Molecular conformation changes along the malignancy revealed by optical nanosensors

An interdisciplinary approach employing functionalized nanoparticles and ultrasensitive spectroscopic techniques is reported here to track the molecular changes in early stage of malignancy. Melanoma tissue tracking at molecular level using both labelled and unlabelled silver and gold nanoparticles has been achieved using surface enhanced Raman scattering (SERS) technique. We used skin tissue from ex vivo mice with induced melanoma. Raman and SERS molecular characterization of melanoma tissue is proposed here for the first time. Optical nanosensors based on Ag and Au nanoparticles with chemisorbed cresyl violet molecular species as labels revealed sensitive capability to tissues tagging and local molecular characterization. Sensitive information originating from surrounding native biological molecules is provided by the tissue SERS spectra obtained either with visible or NIR laser line. Labelled nanoparticles introduced systematic differences in tissue response compared with unlabelled ones, suggesting that the label functional groups tag specific tissue components revealed by proteins or nucleic acids bands. Vibrational data collected from tissue are presented in conjunction with the immunohistochemical analysis. The results obtained here open perspectives in applied plasmonic nanoparticles and SERS for the early cancer diagnostic based on the appropriate spectral databank.     

Chronic Administration of Proanthocyanidins or Docosahexaenoic Acid Reversess the Increase of miR-33a and miR-122 in Dyslipidemic Obese Rats

miR-33 and miR-122 are major regulators of lipid metabolism in the liver, and their deregulation has been linked to the development of metabolic diseases such as obesity and metabolic syndrome. However, the biological importance of these miRNAs has been defined using genetic models. The aim of this study was to evaluate whether the levels of miR-122 and miR-33a in rat liver correlate with lipemia in nutritional models. For this purpose, we analyzed the levels of miRNA-33a and miR-122 in the livers of dyslipidemic cafeteria diet-fed rats and of cafeteria diet-fed rats supplemented with proanthocyanidins and/or ω-3 PUFAs because these two dietary components are well-known to counteract dyslipidemia. The results showed that the dyslipidemia induced in rats that were fed a cafeteria diet resulted in the upregulation of miR-33a and miR-122 in the liver, whereas the presence of proanthocyanidins and/or ω-3 PUFAs counteracted the increase of these two miRNAs. However, srebp2, the host gene of miR-33a, was significantly repressed by ω-3 PUFAs but not by proanthocyanidins. Liver mRNA levels of the miR-122 and miR-33a target genes, fas and pparβ/δ, cpt1a and abca1, respectively, were consistent with the expression of these two miRNAs under each condition. Moreover, the miR-33a and abca1 levels were also analyzed in PBMCs. Interestingly, the miR-33a levels evaluated in PBMCs under each condition were similar to the liver levels but enhanced. This demonstrates that miR-33a is expressed in PBMCs and that these cells can be used as a non-invasive way to reflect the expression of this miRNA in the liver. These findings cast new light on the regulation of miR-33a and miR-122 in a dyslipidemic model of obese rats and the way these miRNAs are modulated by dietary components in the liver and in PBMCs.     

Temporal localization of porcine reproductive and respiratory syndrome virus in reproductive tissues of experimentally infected boars

Porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to be shed in the semen of infected boars. To determine whether the reproductive tissues could be a persistent source of virus and the possible origin of PRRSV found in semen of infected boars, 20 PRRSV-seronegative boars were intranasally inoculated with 5 x 10(6) median tissue culture infective doses (TCID50) of PRRSV and necropsied at different times post-inoculation (p.i.) from Day 2 to Day 37 p.i. Blood samples were collected before experimental inoculation, at necropsy and at different times p.i. At necropsy, epididymal semen and reproductive tissues were collected and the presence of the virus determined by virus isolation. The infection of the boars was demonstrated by the isolation of the virus from the sera of all inoculated boars and by seroconversion. PRRSV was detected in serum samples from Day 2 to Day 23 p.i., although the viremic period was largely dependent on the individual response to infection. Viral replication was proven within different reproductive tissues from Day 2 to Day 23 p.i., being most consistently found in the epididymus. In addition, PRRSV was isolated in semen from Day 4 to Day 10 p.i. The correlation of a diminished viremia and the inability to isolate PRRSV from semen or reproductive tissues may be due to one of two possibilities. First, viremia is responsible for most of the virus isolated from reproductive tissues due to the movement of PRRSV-infected cells out of the blood and into the tissues. Second, viremia may initially seed the reproductive tissues with PRRSV, and then the virus is produced into the reproductive tract and shed into semen at low levels.     

