Environmental exposure to benzene, micronucleus formation and polymorphisms in DNA-repair genes: a pilot study

This report is part of a biomarker study conducted in an Italian population with exposure to environmental benzene ranging from 1.43 to 31.41 μg/m3 (values from personal sampling). DNA damage induced by benzene is the crucial mechanism of its genotoxicity, which leads to chronic benzene poisoning, haematotoxicity and leukaemia. Therefore, genetic variation in DNA-repair genes may modulate susceptibility to benzene-induced DNA damage. In light of this, the effects of polymorphisms in DNA-repair genes (APEX1, hOGG1, NBS1, XPD, XRCC1, and XRCC3) on micronucleus (MN) formation as a biomarker of early biological effects were evaluated. A significantly higher median MN frequency was recorded in traffic wardens than in controls. However, none of the analysed polymorphisms was significantly associated with the median MN frequency. A gene-gender interaction was observed for the APEX1 genotype. The APEX1 variant genotype was associated with significantly lower median MN frequency in men, not in women. Statistical analysis did not reveal any association between the score of the protective alleles - hypothetically pushing the pathway towards optimal DNA-damage repair - and MN. Even though there are some limitations in the study, our results indicate that the general population may be exposed to benzene concentrations higher than the threshold level for air-quality standards in the European Union of 10 μg/m3. Furthermore, urban traffic wardens are exposed to significantly higher levels of benzene than individuals spending most of the time indoors. This higher exposure may contribute to DNA damage, suggesting that benzene might be implicated both as an environmental and occupational risk factor in leukaemia and other haematological diseases. In conclusion, this study suggest the need for (i) regular monitoring of traffic wardens for possible exposure to benzene, as a precautionary step to reduce the associated health risks, and (ii) more comprehensive studies in order to better elucidate the involvement of APEX1 genotypes in benzene genotoxicity.     

Modeling delay to diagnosis for Amyotrophic lateral sclerosis: under reporting and incidence estimates

Background

This paper provides a strategy to obtain a reliable estimate of the incidence rate for Amyotrophic lateral sclerosis based on data from the National Registry of Rare Diseases (NRRD). In fact, unobserved cases may be due to the fact that “a long time” may intercour between the suspect of having the disease (onset) and the date the disease is diagnosed. Potential factors that may influence the probability of experiencing the event (diagnosis) conditionally on the onset (suspected) are investigated. Since we are treating rare diseases, the role of social and economic factors is not that obvious; latent as well as observed factors may influence the delay to diagnosis.

Methods

We use a semiparametric estimator based on the distribution of delay to diagnosis to account for potential underreporting. In particular, we propose to adopt an Horvitz-Thompson based estimator to correct the incidence figure that can be derived for the period 2007-2009 from the NRRD, Italy.

Results

The incidence estimates obtained by adopting the proposed approach are about 1 case per 100000 inhabitants and despite they let recovering a good part of underreporting, they are still far from ALS incidence international ranges between 1.5 and 2.5. However, by looking only at northern Italy, the incidence estimates we can derive are coherent with those known internationally.

Conclusions

These results confirm the existence of substantial differences in reporting accuracy, and point out where the system of data collection must be improved. In particular, when reliable individual characteristics will be available, they could be employed to refine the proposed estimator.

     

G-quadruplex-forming oligonucleotide conjugated to magnetic nanoparticles: synthesis, characterization, and enzymatic stability assays

In the present work, we report the conjugation of superparamagnetic nanoparticles to a fluorescently labeled oligodeoxyribonucleotide (ODN) able to fold into stable unimolecular guanine quadruple helix under proper ion conditions by means of its thrombin-binding aptamer (TBA) sequence. The novel modified ODN, which contained a fluorescent dU(Py) unit at 3'-end and a 12-amino-dodecyl spacer (C(12)-NH(2)) at 5' terminus, was characterized by ESI-MS and optical spectroscopy (UV, CD, fluorescence), and analyzed by RP-HPLC chromatography and electrophoresis. From CD and fluorescence experiments, we verified that dU(Py) and C(12)-NH(2) incorporation does not interfere with the conformational stability of the G-quadruplex. Subsequently, the conjugation of the pyrene-labeled ODN with the magnetite particles was performed, and the ODN-conjugated nanoparticles were studied through optical spectroscopy (UV, CD, fluorescence) and by enzymatic and chemical assays. We found that the nanoparticles enhanced the stability of the TBA ODN to enzymatic degradation. Finally, we evaluated the amount of the TBA-conjugated nanoparticles immobilized on a magnetic separator in view of the potential use of the nanosystem for the magnetic capture of thrombin from complex mixtures.     

Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol

Background aimsWe have recently shown that thymoglobulin (TG) efficiently expands cytokine-induced killer (CIK) cells in combination with interferon (IFN)-γ and interleukin (IL)-2 (ITG2 protocol). It is presently unknown whether the infusion of autologous immune effector cells generated by TG, IFN-γ and IL-2 is feasible and safeMethodsFive patients with advanced and/or refractory solid tumors were enrolled in the present phase I/II study. Peripheral blood mononuclear cells (PBMC) collected by leukapheresis were stimulated under good manufacturing practice (GMP)-conditions with IFN-γ, followed by TG and IL-2. After 2–3 weeks in culture, a median of 4.65 × 10⁶ immune effector cells per kilogram of recipient's body weight was obtained and infused intravenously. The median time from enrollment into the study to infusion of the expanded CIK cells was 30 daysResultsITG2 efficiently expanded immune effector cells that comprised both conventional natural killer (NK) cells and CD3⁺ CD16⁺ CD56⁺ CIK cells. One patient with advanced melanoma died because of disease progression before the infusion of CIK cells. The target dose of at least 2.5 × 10⁶ CIK cells/kg of recipient's body weight was reached in four out of five evaluable patients. CIK cells were administered intravenously without any measurable toxicity. In vitro, CIK cells exerted lytic activity against cervical cancer cells. The median survival was 4.5 months (range 1–13) from the first infusion of CIK cells.ConclusionsThis study has highlighted the feasibility and safety of the administration of CIK cells generated with the ITG2 protocol. Whether CIK cells can help control disease burden in patients with advanced malignancies will be determined in future clinical trials.     

