Preclinical molecular imaging of the translocator protein (TSPO) in a metastases model based on breast cancer xenografts propagated in the murine brain

Previous studies have demonstrated the feasibility of translocator protein (TSPO) imaging to visualize and quantify human breast adenocarcinoma (MDA-MB-231) cells in vivo using a TSPO-targeted near-infrared (NIR) probe (NIR-conPK11195). This study aimed to extend the use of the TSPO-targeted probe to a more biologically relevant and clinically important tumor microenvironment as well as to assess our ability to longitudinally detect the presence and progression of breast cancer cells in the brain. The in vivo biodistribution and accumulation of NIR-conPK11195 and free (unconjugated) NIR dye were quantitatively evaluated in intracranial MDA-MB-231-bearing mice and non-tumor-bearing control mice longitudinally once a week from two to five weeks post-inoculation. The in vivo time-activity curves illustrate distinct clearance profiles for NIR-conPK11195 and free NIR dye, resulting in preferential accumulation of the TSPO-targeted probe in the intracranial tumor bearing hemisphere (TBH) with significant tumor contrast over normal muscle tissue (p < 0.005 at five weeks; p < 0.01 at four weeks). In addition, the TSPO-labeled TBHs demonstrated significant contrast over the TBHs of mice injected with free NIR dye (p < 0.001 at four and five weeks) as well as over the TSPO-labeled non-tumor-bearing hemispheres (NTBHs) of control mice (p < 0.005 at four and five weeks). Overall, TSPO-targeted molecular imaging appears useful for visualizing and quantifying breast cancer xenografts propagated in the murine brain and may assist in preclinical detection, diagnosis and monitoring of metastatic disease as well as drug discovery. Furthermore, these results indicate it should be possible to perform TSPO-imaging of breast cancer cells in the brain using radiolabeled TSPO-targeted agents, particularly in light of the fact that [11C]-labeled TSPO probes such as [11C]-PK 11195 have been successfully used to image gliomas in the clinic.     

Novel loci for adiponectin levels and their influence on type 2 diabetes and metabolic traits: a multi-ethnic meta-analysis of 45,891 individuals

Circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are highly heritable and are inversely associated with type 2 diabetes mellitus (T2D) and other metabolic traits. We conducted a meta-analysis of genome-wide association studies in 39,883 individuals of European ancestry to identify genes associated with metabolic disease. We identified 8 novel loci associated with adiponectin levels and confirmed 2 previously reported loci (P = 4.5×10⁻⁸–1.2×10⁻⁴³). Using a novel method to combine data across ethnicities (N = 4,232 African Americans, N = 1,776 Asians, and N = 29,347 Europeans), we identified two additional novel loci. Expression analyses of 436 human adipocyte samples revealed that mRNA levels of 18 genes at candidate regions were associated with adiponectin concentrations after accounting for multiple testing (p<3×10⁻⁴). We next developed a multi-SNP genotypic risk score to test the association of adiponectin decreasing risk alleles on metabolic traits and diseases using consortia-level meta-analytic data. This risk score was associated with increased risk of T2D (p = 4.3×10⁻³, n = 22,044), increased triglycerides (p = 2.6×10⁻¹⁴, n = 93,440), increased waist-to-hip ratio (p = 1.8×10⁻⁵, n = 77,167), increased glucose two hours post oral glucose tolerance testing (p = 4.4×10⁻³, n = 15,234), increased fasting insulin (p = 0.015, n = 48,238), but with lower in HDL-cholesterol concentrations (p = 4.5×10⁻¹³, n = 96,748) and decreased BMI (p = 1.4×10⁻⁴, n = 121,335). These findings identify novel genetic determinants of adiponectin levels, which, taken together, influence risk of T2D and markers of insulin resistance.     

Minimum convex hull mass estimations of complete mounted skeletons

Body mass is a critical parameter used to constrain biomechanical and physiological traits of organisms. Volumetric methods are becoming more common as techniques for estimating the body masses of fossil vertebrates. However, they are often accused of excessive subjective input when estimating the thickness of missing soft tissue. Here, we demonstrate an alternative approach where a minimum convex hull is derived mathematically from the point cloud generated by laser-scanning mounted skeletons. This has the advantage of requiring minimal user intervention and is thus more objective and far quicker. We test this method on 14 relatively large-bodied mammalian skeletons and demonstrate that it consistently underestimates body mass by 21 per cent with minimal scatter around the regression line. We therefore suggest that it is a robust method of estimating body mass where a mounted skeletal reconstruction is available and demonstrate its usage to predict the body mass of one of the largest, relatively complete sauropod dinosaurs: Giraffatitan brancai (previously Brachiosaurus) as 23200 kg.     

Identification and Characterization of Assembly Proteins of CS5 Pili from Enterotoxigenic Escherichia coli