Lipogenesis Is Decreased by Grape Seed Proanthocyanidins According to Liver Proteomics of Rats Fed a High Fat Diet

Bioactive proanthocyanidins have been reported to have several beneficial effects on health in relation to metabolic syndrome, type 2 diabetes, and cardiovascular disease. We studied the effect of grape seed proanthocyanidin extract (GSPE) in rats fed a high fat diet (HFD). This is the first study of the effects of flavonoids on the liver proteome of rats suffering from metabolic syndrome. Three groups of rats were fed over a period of 13 weeks either a chow diet (control), an HFD, or a high fat diet supplemented for the last 10 days with GSPE (HFD + GSPE). The liver proteome was fractionated, using a Triton X-114-based two-phase separation, into soluble and membrane protein fractions so that total proteome coverage was considerably improved. The data from isobaric tag for relative and absolute quantitation (iTRAQ)-based nano-LC-MS/MS analysis revealed 90 proteins with a significant (p < 0.05) minimal expression difference of 20% due to metabolic syndrome (HFD versus control) and 75 proteins due to GSPE treatment (HFD + GSPE versus HFD). The same animals have previously been studied (Quesada, H., del Bas, J. M., Pajuelo, D., Díaz, S., Fernandez-Larrea, J., Pinent, M., Arola, L., Salvadó, M. J., and Bladé, C. (2009) Grape seed proanthocyanidins correct dyslipidemia associated with a high-fat diet in rats and repress genes controlling lipogenesis and VLDL assembling in liver. Int. J. Obes. 33, 1007–1012), and GSPE was shown to correct dyslipidemia observed in HFD-fed rats probably through the repression of hepatic lipogenesis. Our data corroborate those findings with an extensive list of proteins describing the induction of hepatic glycogenesis, glycolysis, and fatty acid and triglyceride synthesis in HFD, whereas the opposite pattern was observed to a large extent in GSPE-treated animals. GSPE was shown to have a wider effect than previously thought, and putative targets of GSPE involved in the reversal of the symptoms of metabolic syndrome were revealed. Some of these novel candidate proteins such as GFPT1, CD36, PLAA (phospholipase A₂-activating protein), METTL7B, SLC30A1, several G signaling proteins, and the sulfide-metabolizing ETHE1 and SQRDL (sulfide-quinone reductase-like) might be considered as drug targets for the treatment of metabolic syndrome.An increase in high calorie diets and a sedentary lifestyle are considered the key factors in explaining the epidemic rise in obesity in developed countries (1). Obese patients, especially those with abdominal obesity due to visceral adipose tissue accumulation, run a higher risk of impaired glucose tolerance, which frequently evolves into insulin resistance (2). Obesity and insulin resistance are frequently associated with hypertension, proatherogenic dyslipidemia, chronic inflammation, a prothrombotic state, and recently also fatty liver (3), conditions that together make up what is known as metabolic syndrome and lead to an increased risk of developing cardiovascular disease (CVD)¹ and type 2 diabetes (4). Conversely, some dietary patterns and specific food components have been associated with a lower prevalence of obesity, type 2 diabetes, and CVD. In this sense, the traditional Mediterranean diet (characterized by a high fiber content, low glycemic index carbohydrates, unsaturated fats, vitamins, and antioxidant polyphenols) has been linked to a lower incidence of CVD, obesity, and type 2 diabetes (5–8). Moreover, the French population presents a very low prevalence of death due to CVD despite consuming a diet rich in saturated fats and cholesterol. This phenomenon, known as “the French paradox” (9), has been ascribed to the moderate consumption of red wine and specifically to its content of polyphenols (10–12).Polyphenols include flavonoids of which flavan-3-ols and their oligomeric forms (proanthocyanidins) have been reported to exhibit several beneficial health effects by acting