 4. The resulting binary three-dimensional reconstruction of the vasculature was then subdivided into MR voxel size sections using in-house Matlab codes  and used as an input for the FPFDM simulation.V, the B0 field components, and the susceptibility difference (Δχ) between tissue compartments. The magnetic field shift computed by the FPM is:V is a binary tissue structure, The magnetic field perturbations induced by susceptibility variations within the simulated volume were computed using the FPM N3 voxels the discretized solution of the Bloch-Torrey equation for transverse magnetization (M) using the matrix-based FDM is described by:Estimation of the MR signal relaxation induced by the inhomogeneous field gradients requires simulation of proton diffusion. To track the Brownian motion of thousands of protons over a large number of time steps and calculate their phase accumulation, a Monte Carlo (MC) simulation is frequently used A is an N3×N3 diffusion transition matrix containing the tissue structural information given in terms of the jump probabilities , I is an identity matrix with the same size of A, and ∶ represents element-by-element vector multiplication. The Φ(t) term is a 1×N3 vector containing the phase accumulation and relaxation for each voxel at each time step and is given by:6 rad s−1 T−1), Δt is the simulation time step, the subscript k indicates a spatial index, k, and2,kT is the transverse relaxation time at location k within the simulation grid. When a GE sequence is used 2,kT represent the intrinsic tissue 2*T, and in the case of SE it represents the intrinsic 2T. In general, the jump probability from simulation grid a to b is described by:a and b, mP is the permeability of the membrane when a and b are in different compartments, fc is the free concentration of water. The explicit form of the 1D transition matrix can be found in 0M is estimated by summing the magnetizations over all grid points at a particular t and is given by:t = TE using:t = TE/2. This model is designed to handle cases where the three tissue compartments within a voxel can have different intrinsic transverse relaxation times. By updating 2,kT in For spin echo imaging, the phase was inverted at 1T, 2T, etc) were selected from values measured in previous MRI, PET and CT brain tumor studies as previously described To demonstrate the potential of the FPFDM to simulate DSC-MRI signals arising from the dynamic passage of contrast agent through the vascular and extravascular spaces, such as would occur in brain tumors with a breakdown of the blood brain barrier, we used tissue structures composed of ellipsoids packed around fractal based vascular network. Concentration time curves were sampled using 150 time points for a total of 9 minutes. The arterial input function (AIF) was generated as previously described 2*) and spin-echo (ΔR2) relaxivity on perturber (vessel) size has previously been characterized using Monte Carlo techniques −7, cylinder volume fraction (pV) = 2%, B0 = 1.5T, water diffusion coefficient D = 10−5 cm2/s, simulation time step Δt = 0.2 ms, GE TE = 60 ms and SE TE = 100 ms), we computed the vessel size dependence of ΔR2* and ΔR2 averaged over all cylinder arrangements. The computed ΔR2* and ΔR2 values show negligible changes as the number of averaged structures increases beyond 10. As shown in For validation, FPFDM and Monte Carlo based MRI signals were computed and compared for models consisting of randomly orientated cylinders and packed spheres. The dependence of gradient-echo  volume fraction for the FPFDM was compared to that for the MC method 2* for each of the two sphere packing techniques computed by both the MC and FPFDM, using Δχ = 5×10−8, B0 = 1.5T, D = 10−5 cm2/s, Δt = 0.2 ms, GE TE = 40 ms, and simulation universe size = (0.5 mm)3. The FPFDM results were obtained by averaging the MR signal for 5 different sphere distributions for each packing and cell volume fraction using a simulation grid size of 1283. In contrast, the MC method involves tracking 15,000 random walks for each cell volume fraction and the redistribution of the spheres prior to each random walk. The FPFDM results are in excellent agreement to those produced from the MC methodology.To further validate the accuracy of the FPFDM we also computed ΔR2* values obtained from 2* values. For both vessel sizes the computed FPFDM ΔR2* values converge to the corresponding reported values To investigate the convergence of the FPFDM for randomly distributed structures such as those used above, ΔR2*and ΔR2 for 3D cellular models consisting of packed spheres or ellipsoids (9 µm radius). To model contrast agent leakage effects relevant contrast agent levels corresponding to a Δχ value that is half the peak value of single dose Gd-DTPA injection was assumed. 2* and ΔR2 values for Δχ = 5×10−8, B0 = 1.5T, D = 10−5 cm2/s, simulation time step Δt = 0.2 ms, GE TE = 40 ms and SE TE = 80 ms.To demonstrate the potential of the FPFDM for investigating the complex susceptibility effects that occur when contrast agent extravasates and compartmentalizes around cells, we computed the volume fraction dependence of ΔR2* and ΔR2 values associated with the ellipsoid-based structures were greater in magnitude than those found with spheres.The highly ordered FCC packing of spheres resulted in the smallest relaxivity, reflecting the more homogeneous magnetic field perturbations and proton phase distributions. Randomly distributed spheres yielded slightly greater relaxivities with a non-linear relationship with packing fraction. Finally, the packed ellipsoids, which better approximate cell shape in vivo, enable higher random non-overlapping packing fractions (>65%), are less ordered and also yielded a non-linear relationship between relaxivity and cell volume fraction. For all cell volume fractions, the ΔR2 and ΔR2* for typical DSC-MRI contrast agent concentrations. For these simulations we generated three different vascular networks within a 1 mm3 volume containing 1283 voxels. 