This study investigated the role of three genes comprising part of the operon which encodes CS5 pili from enterotoxigenic Escherichia coli. In-frame gene deletions were constructed, and the effects on biogenesis of the pili were examined. A deletion in csfB abolished CsfA major subunit accumulation in the periplasm, which could be restored by trans-complementation with a complete copy of the csfB gene. Localization studies using an antibody against CsfB showed that this protein was periplasmically located, and thus CsfB is likely to function as the specific chaperone for CsfA. An in-frame deletion mutation in the csfE gene resulted in pili approximately three times longer than those of the wild-type strain, thereby indicating a role for CsfE in pilus length regulation. Localization studies using an antibody generated against CsfE showed low-level CsfE accumulation in the outer membranes. Modulation of csfE expression in trans did not reduce the mean length of the pilus below that of the wild type, which indicated that CsfE is not rate-limiting for termination of pilus assembly. Interestingly, a deletion in the csfF gene also resulted in an elongated pilus morphology identical to that of the csfE deletion strain. However, unlike CsfE, CsfF was shown to be rate-limiting for termination of assembly, since overexpression of CsfF in a csfF deletion strain resulted in a significant decrease in the mean length of the pilus compared to that of the wild type. When the same construct was introduced into the wild-type strain, pilus expression was abolished. Since CsfF bears significant homology to the proposed CsfB chaperone, CsfF was predicted to act as the specific chaperone for CsfE. A double deletion in the csfB and csfF genes was shown to abolish the periplasmic accumulation of both CsfA and CsfD pilins, which could be restored individually only when the strain was trans-complemented with a wild-type copy of csfB or csfF, respectively. Therefore, CsfF may chaperone not only CsfE but also CsfD. A model for CS5 biogenesis is also proposed based on these and previous observations.     

Haematology of the turbot, Psetta maxima (L.): ultrastructural, cytochemical and morphological properties of peripheral blood leucocytes

Optimum procedures for fish handling and sample processing for use when employing haematological parameters as health indicators in turbot, Psetta maxima (L.), have been established. We found thrombocytes to be the most abundant blood cell, representing approximately 52% of circulating leucocytes (lymphocytes, 40.8%; granulocytes, 5.6%; monocytes, 1.6%; total number of leucocytes=1.3 × 10⁵ ml⁻¹; packed cell volume=22.7%). The light- and electron-microscopical characteristics of these cell types are described, together with their cytochemical properties using Sudan Black B, Periodic Acid Schiff, Non-specific Esterase, and Acid and Alkaline Phosphatase. Turbot thrombocytes showed a high degree of shape alterations when observed in live preparations using phase contrast microscopy, while ultrastructural observations following the in vitro uptake of carbon particles supported an active process of phagocytosis by the thrombocytes, rather than passive entrapment. The lymphocytes of turbot are structurally similar to mammalian lymphocytes with the highest nuclear:cytoplasmic ratio of all the leucocytes observed. Small lymphocytes predominated, large lymphocytes forming less than 1% of the total white blood cell population. The most frequent granulocyte type was a neutrophil-like cell with an eccentric nucleus, only rarely seen in segmented form. In vitro uptake of carbon particles by granulocytes was not observed under the conditions of the experiment, although turbot granulocytes are capable of phagocytosis under different circumstances. These are discussed, along with other physiological and technical factors which can influence the blood parameter findings in fish.     

Multilocus Sequence Typing for Comparison of Veterinary and Human Isolates of Campylobacter jejuni

Multilocus sequence typing (MLST) has been applied to 266 Campylobacter jejuni isolates, mainly from veterinary sources, including cattle, sheep, poultry, pigs, pets, and the environment, as well as isolates from human cases of campylobacteriosis. The populations of veterinary and human isolates overlap, suggesting that most veterinary sources should be considered reservoirs of pathogenic campylobacters. There were some associations between source and sequence type complex, indicating that host or source adaptation may exist. The pig isolates formed a distinct group by MLST and may well represent a potential pig-adapted clone of C. jejuni. A subset (n = 82) of isolates was reanalyzed with a second MLST scheme which provided a unique set of isolates that had been analyzed at a total of 12 loci. The distribution of isolates among the complexes in each of the two schemes was similar but not identical. In addition to isolates from human outbreaks, one group of isolates that were not epidemiologically linked was also identical at all 12 loci. This group of isolates is believed to represent another stable strain of C. jejuni.     

Receptor and G protein-mediated responses to thrombin in HEL cells.

Thrombin is believed to activate platelets via cell surface receptors coupled to G proteins. In order to better understand this process, we have examined the interaction of thrombin with HEL cells, a leukemic cell line that has served as a useful model for studies of platelet structure and function. In HEL cells, as in platelets, thrombin stimulated inositol trisphosphate (IP3) formation and suppressed cAMP synthesis. Both events were inhibited by pertussis toxin with 50% inhibition occurring at a toxin concentration that ADP-ribosylated 50% of the Gi alpha subunits present in HEL cells. IP3 formation was also stimulated by a second serine protease, trypsin. The trypsin response was identical to the thrombin response in time course, magnitude, and pertussis toxin sensitivity, suggesting that a similar mechanism is involved. Agonist-induced changes in the cytosolic-free Ca2+ concentration were used to test this hypothesis. Both proteases caused a transient increase in intracellular calcium [Ca2+]i that could be inhibited with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone thrombin. Exposure to either protease desensitized HEL cells against subsequent increases in [Ca2+]i and IP3 caused by the other, although responses to other agonists were retained. This loss of responsiveness persisted despite repeated washing of the cells and the addition of hirudin. Complete recovery occurred after 20 h and could be prevented with cycloheximide. These observations suggest that 1) HEL cell thrombin receptors, like those on platelets, are coupled to phospholipase C and adenylylcyclase by pertussis toxin-sensitive G proteins, 2) the G proteins involved are equally accessible to pertussis toxin in situ, 3) when access is limited to the outside of the cell the response mechanisms for thrombin and trypsin are similar, if not identical, despite the broader substrate specificity of trypsin, 4) both proteases cause persistent changes that may involve proteolysis of their receptors or associated proteins, and 5) desensitization of the thrombin response occurs at a step no later than the activation of phospholipase C and requires protein synthesis for recovery.