as antioxidant, anticarcinogen, cardioprotective, antimicrobial, antiviral, and neuroprotective agents (for a review, see Ref. 13). Specifically, grape and wine proanthocyanidins have a cardioprotective effect through increasing plasma high density lipoprotein cholesterol, decreasing low density lipoprotein-derived atherosclerotic foam cell lesions, attenuating oxidant formation by quenching harmful radicals, increasing endothelium-dependent vasorelaxation, etc. (13). In this context, our group has been working for years on the effect of a grape seed proanthocyanidin extract (GSPE) (containing monomers and oligomers of flavan-3-ols) in relation to metabolic syndrome. In previous works, we have found that GSPE prevents oxidative injury (14), has an insulinomimetic effect on adipocytes and adipose tissue (15), modulates glucose homeostasis (16), decreases plasma levels of triglycerides (TGs) and apolipoprotein B in normolipidemic rats (17), and acts as an in vitro (18, 19) and in vivo (20) anti-inflammatory. We have also shown that GSPE decreases postprandial plasma TG and apolipoprotein B in mice through a hepatic induction of a farnesoid X receptor (FXR) and the small heterodimer partner (SHP) that in turn down-regulates SREBP1c and other lipogenic genes in the liver (21, 22). Furthermore, we have demonstrated that the molecules responsible for the reduced TG synthesis in HepG2 cells treated with GSPE are the sum of a proanthocyanidins trimer and a dimer gallate because they reproduce the GSPE effect (23).The effect of GSPE on metabolic syndrome has been studied in our laboratory by feeding rats a “cafeteria diet.” This diet is an experimental model of a western high sugar and high fat diet extensively used to produce obesity in rats because its palatability induces the animals to increase their energy intake (24). In a recent study conducted by our group (25) as well as this study, the rats were fed a high fat diet (HFD) (cafeteria diet) for 13 weeks, and one group of the animals was treated with a daily dose of GSPE (25 mg/kg of body weight) for the last 10 days (HFD + GSPE). In that study, HFD was shown to cause the animals to be overweight and to suffer from fatty liver, dyslipidemia, and hepatic overexpression of key genes involved in lipogenesis and VLDL assembly, whereas GSPE treatment corrected dyslipidemia and down-regulated some of the genes up-regulated by HFD (25).To better investigate the mechanism behind the changes observed in HFD- and HFD + GSPE-fed rats, we analyzed protein expression in the liver. Because GSPE treatment and obesity have multiple effects, a proteome-wide approach is needed to map proteins from different pathways. Proteomics studies related to obesity, metabolic syndrome, fatty liver, or insulin resistance have previously been performed on the liver (26–32). Two such studies looked into the effects of flavonoids in mouse livers (33, 34), but to our knowledge, this is the first hepatic proteome analysis of the effect of flavonoids in rats suffering from metabolic syndrome. To improve the proteome coverage of the complex liver samples, we performed a proteome fractionation according to protein solubility using a two-phase detergent protocol (35). This strategy was advantageous because it captured membrane proteins that otherwise would have been difficult to detect. The resulting soluble and membrane protein fractions were digested, iTRAQ-labeled, fractionated according to isoelectric point, and analyzed by nano-LC-MS/MS. The proteomics study presented here reports a differential expression due to HFD or HFD + GSPE for approximately 140 proteins, indicating that both conditions were potent modifiers of the liver proteome. We have focused on the sugar and lipid metabolism data, which confirmed the repression of hepatic lipogenesis in HFD + GSPE rats. Additionally, new proteins have been revealed as putative GSPE targets.     

A beta-tubulin leucine cluster involved in microtubule assembly and paclitaxel resistance.