2* and ΔR2 for the three θs considered. For these simulations, Δχ = χm. CA, where χm is the CA molar susceptibility (0.027×10−6 mM−1) 0 = 4.7T, D = 10−5 cm2/s, Δt = 0.2 ms, GE TE = 40 ms and SE TE = 80 ms. All simulated vascular structures incorporate vessel radii ranging from 12 µm to 80 µm with a 2% target vascular volume fraction (vp). The computed relaxation rates were averaged over five different orientations for each simulated vascular network. Using the slope of the ΔR2* dose response curves, kp values ranging from 100–295 (mM–sec) −1 were obtained. For this low vascular volume fraction, the kp values for the more tumor-like vascular trees (higher Δθ) were higher than those in normal trees, up to three fold for this simplified simulation. Similar dependency on branching angle with a reduced susceptibility effect was observed for ΔR2 dose response curves.To illustrate the potential of the FPFDM for modeling the complex geometries of the microvasculature, we used fractal-based branching networks as input to the FPFDM. pC) and extravascular (eC) contrast agent concentration time curves are shown in , both in the presence (KTrans = 0.2 min−1) and absence (KTrans = 0 min−1) of contrast agent extravasation. T10 = 500 ms, T10 = 1000 ms and T10 = 1500 ms, respectively. The corresponding time series for the tissue structures modeled with 15 µm cellular radii are shown in  even at T10 = 1500 ms  −1, respectively.To further illustrate the versatility of the FPFDM, micro-CT images of perfused mouse kidney vasculature  were used as source data for multiple 1 mmin vivo. The FPM can compute the induced magnetic fields around arbitrary microvasculature structures without necessitating any assumptions about the underlying vessel geometry A is either invariant or requires only partial updating for most tissues under consideration, further increasing the computing efficiency of matrix-based FDM that also benefit from optimized MATLAB packages for computations involving large matrices The FPFDM is a novel efficient computational tool combining features of the FP and FD techniques for calculating susceptibility-induced relaxivity changes for realistic simulated or imaging-based 3D vascular and cellular geometries that might be observed A. Since A can be determined at the start of the simulation, a library of diffusion transition matrices, for a range of tissue structures, can be established to increase the computing efficiency. For example, computing a dynamic signal for the same structure only requires loading the transition matrix corresponding to the structure once from the library of diffusion transition matrices.A Gaussian diffusion kernel convolution can also be used to model CA and water diffusion 2* and ΔR2  calculated using appropriate CA diffusion coefficients and permeability across membranes from literature Although simulations in this study are based on a simple 2-compartment model, at the expense of computational time, the same approach to model water diffusion and exchange can be used to model CA diffusion and transport across compartments, yielding a more realistic heterogeneous CA distribution within a voxel. This can be achieved by updating CA concentration for each voxel at each simulation time step using a CA diffusion transition matrix  on relaxation rates. Fractal-based vascular trees better approximate the microvascular network in vivo, but this complex geometry with variable vessel rotation, size distribution, branching angles, and diameters of daughter vessels is very difficult to model and require high resolution to achieve structural details. Our results demonstrate the feasibility of using FPFDM for complex geometries, and suggest that although the generally accepted linear relationship between relaxation rate and CA concentration is reasonable, the proportionality constant The systematic evaluation of fractal-based vascular trees using the FPFDM could shed new insights into the relationship between DSC-MRI relaxation rate and vascular geometry. Furthermore, the use of fractal trees enables the application of well-established flow models 1 and T2* effects as the CA traverses the extravascular extracellular space. For a given T10, the structure with smaller sized cells exhibited higher signal intensity recoveries as compared to that with larger sized cells. The compartmentalization of CA around the larger cells creates stronger magnetic field perturbations and greater relaxation rate changes (T2* effects). In general, as T10 increases, T1 leakage effects will be more pronounced and may dominate any T2* leakage effects, as is the case for the smaller-sized cells. In such cases, the characteristic signal overshoot may be observed (2* leakage effects (2* leakage effects decreasing (and T1 leakage effects increasing) as the cell density increases. Consequently, DSC-MRI data from tumors with tightly packed, smaller-sized cells