Tissue mineral density measured at the sub-millimetre scale can provide reliable statistics of elastic properties of bone matrix

Reliability of multiscale models of bone is related to the accuracy of the experimental information available on bone microstructure. X-ray-based imaging techniques allow to inspect bone structure and mineralization in vitro at the micrometre scale. However, spatial resolution achievable in vivo is much coarser and can produce blurry, uncertain information on bone microstructure. Working with uncertain data calls for new modelling paradigms able to propagate uncertainty through the scales. In this paper we investigate the effects of uncertain bone mineralization on the elastic coefficients of the bone matrix. To this aim, some stochastic concepts were developed and compared with one another in order to identify the best way to account for uncertain input data. These concepts step from a deterministic micromechanical model of bone matrix which was extended in order to account for uncertain bone composition. Uncertainty was introduced by assuming to know only mean value and dispersion of the parameters describing bone composition. Thus, these parameters were modelled as random variables and their distribution functions were obtained using the maximum entropy principle. Either the tissue mineral density (TMD) or the ensuing volume fractions of collagen and mineral were used to describe uncertain bone composition. Moreover, mean value and dispersion were estimated at the scales of either 10 or a few 100 \(\upmu \)m, representative of standard in vitro and in vivo spatial resolutions, respectively. Analysis of these modelling concepts suggests that TMD measured at the sub-millimetre scale can be used to obtain reliable statistical information about the elastic coefficients of bone matrix.     

The somatostatin subtype-2 receptor antagonist, BIM-23627, improves the catabolic effects induced by long-term glucocorticoid treatment in the rat

BIM-23627 is a synthetic peptide with "in vitro" and "in vivo" properties consistent with a pure sst2 antagonist. The aim of the present study was to evaluate the effects of long-term administration of BIM-23627 and the combined effects of BIM-23627 and dexamethasone (DEX) on the somatotropic axis, including growth, epididymal fat accumulation, glucose homeostasis and insulin activity, in young male rats. Beginning on day 23 of age, 16 animals were treated daily with saline or DEX (40 microg/kg/daily). Each group was subdivided into two paired groups and treated with either vehicle or BIM-23627 (0.5 mg/kg, t.i.d.). The treatment period lasted 31 days. The animals were killed by decapitation; trunk blood and pituitaries were collected for the determination of hormone concentrations and GH mRNA expression, respectively. Based on plasma GH and IGF-I concentrations and GH mRNA expression in the pituitary, BIM-23627 was able to counteract the inhibitory effects of DEX on the somatotropic axis; however, only a partial reversal of somatic growth inhibition was observed. DEX-treated rats remained euglycemic, but their insulin levels were significantly increased, indicating an incipient insulin resistance. Although BIM-23627 itself tended to increase insulin concentration in saline-treated rats, its administration to DEX-treated rats reduced insulin levels (saline: 25+/-3; DEX: 55+/-16*; DEX+BIM-23627: 34+/-5; BIM-23627: 38+/-7 microIU/ml; *P<0.05 vs. saline), apparently improving the degree of insulin sensitivity. DEX administration significantly reduced circulating ghrelin, whereas the sst2 antagonist had no significant effect. An inverse correlation was found between ghrelin concentrations and plasma insulin levels. Both rats receiving DEX and rats receiving BIM-23627 had decreased plasma concentration of total testosterone (P<0.05); however, the effects of DEX and BIM-23627 were not additive. In conclusion, BIM-23627 may represent a new pharmacological agent to reduce the suppression of the GH-IGF-I axis in long-term GC treated patients and enhance insulin sensitivity. Further studies are required in order to fully optimize the SSTR-2 antagonist-induced reversal of DEX-induced somatic growth inhibition.     

Effects of pexiganan alone and combined with betalactams in experimental endotoxic shock

To investigate the efficacy of pexiganan, a 22-residue magainin analog, alone and combined with betalactmas antibiotics in three experimental rat models of Gram-negative septic shock. Adult male Wistar rats were given (i) an intraperitoneal injection of 1 mg Escherichia coli 0111:B4 LPS; (ii) 2x10(10)CFU of E. coli ATCC 25922; and (iii) intra-abdominal sepsis induced via cecal ligation and puncture. For each model, all animals were randomized to receive intraperitoneally isotonic sodium chloride solution, 1 mg/kg pexiganan, 1 mg/kg polymyxin B, 20 mg/kg imipenem, 60 mg/kg piperacillin alone and combined with 1 mg/kg pexiganan. Each group included 15 animals. Lethality, bacterial growth in blood or intra-abdominal fluid, endotoxin and TNF-alpha concentrations in plasma. All compounds reduced the lethality when compared to controls. Piperacillin and imipenem significantly reduced the lethality and the number of E. coli in abdominal fluid compared with saline treatment. Pexiganan showed a slightly lower antimicrobial activity than betalactams even though it achieved a substantial higher decrease in endotoxin and TNF-alpha plasma concentrations than imipenem and piperacillin. No statistically significant differences were noted for antimicrobial and antiendotoxin activities between pexiganan and polymyxin B. Combination between pexiganan and betalactams showed to be the most effective treatment in reducing all variables measured. The use of a novel antimicrobial compound able to bind to LPS associated to potent antibiotics such as betalactams may become an important future consideration for sepsis treatment.     