Analysis of beta-tubulin alleles from nine paclitaxel-resistant Chinese hamster ovary cell lines revealed an unexpected cluster of mutations affecting Leu-215, Leu-217, and Leu-228. Six of the mutant alleles encode a His, Arg, or Phe substitution at Leu-215; another mutant allele has an Arg substitution at Leu-217; and the final two mutant alleles have substitutions of His or Phe at Leu-228. Using plasmids that allow tetracycline regulated expression, the L215H, L217R, and L228F mutations were introduced into a hemagglutinin antigen-tagged beta-tubulin cDNA and transfected into wild-type Chinese hamster ovary cells. In all three cases, low to moderate expression of the transfected mutant gene conferred paclitaxel resistance. Higher levels of expression caused disruption of microtubule assembly, cell cycle arrest at mitosis, and failure to proliferate. Consistent with reduced microtubule stability, cells expressing mutant hemagglutinin beta-tubulin had fewer acetylated microtubules than nonexpressing cells in the same population. These data, together with previous studies showing that the paclitaxel-resistant mutant cell lines have less stable microtubules, indicate that the leucine cluster represents an important structural motif for microtubule assembly.     

Inner structural organization of the distal humerus in Paranthropus and Homo

The taxonomic attribution of isolated hominin distal humeri has been a matter of uncertainty and disagreement notwithstanding their relative abundance in the fossil record. Four taxonomically-based morphotypes, respectively representing P. boisei, P. robustus, non-erectus early Homo and H. erectus, have been identified based on the cross-sectional outer shape variation of an assemblage of Plio-Pleistocene eastern and southern African specimens (Lague, 2015). However, the existence of possible differences between Paranthropus and Homo in the inner structural organisation at this skeletal site remains unexplored. We used noninvasive imaging techniques to tentatively characterize the endostructural organization of five early Pleistocene distal humeri from South Africa (TM 1517g, SK 24600, SKX 10924, SKX 34805) and Ethiopia (Gombore IB), which have been variably attributed to Paranthropus or Homo. While the investigated specimens reveal diverse degrees of inner preservation related to their taphonomic and diagenetic history, in all but SK 24600 from Swartkrans we could comparatively assess some geometric properties at the most distal cross-sectional level (%CA, I_x/I_y, I_max/I_min) and quantify cortical bone thickness topographic variation across the preserved shaft portions by means of a 2-3D Relative Cortical Thickness index. Whenever possible, we also provided details about the site-specific organization of the cancellous network and measured the same parameters in a comparative sample of twelve adult extant humans. For most features, our results indicate two main patterns: the first includes the specimens TM 1517g, SKX 10924 and SKX 34805, while the second endostructural morphotype sets apart the robust Homo aff. erectus Gombore IB specimen from Melka Kunture, which more closely resembles the condition displayed by our comparative human sample. Notably, marked differences in the amount and pattern of proximodistal cortical bone distribution have been detected between Gombore IB and SKX 34805 from Swartkrans. Given its discordant outer and inner signatures, we conclude that the taxonomic status of SKX 34805 deserves further investigations.     

Tomato belowground–aboveground interactions: <Emphasis Type="Italic">Rhizophagus irregularis</Emphasis> affects foraging behavior and life history traits of the predator <Emphasis Type="Italic">Macrolophus pygmaeus</Emphasis> (Hemiptera: Miridae)

In recent years, studies on arbuscular mycorrhizal fungi (AMF) have been revealing that the belowground symbiosis can influence the performance of aboveground herbivores and their natural enemies through its effects on the host plant. In this study, we tested whether the colonization of tomato plants by the arbuscular mycorrhizal fungus Rhizophagus irregularis (Syn. Glomus intraradices Schenk and Smith) (Glomeromycota: Glomeraceae) affects the performance of the zoophytophagous mirid bug Macrolophus pygmaeus Rambur (Hemiptera: Miridae). Mycorrhizal colonization in tomato plants positively influenced the predator host-plant acceptance for feeding and oviposition, as well as nymphal survival and female weight. We hypothesize that AMF can modify mirid bug foraging behavior and performance.