would likely present with pronounced T1 leakage effects . Given the clinical importance of DSC-MRI signal recovery characteristics to help differentiate among tumor types in silico study of DSC-MRI signal recovery and its dependence on physiological, pulse sequence and physical parameters is currently under investigation.The FPFDM also provides a computationally reasonable approach for simulating DSC-MRI derived transverse relaxation rates both in the presence and absence of CA extravasation, and restricted water diffusion induced by membrane permeability . The resobserved . For the effects . As showex vivo micro-CT scans of perfused kidney vasculature. The dose-response curves from MRI voxel-sized regions of the kidney vasculature were used to determine the distribution of vascular susceptibility calibration factors, kp, within the kidney. For vascular volume fractions up to 30%, kp values were very heterogeneous .Prior studies have shown the potential and value of incorporating image-based vascular structures into susceptibility simulations ogeneous , with deOne of the limitations of the FPM is the use of FFT to calculate the spatial convolution of the vascular structure with the finite perturber magnetic field perturbation. As demonstrated in The FPM was designed to compute the magnetic field changes from a single finite perturber convolved with a digitized tissue structure array, and hence this approach cannot be used for arbitrary magnetic susceptibility distributions. While methods capable of computing arbitrary susceptibility distributions are more comprehensive and should be explored t) due to increased resolution or decreased perturber size (Δx), in order to satisfy the constraint that the jump probability (see Eq. 6) should be ≤1/6. This is because when the number of spins leaving a given node exceeds the number that was present, the FDM becomes unstable The sampling of tissue structures at higher resolution increases the computational accuracy of the FPM but it comes at the expense of computational time. Such increases in resolution would also add to computational time needed to compute the MR signal using the FDM. This is particularly true if a need arises to reduce the simulation time step (ΔThe FPFDM is an alternative computational tool for efficiently modeling susceptibility induced MR signal relaxation from complex perturber geometries. In general, the proposed FPFDM could be used to investigate the influence of realistic tissue microstructure on any susceptibility based contrast mechanism such as vessel size imaging, BOLD contrast, single cell imaging, and quantitative susceptibility mapping. Currently, the proposed method is being utilized to assess the influence of geometrical, morphological and physiological parameters of microvessels and cells on susceptibility induced MR relaxation rate changes. Such studies should shed new insights into DSC-MRI contrast mechanisms and enable the systematic evaluation of how acquisition and analysis methods influence the measurement of reliable perfusion parameters in brain and tumor tissue.


Alternaria alternata. This pathogen produces ACT-toxin, which induces necrotic lesions on fruit and young leaves, defoliation and fruit drop in susceptible genotypes. It is a strong concern for triploid breeding programs aiming to produce seedless mandarin cultivars. The monolocus dominant inheritance of susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. Bulk segregant analysis coupled with genome scan using a large set of genetically mapped SNP markers and targeted genetic mapping by half tetrad analysis, using SSR and SNP markers, allowed locating a 3.3 Mb genomic region linked to ABS resistance near the centromere of chromosome III. Clusters of resistance genes were identified by gene ontology analysis of this genomic region. Some of these genes are good candidates to control the dominant susceptibility to the ACT-toxin. SSR and SNP markers were developed for efficient early marker-assisted selection of ABS resistant hybrids.Genetic analysis of phenotypical traits and marker-trait association in polyploid species is generally considered as a challenge. In the present work, different approaches were combined taking advantage of the particular genetic structures of 2n gametes resulting from second division restitution (SDR) to map a genome region linked to Alternaria brown spot (ABS) resistance in triploid citrus progeny. ABS in citrus is a serious disease caused by the tangerine pathotype of the fungus  Alternaria alternata (Fr.) Keissl, which carries a gene cluster (ACTT) located in a small (<2.0 Mb) conditionally dispensable chromosome responsible for ACT-toxin biosynthesis  can be C. clementina  and three triploid populations derived from 2x × 4x crosses  were phenotyped to compare the expected and observed proportions of resistant and susceptible genotypes, to confirm the monolocus inheritance and dominance of the ABS susceptibility. For the ‘Aa’ × ‘AA’ cross, all segregation progeny are expected to be susceptible to ABS , whereas for the ‘aa’ × ‘aa’ cross, all segregation progeny are expected to be ABS resistant (‘aaa’). In case of the ‘aa’ × ‘AAaa’ and ‘Aa’ × ‘AAaa’ crosses, the resistant and susceptible proportions depend on heterozygosity restitution (HR) from the tetraploid parent to the progeny, which varies between 0.55 and 0.66 depending on the double-reduction frequency .(DOCX)Click here for additional data file.