Redox proteomics analysis of oxidatively modified proteins in G93A-SOD1 transgenic mice--a model of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease characterized by the loss of neuronal function in the motor cortex, brain stem, and spinal cord. Familial ALS cases, accounting for 10-15% of all ALS disease, are caused by a gain-of-function mutation in Cu,Zn-superoxide dismutase (SOD1). Two hypotheses have been proposed to explain the toxic gain of function of mutant SOD (mSOD). One is that mSOD can directly promote reactive oxygen species and reactive nitrogen species generation, whereas the other hypothesis suggests that mSODs are prone to aggregation due to instability or association with other proteins. However, the hypotheses of oxidative stress and protein aggregation are not mutually exclusive. G93A-SOD1 transgenic mice show significantly increased protein carbonyl levels in their spinal cord from 2 to 4 months and eventually develop ALS-like motor neuron disease and die within 5-6 months. Here, we used a parallel proteomics approach to investigate the effect of the G93A-SOD1 mutation on protein oxidation in the spinal cord of G93A-SOD1 transgenic mice. Four proteins in the spinal cord of G93A-SOD1 transgenic mice have higher specific carbonyl levels compared to those of non-transgenic mice. These proteins are SOD1, translationally controlled tumor protein (TCTP), ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1), and, possibly, alphaB-crystallin. Because oxidative modification can lead to structural alteration and activity decline, our current study suggests that oxidative modification of UCH-L1, TCTP, SOD1, and possibly alphaB-crystallin may play an important role in the neurodegeneration of ALS.     

Proteomic analysis of 4-hydroxy-2-nonenal-modified proteins in G93A-SOD1 transgenic mice--a model of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an age-related, fatal motor neuron degenerative disease occurring both sporadically (sALS) and heritably (fALS), with inherited cases accounting for approximately 10% of diagnoses. Although multiple mechanisms likely contribute to the pathogenesis of motor neuron injury in ALS, recent advances suggest that oxidative stress may play a significant role in the amplification, and possibly the initiation, of the disease. Lipid peroxidation is one of the several outcomes of oxidative stress. Since the central nervous system (CNS) is enriched with polyunsaturated fatty acids, it is particularly vulnerable to membrane-associated oxidative stress. Peroxidation of cellular membrane lipids or circulating lipoprotein molecules generates highly reactive aldehydes, among which is 4-hydroxy-2-nonenal (HNE). HNE levels are increased in spinal cord motor neurons of ALS patients, indicating that lipid peroxidation is associated with the motor neuron degeneration in ALS. In the present study, we used a parallel proteomic approach to identify HNE-modified proteins in the spinal cord tissue of a model of fALS, G93A-SOD1 transgenic mice, in comparison to the nontransgenic mice. We found three significantly HNE-modified proteins in the spinal cord of G93A-SOD1 transgenic mice: dihydropyrimidinase-related protein 2 (DRP-2), heat-shock protein 70 (Hsp70), and possibly alpha-enolase. These results support the role of oxidative stress as a major mechanism in the pathogenesis of ALS. Structural alteration and activity decline of functional proteins may consistently contribute to the neurodegeneration process in ALS.     

Isolation, Characterization, and Heterologous Expression of a Carboxylesterase of Pseudomonas aeruginosa PAO1

We purified to homogeneity an intracellular esterase from the opportunistic pathogen Pseudomonas aeruginosa PAO1. The enzyme hydrolyzes p-nitrophenyl acetate and other acetylated substrates. The N-terminal amino acid sequence was analyzed and 11 residues, SEPLILDAPNA, were determined. The corresponding gene PA3859 was identified in the P. aeruginosa PAO1 genome as the only gene encoding for a protein with this N-terminus. The encoding gene was cloned in Escherichia coli, and the recombinant protein expressed and purified to homogeneity. According to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis and analytical gel filtration chromatography, the esterase was found to be a monomer of approximately 24 kDa. The experimentally determined isoelectric point was 5.2 and the optimal enzyme activity was at 55°C and at pH 9.0. The esterase preferentially hydrolyzed short-chain fatty acids. It is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by ethylendiaminotetraacetic acid (EDTA). Native enzyme preparations typically showed a Michaelis constant (K_m) and V_max of 0.43 mM and 12,500 U mg^–1, respectively, using p-nitrophenyl acetate as substrate. Homology-based database searches clearly revealed the presence of the consensus GXSXG signature motif that is present in the serine-dependent acylhydrolase protein family.     