The role of superior temporal cortex in auditory timing

Recently, there has been upsurge of interest in the neural mechanisms of time perception. A central question is whether the representation of time is distributed over brain regions as a function of stimulus modality, task and length of the duration used or whether it is centralized in a single specific and supramodal network. The answers seem to be converging on the former, and many areas not primarily considered as temporal processing areas remain to be investigated in the temporal domain. Here we asked whether the superior temporal gyrus, an auditory modality specific area, is involved in processing of auditory timing. Repetitive transcranial magnetic stimulation was applied over left and right superior temporal gyri while participants performed either a temporal or a frequency discrimination task of single tones. A significant decrease in performance accuracy was observed after stimulation of the right superior temporal gyrus, in addition to an increase in response uncertainty as measured by the Just Noticeable Difference. The results are specific to auditory temporal processing and performance on the frequency task was not affected. Our results further support the idea of distributed temporal processing and speak in favor of the existence of modality specific temporal regions in the human brain.     

Evaluation of microvascular density in tumors: pro and contra

Microvascular density (MVD) counting protocols have become the morphological gold standard to assess the neovasculature in human tumors. This method requires the use of specific markers to vascular endothelium and of immunohistochemical procedures to visualize microvessels. MVD determined in primary tumors is significantly associated with metastasis and prognosis in several tumors and is most predictive in those tumors that induce significant angiogenesis, namely carcinomas of breast and prostate, and haematological malignancies. There is such a wide range of antibodies and suppliers, antigen retrieval methods, designation of high and low vessel count groups, patient study groups and data interpretation, that it is exceedingly difficult to compare results.     

Patterns of molecular evolution associated with two selective sweeps in the Tb1-Dwarf8 region in maize

We focused on a region encompassing a major maize domestication locus, Tb1, and a locus involved in the flowering time variation, Dwarf8 (D8), to investigate the consequences of two closely linked selective sweeps on nucleotide variation and gain some insights into maize geographical diffusion, through climate adaptation. First, we physically mapped D8 at approximately 300 kb 3' of Tb1. Second, we analyzed patterns of nucleotide variation at Tb1, D8, and seven short regions (400-700 bp) located in the Tb1-D8 region sequenced on a 40 maize inbred lines panel encompassing early-flowering temperate and late-flowering tropical lines. The pattern of polymorphism along the region is characterized by two valleys of depleted polymorphism while the region in between exhibits an appreciable amount of diversity. Our results reveal that a region approximately 100 kb upstream of the D8 gene exhibits hallmarks of divergent selection between temperate and tropical lines and is likely closer than the D8 gene to the target of selection for climate adaptation. Selection in the tropical lines appears more recent than in the temperate lines, suggesting an initial domestication of early-flowering maize. Simulation results indicate that the polymorphism pattern is consistent with two interfering selective sweeps at Tb1 and D8.     

Levels of uroporphyrinogen decarboxylase (URO-D) in erythrocytes of Italian porphyria cutanea tarda patients.

Porphyria cutanea tarda (PCT) is a human metabolic disorder due to the acquired or genetic impairment of uroporphyrinogen decarboxylase (URO-D) activity, the fifth enzyme of the heme biosynthetic pathway. A classification of inherited and non-inherited forms is based on the enzyme activity levels in red blood cells (RBC). Clinical manifestations of PCT are often precipitated by triggering factors such as alcohol, drug abuse, estrogens, virus infections, hepatotoxic chemicals and hepatic siderosis. We measured URO-D activity in RBC from a large sample of Italian PCT patients in order to define the enzyme activity distribution and to attempt a correlation among activity, risk factors and clinical outcome. Three classes of patients with low, normal and over-normal URO-D activity were defined according to control values. Low URO-D levels were present in 25.8% of patients, suggesting the familial form of PCT (type II). In this group, the outcome of PCT seems to be less influenced by risk factors. Patients with over-normal URO-D activity in RBC deserve further investigation.     

Synthesis and evaluation of new endomorphin analogues modified at the Pro2 residue

Six new endomorphin analogues, incorporating constrained amino acids in place of native proline have been synthesized. Residues of (S)-azetidine-2-carboxylic acid (Aze), 3,4-dehydro-(S)-proline (Δ³Pro), azetidine-3-carboxylic acid (3Aze) and dehydro-alanine (ΔAla) have been used to prepare [Δ³Pro²]EM-2 (1), [Aze²]EM-1 (2), [Aze²]EM-2 (3), [3Aze²]EM-1 (4), [3Aze²]EM-2 (5) and [ΔAla²]EM-2 (6). Binding assays and functional bioactivities for μ- and δ-receptors are reported. The highest affinity, bioactivity and selectivity are shown by peptides 2 and 3 containing the Aze residue.