Euphausia superba is a predominant species in the Southern Ocean, it is very sensitive to climate change, and it supports large stocks of fishes, seabirds, seals and whales in Antarctic marine ecosystems. Modern krill stocks have been estimated directly by net hauls and acoustic surveys; the historical krill density especially the long-term one in the Southern Ocean, however, is unknown. Here we inferred the relative krill population changes along the West Antarctic Peninsula (WAP) over the 20th century from the trophic level change of Antarctic fur seal Arctocephalus gazella using stable carbon (δ13C) and nitrogen (δ15N) isotopes of archival seal hairs. Since Antarctic fur seals feed preferentially on krill, the variation of δ15N in seal hair indicates a change in the proportion of krill in the seal's diets and thus the krill availability in local seawater. For the past century, enriching fur seal δ15N values indicated decreasing krill availability. This is agreement with direct observation for the past ∼30 years and suggests that the recently documented decline in krill populations began in the early parts of the 20th century. This novel method makes it possible to infer past krill population changes from ancient tissues of krill predators.Antarctic krill  Over the past 50 years, the West Antarctic Peninsula (WAP) has experienced rapid regional warming associated with significant sea-ice and krill stock reductions 13C) and nitrogen (δ15N) isotope signatures of hair and whisker have been used to infer animal diets Stable isotope analyses of animal tissues are increasingly recognized as a powerful tool for quantifying animal's foraging habitat and trophic level 15N of Antarctic fur seal hair. Since Antarctic fur seals feed preferentially on krill 15N) is controlled by their dietary compositions. Therefore, a shift in δ15N of seal hair indicates a change in the proportion of krill in the seal's diet, and such a change should be a reflection of the availability of krill in local seawater.In this study, we propose a novel stable isotope methodology for deducing long-term relative krill population dynamics based upon the trophic level of krill predators, and inferred the relative krill population change of the 20th century using the δThe authors have declared that there is no ethics problem because our samples were extracted from lake sediment.137Cs dating on sediments, which was verified as a reliable dating method in previous studies 137Cs signal in sediments, we determined the depth of 15.5 cm corresponds to 1954 AD, the beginning of the 137Cs sedimentation. The 137Cs peaks at depths of 11.5, 8.5, and 4.5 cm correspond to 1965 AD, 1977 AD and 1988 AD, respectively . The sediment core HN1, 35.5 cm long, was retrieved from a lake catchment near a large fur seal colony. Usually, there are many female fur seals and only one male seal in the colony in summer, and seal remains such as excrements and hairs were shed to the lake and deposited into the sediments. The top 25.5-cm layer of HN1 contains seal excrement and seal hairs and is identified as seal excrement deposition; the section below 25.5 cm is littoral deposition with black basaltic sand. Here we focus on the top 25.5 cm. The chronology of HN1 was established by ectively . The botectively . These r13C and δ15N by continuous-flow direct combustion and mass spectrometry using a Europa 20/20 SL isotope-ratio mass spectrometer at Utah State University. Precision is ±0.15‰ for δ15N and ±0.10‰ for δ13C. δ15N in sediments were determined by Finnigan-MAT-251 mass spectrometer at Institute of Soil Science, Chinese Academy of Sciences, and the precision is ±0.20‰. Results are presented in δ (‰) and expressed relative to air for δ15N and Vienna Pee Dee Belemnite (VPDB) for δ13C according to the equation: δ (‰) = [(Rsample−Rstandard)/Rstandard] ×103, where δ (‰) represents the δ15N or δ13C value, Rsample is the isotopic ratio of the sample, and Rstandard of the air and VPDB.HN1 was sectioned at 0.5-cm intervals. Seal hairs were hand picked, cleaned using 2∶1 chloroform: methanol solution, dried at 40°C, and then weighed accurately into tin capsules. Seal hairs were analyzed for δRegression analysis and t-test for the null hypothesis of a zero slope were performed on the time-series data of isotope values (n = 50) obtained from sediments and seal hairs in HN1 using SPSS 16.0.13C values of seal hairs range from −22.87‰ to −20.17‰ with a mean of −21.18‰  is larger than that in 1958–1997 . The δ15N values of sediments are as enriched as 19.37‰, exceeding the normal δ15N values of marine mammals; the abnormally enriched δ15N values of sediments could be the result of large fractionation effects during ammonia volatilization from seal excrement 15N signature in hair, however, is not subject to this fractionation effect because hair is keratinized. The δ15N signatures of both sediments and hairs become significantly enriched over time . The 1.68‰ enrichment of δ15N in seal hairs indicates an obvious change of fur seal diets over time.The δ −21.18‰  and showf 14.67‰ . The δ15f 11.24‰ ; There i13C values vary with latitudes and along an inshore/offshore gradient, and typical values for Antarctica fur seal are −23‰ ∼ −19‰ in Antarctica and −19‰ ∼ −16‰ in subantarctica 13C values in HN1 clearly indicate foraging of fur seal in Antarctica during breeding seasons in the study area although we cannot rule out possible inshore/offshore influence.Due to the different metabolic rates of various tissues, stable isotope values reflect trophic levels at different time scales, from days for plasma and excrement to weeks and months for feathers and hairs 15N values in seal hair with various percentages of krill vs. fish in seal diet using endmember bulk nitrogen isotope values, and the result shows that seal δ15N becomes enriched as krill proportion in seal diet decreases  anomaly in