Micronuclei in humans induced by exposure to low level of ionizing radiation: influence of polymorphisms in DNA repair genes

Understanding the risks deriving from protracted exposure to low doses of ionizing radiation has remarkable societal importance in view of the large number of work settings in which sources of IR are encountered. To address this question, we studied the frequency of micronuclei (MN), which is an indicator of DNA damage, in a population exposed to low levels of ionizing radiation and in matched controls. In both exposed population and controls, the possible influence of single nucleotide polymorphisms in XRCC1, XRCC3 and XPD genes on the frequency of micronuclei was also evaluated. We also considered the effects of confounding factors, like smoking status, age and gender. The results indicated that MN frequency was significantly higher in the exposed workers than in the controls [8.62+/-2.80 versus 6.86+/-2.65; P=0.019]. Radiological workers with variant alleles for XRCC1 or XRCC3 polymorphisms or wild-type alleles for XPD exon 23 or 10 polymorphisms showed a significantly higher MN frequency than controls with the same genotypes. Smoking status did not affect micronuclei frequency either in exposed workers or controls, while age was associated with increased MN frequency in the exposed only. In the combined population, gender but not age exerted an influence on the yield of MN, being higher in females than in males. Even though there is a limitation in this study due to the small number of subjects, these results suggest that even exposures to low level of ionizing radiation could have genotoxic effects and that XRCC3, XRCC1 and XPD polymorphisms might contribute to the increased genetic damage in susceptible individuals occupationally exposed to chronic low levels of ionizing radiation. For a clear conclusion on the induction of DNA damage caused by protracted exposure to low doses of ionizing radiation and the possible influence of genetic polymorphism in DNA repair genes larger studies are needed.     

Characterisation of gastric ghrelin cells in man and other mammals: studies in adult and fetal tissues

Ghrelin is a new gastric peptide involved in food intake control and growth hormone release. We aimed to assess its cell localisation in man during adult and fetal life and to clarify present interspecies inconsistencies of gastric endocrine cell types. A specific serum generated against amino acids 13-28 of ghrelin was tested on fetal and adult gastric mucosa and compared with ghrelin in situ hybridisation. Immunogold electron microscopy was performed on normal human, rat and dog adult stomach. Ghrelin cells were detected in developing gut, pancreas and lung from gestational week 10 and in adult human, rat and dog gastric mucosa. By immunogold electron microscopy, gastric ghrelin cells showed distinctive morphology and hormone reactivity in respect to histamine enterochromaffin-like, somatostatin D, glucagon A or serotonin enterochromaffin cells. Ghrelin cells were characterised by round, compact, electron-dense secretory granules of P/D(1) type in man (mean diameter 147+/-30 nm), A-like type in the rat (183+/-37 nm) and X type in the dog (273+/-49 nm). It is concluded that, ghrelin is produced by well-defined cell types, which in the past had been labelled differently in various mammals mostly because of the different size of their secretory granule. In man ghrelin cells develop during early fetal life.     

Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I

During meiosis, two rounds of chromosome segregation occur after a single round of DNA replication, producing haploid progeny from diploid progenitors. Three innovations in chromosome behaviour during meiosis I accomplish this unique division. First, crossovers between maternal and paternal sister chromatids (detected cytologically as chiasmata) bind replicated maternal and paternal chromosomes together. Second, sister kinetochores attach to microtubules from the same pole (mono-polar orientation), causing maternal and paternal centromere pairs (and not sister chromatids) to be separated. Third, sister chromatid cohesion near centromeres is preserved at anaphase I when cohesion along chromosome arms is destroyed. The finding that destruction of mitotic cohesion is regulated by Polo-like kinases prompted us to investigate the meiotic role of the yeast Polo-like kinase Cdc5. We show here that cells lacking Cdc5 synapse homologues and initiate recombination normally, but fail to efficiently resolve recombination intermediates as crossovers. They also fail to properly localize the Lrs4 (ref. 3) and Mam1 (ref. 4) monopolin proteins, resulting in bipolar orientation of sister kinetochores. Cdc5 is thus required both for the formation of chiasmata and for cosegregation of sister centromeres at meiosis I.     

Both of us disgusted in My insula: the common neural basis of seeing and feeling disgust

What neural mechanism underlies the capacity to understand the emotions of others? Does this mechanism involve brain areas normally involved in experiencing the same emotion? We performed an fMRI study in which participants inhaled odorants producing a strong feeling of disgust. The same participants observed video clips showing the emotional facial expression of disgust. Observing such faces and feeling disgust activated the same sites in the anterior insula and to a lesser extent in the anterior cingulate cortex. Thus, as observing hand actions activates the observer's motor representation of that action, observing an emotion activates the neural representation of that emotion. This finding provides a unifying mechanism for understanding the behaviors of others.