Southern Ocean (50°S) also shows an obvious increasing trend for the 20th century 2 = 0.82, p<0.001) suggests that the inferred decreasing krill population is linked with warming ocean and declining sea ice extent.The significantly enriching trend of the δ15N values in the sediment core HN1 could be excluded. First, an enriching baseline (δ15N of the primary producer) may have contributed to the enriching seal δ15N values. The δ15N values of the Southern Ocean primary producer (diatom) are enriched during cold periods and depleted during warm periods 15N values of local primary producer were expected to be depleted. Therefore, the enriching baseline explanation seems unlikely. Second, an increasing intra-specific competition could also have led to the rising trophic level of seal in this study. The seal populations inferred from HN1 show rapid increase between 1955 and 1965 due to the ban of sealing 15N values do not show corresponding ‘rapid’ enrichment, indicating that the intra-specific competition is not responsible for seal dietary changes. Or it is also possible that the increases in seal populations might still have been too low during that time for krill stocks to be limited. Thus, the rising fur seal trophic levels are caused mainly by reduced krill availability and dietary changes.Two other plausible explanations for the enriching δ15N values in Antarctic fur seal hair can be used as an indicator for seal trophic level changes and the potential relative krill population dynamics. And long-term δ15N series from krill predators could provide vital information about the relative krill abundance in the Holocece epoch and its responses to abrupt or transitional climatic and environmental changes.Krill is the major consumer of diatom primary producers, the major food source for upper trophic predators, and the major fishery resource in the Southern Ocean. Krill density is of critical importance for the apex predator, and its massive biomass underpins the entire Southern Ocean ecosystems. Therefore finding a potential method for inferring krill populations change would be highly beneficial for research and management purposes. We could provide only a single core (HN1) for stable isotope analyses at present due to the limits of logistics. Further core analysis in other areas in Antarctica could be performed in future by international collaborations in field. Nevertheless, the results from HN1 do show that δ


Two vertebrate UNC-45 homologues, unc45a and unc45b, affect distinct stages of muscle differentiation when knocked down in cell culture and are functionally redundant in vitro. UNC-45 proteins are members of the UCS (UNC-45/CRO1/She4p) protein family that has been shown to regulate myosin-dependent functions from fungi to vertebrates through direct interaction with the myosin motor domain. To test whether the same functional relationship exists between these unc45 paralogs in vivo, we examined the developmental phenotypes of doubly homozygous −/−unc45b; −/−unc45a mutant zebrafish embryos. We focused specifically on the combined effects on morphology and gene expression resulting from the zygotic lack of both paralogs. We found that −/−unc45b and −/−unc45b; −/−unc45a embryos were phenotypically indistinguishable with both mutants displaying identical cardiac, skeletal muscle, and jaw defects. We also found no evidence to support a role for zygotic Unc45a function in myoblast differentiation. In contrast to previous in vitro work, this rules out a model of functional redundancy between Unc45a and Unc45b in vivo. Instead, our phylogenetic and phenotypic analyses provide evidence for the role of functional divergence in the evolution of the UCS protein family.Since the majority of protein-coding genes in vertebrates have intra-genomic homologues, it has been difficult to eliminate the potential of functional redundancy from analyses of mutant phenotypes, whether produced by genetic lesion or transient knockdown. Further complicating these analyses, not all gene products have activities that can be assayed  UNC-45/CRO1/She4p) protein family are present in single-celled eukaryotes and metazoa in vitro as both a direct myosin chaperone and an Hsp90 co-chaperone Members of the UCS  is found only in the central 2 µm of the filament, while MHC B is found along the majority of the lateral arms unc-54 null mutants), C. elegans muscle thick filaments can be induced to form using only MHC A unc-45 have no effect on thick filament assembly UNC-45 is essential for muscle assembly and function in unc45a and unc45b, whose expression patterns differ dramatically unc45b and hsp90a in zebrafish or Xenopus produce similar phenotypes, including cardiac dysfunction, disordered sarcomeres and paralysis, and regulation of these two genes is interdependent unc45b have been identified in zebrafish (steif) and Xenopus (dicky ticker) unc45b mutant steif has defective myofibrils containing disorganized thick and thin filaments and reduced levels of muscle myosin protein C. elegans for unc-45, overexpression of unc45b in zebrafish leads to severely disorganized myosin filaments, indicating the need for tight regulation of unc45b expression Vertebrates have two UNC-45 genes, In vitro, both Unc45a and Unc45b have the capacity to fold the myosin motor domain in an Hsp90/ATP-dependent manner unc45a has a broad expression pattern with transcripts present in the brain, pharyngeal arches, and retina, although no significant expression is seen in the striated muscles of the trunk or heart unc45a (kurzschluss) results in aortic arch defects, which is not easily explained via a role in myosin regulation unc45a mutants develop an arteriovenous malformation involving aortic arches 5 and 6 that causes blood to shunt from the primary head sinus back into the heart. In contrast to unc45b, myofibril organization and thick filament assembly appear normal in unc45a mutants. These observations have led to the hypothesis that Unc45a may either regulate myosin assembly in non-muscle cells, or contribute to muscle myosin assembly solely in muscle groups other than the trunk or heart.Both Unc45a and Unc45b are expressed in differentiating C2C12 myoblasts and such cells show defects in myogenesis when expression is reduced C. elegans, the single unc-45 gene is responsible for both striated muscle assembly and function through the muscle myosin UNC-54/MHC-B, as well as cytokinesis and cell polarity via the type II non-muscle myosin NMY-2 unc45 mutants in zebrafish in vitro biochemical redundancy toward myosin folding and the unc45a aortic arch phenotype. First, functional redundancy between the two vertebrate Unc45 proteins could be masking a cytokinesis phenotype in either of the unc45 zebrafish mutants. Second, Unc45a contributes to muscle myosin assembly only in the tissues in which it is expressed and only during later stages of development. A subset of specific muscles lacking both isoforms would express a phenotype more severe than that seen in the single unc45b mutant. Finally, given the striking lack of a muscle phenotype in the zebrafish unc45a mutant, Unc45a function is not required for myogenesis in zebrafish, but is required in mouse C2C12 cells. The availability of putative null mutants in both zebrafish homologues provides us a unique opportunity to test these hypotheses and examine developmental redundancy between two members of a highly conserved protein family.In −/−; unc45a−/−unc45b zebrafish embryos with an emphasis on the combined effects resulting from the loss of both paralogs. Of special interest were regions where the expression domains of unc45a and unc45b may overlap, such as in the pharyngeal arches at later stages of development. Our results effectively rule out any functional redundancy between Unc45a and Unc45b in vivo and provide evidence for the role of functional divergence between the UNC-45 proteins during vertebrate evolution.Here we describe the phenotypic characterization of doubly mutant This study was carried out in strict accordance with the recommendations from the Canadian Council for Animal Care. The protocol was approved by the Animal Care and Use Committee of the University of Alberta.steif/unc45b+/sb60 and kus/unc45a+/tr12 strains have been previously described and were generously shared with us steif/unc45b+/sb60, homozygous kus/unc45a+/tr12, and the trans-heterozygous unc45b+/sb60; unc45a+/tr12 animals were used for all experiments. Genotypes were determined using derived cleaved amplified polymorphic sequence (dCAPS) analysis for all experiments that required the identification of either homozygous or heterozygous double mutants, or was conducted at a developmental stage prior to when mutants could be scored phenotypically unc45a−/−, unc45b−/−, and unc45b−/−; unc45a−/− to refer to single and double mutants of the alleles above.Zebrafish were maintained according to standard procedures http://www.ensembl.org/index.html) and aligned using ClustalW2 Phylogenetic analysis was performed using the MEGA5 program in vitro transcription using T7 RNA polymerase (Ambion) and digoxigenin-11-UTP RNA labeling (Roche). Probes  were made from PCR template amplified using gene-specific primers. The 5′ ends of the reverse primers incorporated an RNA polymerase promoter sequence (T3 or T7) to enable RNA production unc45a probe and the unc45b mRNA sequence (the largest span of identity is 11 bases).Antisense RNA probes were synthesized by in situ hybridization analysis was performed essentially as described Whole-mount Embryos were fixed in 2% TCA and washed four times with 0.8% Triton X-100 in PBS (PBS-Tx) for 5 minutes at room temperature. Samples were blocked in 5% BSA in PBS-Tx for 1 hour at room temperature and incubated overnight at 4°C in a 1∶10 dilution of monoclonal antibody MF-20  in blocking solution. Embryos were washed five times with PBS-Tx for 5 minutes at room temperature. Subsequent to a single 5 minute PBS wash, embryos were incubated overnight at 4°C in a 1∶1,000 dilution of anti-mouse Alexa 488 (Molecular Probes) secondary antibody. Antibody was removed by three washes in PBS-Tx for 10 minutes each at room temperature prior to mounting embryos in 3% methylcellulose. Images were captured using a Nikon Eclipse 80i confocal.2, and 70% ethanol. Following a rinse in water, embryos were bleached for 20 minutes in equal volumes of 3% H2O2 and 2% KOH in open tubes on the bench top. To visualize cartilage more readily, tissues were cleared for 20 minutes with gentle rocking using 1 mg/mL trypsin dissolved in saturated sodium tetraborate Alcian Blue stains proteoglycan components of the extracellular matrix −/−; unc45a−/−unc45b mutant . Specifically, no underrepresentation of the −/−; unc45a−/−unc45b genotype was observed, which might have suggested lethality during early developmental stages. Additionally, no evidence for a heterozygous effect was observed such that unc45+/− and unc45+/+ genotypes appeared phenotypically identical in all combinations.We used a genetic approach to examine the functional relationship between −/−unc45b; −/−unc45a and −/−unc45b embryos appear identical  and yolk sac edema by 5 dpf. To a lesser extent, the −/−unc45a mutants also develop cardiac edema, but yolk sac edema does not advance to the degree seen in the other mutants.In regard to gross morphology, the dentical .. Both munc45a and unc45b transcripts have been reported, but expression was not examined in the reciprocal unc45 mutant, which might suggest compensatory regulation between the two homologues unc45a expression in the brain and pharyngeal arch region  display improper angling of the ceratobranchial and ceratohyal cartilages and shortening of the palatoquadrates and Meckel's cartilage , pharyngeal endodermal pouches (nkx2.3), or pharyngeal mesenchyme (nkx3.2)  is based on the observation that many genes required for development have a number of independent functions based on their spatial and temporal expression patterns C. elegans and D. melanogaster. Both genes would have been redundant until mutations accumulated following the duplication event. Duplicate gene pairs have a number of potential fates: become a pseudogene, subdivide the ancestral function at the regulatory or protein level , develop a new function , or a combination of the above. Both vertebrate Unc45b and C. elegans UNC-45 are required for myosin motor domain folding and thick filament assembly and are expressed in similar tissues. This suggests that few changes have occurred in the protein domains and regulatory elements responsible for producing the muscle myosin functions of UNC-45 and Unc45b. As the expression pattern and functions of Unc45a appear to have diverged significantly from those of Unc45b in vertebrates, it is likely that expression domain subfunctionalization and neofunctionalization occurred in the Unc45a copy prior to the branching of the bony fish lineage. Had the new Unc45 function not been established after the divergence of lobe and ray-finned fish, either mammals or teleosts, but not both, would have the gene copy encoding a new Unc45 function. The differential distribution of unc45a and unc45b transcripts suggests that subfunctionalization, which occurred following their duplication, is the likely mechanism for the preservation of Unc45 duplicates in vertebrate genomes.Since mammals and teleosts have two −/−unc45b; −/−unc45a and −/−unc45b mutants are phenotypically indistinguishable both morphologically and at the level of gene expression. No phenotypes were observed in the −/−unc45b; −/−unc45a mutant that were novel and/or more severe than any of those found in the single unc45 mutants as would be expected if unc45a and unc45b were functionally redundant in vivo. Nor did we see any amelioration in the −/−unc45b; −/−unc45a embryos compared to −/−unc45b siblings, suggesting that unc45a is not epistatic to unc45b. Interestingly, our results highlight major differences between mouse cell culture and zebrafish unc45a and unc45b. Unlike findings in mouse C2C12 cells, zebrafish lacking unc45a display no defects in myocyte function or differentiation suggesting that Unc45a function is not required for myogenesis in vivo.In conclusion, we have shown that the zygotic-lethal Figure S1Morphology of 4 dpf unc45 mutants. Wild type siblings ; −/−unc45a ; −/−unc45b ; and −/−unc45b; −/−unc45a  mutants. Blood circulates through the hearts of wild type siblings  and −/−unc45a mutants  but not in those of the −/−unc45b mutants . Cardiac edema is most pronounced in the −/−unc45b  and −/−unc45b; −/−unc45a  mutants and absent in wild type siblings (arrowheads). A fully inflated swim bladder is present only in wild type embryos, absent in −/−unc45b and −/−unc45b; −/−unc45a mutants, and minimally inflated in −/−unc45a mutants (arrows). Somite birefringency is reduced in −/−unc45b and −/−unc45b; −/−unc45a mutants compared to wild type and −/−unc45a embryos. Apostrophes following a letter denote an increased magnification of the same embryo.(TIF)Click here for additional data file.Figure S2Pharyngeal arch formation and patterning of unc45 mutants is consistent with that of wild type siblings. Wild type siblings , −/−unc45a , −/−unc45b , and −/−unc45b; −/−unc45a  embryos. Expression of: hand2 at 30 hpf (a–d), dlx2a at 30 hpf (e–h), nkx2.3 at 36 hpf (i–l), nkx3.2 at 52 hpf (m–p), and sox9a at 48 hpf (q–t). Dorsal , ventral (m–p) and lateral (q–t) views. cb, ceratobranchial; p, pharyngeal arch. Numbers denote pharyngeal arch number.(TIF)Click here for additional data file.Table S1UNC-45 sequences used in phylogenetic analysis.(DOC)Click here for additional data file.


Owing to their unique functions in regulating glucose, lipid and cholesterol metabolism, PPARs (peroxisome proliferator-activated receptors) have drawn special attention for developing drugs to treat type-2 diabetes. By combining the lipid benefit of PPAR-alpha agonists (such as fibrates) with the glycemic advantages of the PPAR-gamma agonists (such as thiazolidinediones), the dual PPAR agonists approach can both improve the metabolic effects and minimize the side effects caused by either agent alone, and hence has become a promising strategy for designing effective drugs against type-2 diabetes. In this study, by means of the powerful “core hopping” and “glide docking” techniques, a novel class of PPAR dual agonists was discovered based on the compound GW409544, a well-known dual agonist for both PPAR-alpha and PPAR-gamma modified from the farglitazar structure. It was observed by molecular dynamics simulations that these novel agonists not only possessed the same function as GW409544 did in activating PPAR-alpha and PPAR-gamma, but also had more favorable conformation for binding to the two receptors. It was further validated by the outcomes of their ADME  predictions that the new agonists hold high potential to become drug candidates. Or at the very least, the findings reported here may stimulate new strategy or provide useful insights for discovering more effective dual agonists for treating type-2 diabetes. Since the “core hopping” technique allows for rapidly screening novel cores to help overcome unwanted properties by generating new lead compounds with improved core properties, it has not escaped our notice that the current strategy along with the corresponding computational procedures can also be utilized to find novel and more effective drugs for treating other illnesses. Diabetes mellitus is a group of metabolic diseases that has been classified as a disease of glucose overproduction by tissues without enough insulin production, or a disease resulting from cells not responding to the insulin in human body Peroxisome Proliferator-Activated Receptor (PPAR) has drawn increased attention as a drug discovery target by regulating glucose and lipid metabolism Recently, new dual agonists have received considerable attention for developing powerful drugs against diabetes. The strategy of dual agonists was aimed to treat both insulin resistance and dyslipidemia Many studies have indicated that computational approaches, such as structural bioinformatics Encouraged by the aforementioned researches in successfully utilizing various computational approaches for drug development, the present study was initiated in an attempt to screen the fragment database for finding new PPAR dual agonists for treating type-2 diabetes. To realize this, the techniques of “core hopping” with glide docking The L-tyrosine analogue GW409544 was obtained by modifying the structure of farglitazar, a dual agonist for both PPARα and PPARγ The representative complex crystal structures of PPARα (PDB ID 1k7l) and PPARγ (PDB ID 1k74) with the same ligand GW409544 All the calculations were carried out on Dell Precision™ T5500 computer with Schrodinger software package The proteins with PDB codes 1k71 and 1k74 were chosen for modeling. In addition to the available knowledge of their 3D  structures, the reasons of selecting the two proteins as receptors are as follows. (1) The two proteins contain the same ligand GW409544 as PPARα and PPARγ do, and their binding affinities with the ligand are also quite similar; however, the former selectivity is about 10-fold weaker than the latter In the process of preparing receptors for modelling, the protein preparation facility www.schrodinger.com) as described in The protein binding-site was identified by the SiteMap tool embedded in Schrodinger Suite 2009 . The 2nd step is to define the “receptor grid file”, which was done in the “Receptor Preparation” panel. The 3rd step is the cores preparation that was operated with the “Protocore Preparation” module to find the cores attaching to the scaffold using the fragment database derived from ZINC During the process of core hopping, the 1st step is to define the possible points at which the cores are attached. It is performed in the “Define Combinations” step from the Combinatorial Screening panel in Schrodinger2009  system minimization and relaxation, the operation was followed by running the 10 ns NPT production simulation and saving the configurations thus obtained in 2ps (picosecond or 10−12 of a second) intervals. All the molecular dynamics simulations were performed with a time step of 2fs (femtosecond or 10−15 of a second).Here, the “Desmond 2.4 Package” The QikProp b ), and apparent MDCK permeability (QPP MDCKc) All the compounds investigated need not the treatment for neutralization before using QikProp because it will be automatically done in QikProp. The normal mode was applied in the program. The property analyses for the partition coefficient (QP logP o/w), van der Waals surface area of polar nitrogen and oxygen atoms (PSA), predicted aqueous solubility  the crystal complex in PPARα (1k7l) and (B) the crystal complex in PPARγ (1k74), respectively.Shown in As described in The hydrophobic tail of both Core A and Core C of agonists are buried well in the hydrophobic arm I and arm II that are formed by hydrophobic residues as shown by the green surface in A and C).Molecular dynamics can provide useful information for characterizing the internal motions of biomacromolecules with time. For a comparison study, the 10 ns molecular dynamics simulations were performed, respectively, for the crystal structures of PPARα (1k7l), PPARγ (1k74), as well as their complexes with GW409544 and Comp#1, i.e., PPARα-GW409544, PPARγ-GW409544, PPARα-Comp#1, and PPARγ-Comp#1. As we can see from Meanwhile, the corresponding root mean square deviation (RMSD) value curves of the protein backbone for PPARα, PPARγ, PPARα-GW409544, PPARγ-GW409544, PPARα-Comp#1, and PPARγ-Comp#1 were also computed, respectively. It is interesting to see that the RMSD curves for PPARα-Comp#1 and PPARγ-Comp#1 are remarkably more stable than those of PPARα-GW409544 and PPARγ-GW409544, particularly for the case of PPARα (1k7l) system, where only a fluctuation of around 0.3 nm was observed when the complex system reached the plateau.B and D of The detailed fluctuations of the aforementioned six different structures, as well as the root mean square fluctuations (RMSF) of their side-chain atoms, were also computed within 10 ns molecular dynamics simulations , which is ranked number 25 according to the strength of binding affinity with PPARα and PPARγ cf. Table 1.3) than any other nuclear hormone receptors Some pharmaceutically relevant properties of the new designed agonist derivatives as well as the original GW409544 compound, such as the “partition coefficient” (logPo/w), “van der Waals surface area of polar nitrogen and oxygen atoms” (PSA), “aqueous solubility” (logS), and “apparent MDCK permeability” (PMDCK), were predicted by means of the QP program embedded in the “Schrodinger2009 Software Package”. The results thus obtained are also listed in the As shown in The goal of this study was to find new and more powerful dual agonists for PPARα and PPARγ. The new technique of “core hopping” adopted in this study allows for the rapid screening of novel cores to help overcome unwanted properties by generating new lead compounds with improved core properties. A set of 10 novel compounds were found in this regard. Compared with the existing dual agonist, the new agonists not only had the similar function in activating PPARα and PPARγ, but also assumed the conformation more favorable in binding to PPARα and PPARγ. It is anticipated that the new agonists may become potential drug candidates. Or at the very least, they may stimulate new strategy for developing novel dual agonists against type-2 diabetes.