Sortilin Expression Is Essential for Pro-Nerve Growth Factor-Induced Apoptosis of Rat Vascular Smooth Muscle Cells

Background

Sortilin, a member of the Vps10p-domain receptor family, has been demonstrated a key regulator in mediating cellular response to pro-neurotrophins. In the present study, we investigated the role of sortilin in the apoptotic pathway of vascular smooth muscle cells.

Methods and Principal Findings

Immunohistochemistry revealed that sortilin was barely detectable in human and rat normal young vessels, while its expression was increased in human fibroatheromatous plaques. Sortilin immunodetection was also marked in the neointima of the rat aorta fifteen days after ballooning. In vitro, rat aortic intimal cells expressed higher sortilin levels than normal media SMCs; sortilin was distributed in the cytoplasm and in correspondence of the cell membrane. After 48 h, pro-nerve growth factor (proNGF) induced the strong dose-dependent increase of intimal cell apoptosis and the accumulation of sortilin protein. ProNGF was a more potent apoptotic inducer than equimolar or even higher concentration of NGF, whereas brain derived neutrotrophic factor was ineffective. Targeted interfering RNA-mediated sortilin reduction counteracted proNGF-induced apoptosis without affecting p75^NTR expression. ProNGF-induced apoptosis was associated to NF-κB down-regulation and bax increase. Inhibition of NF-κB activity increased intimal cell apoptosis that did not further increase with the addition of proNGF.

Conclusions

Our results indicate that sortilin expression characterizes human atheromatous lesions and rat aortic post-injury neointima, and suggest that sortilin represents an important regulator of proNGF-induced SMC apoptosis and arterial remodeling.     

Deletion in the EVC2 Gene Causes Chondrodysplastic Dwarfism in Tyrolean Grey Cattle

During the summer of 2013 seven Italian Tyrolean Grey calves were born with abnormally short limbs. Detailed clinical and pathological examination revealed similarities to chondrodysplastic dwarfism. Pedigree analysis showed a common founder, assuming autosomal monogenic recessive transmission of the defective allele. A positional cloning approach combining genome wide association and homozygosity mapping identified a single 1.6 Mb genomic region on BTA 6 that was associated with the disease. Whole genome re-sequencing of an affected calf revealed a single candidate causal mutation in the Ellis van Creveld syndrome 2 (EVC2) gene. This gene is known to be associated with chondrodysplastic dwarfism in Japanese Brown cattle, and dwarfism, abnormal nails and teeth, and dysostosis in humans with Ellis-van Creveld syndrome. Sanger sequencing confirmed the presence of a 2 bp deletion in exon 19 (c.2993_2994ACdel) that led to a premature stop codon in the coding sequence of bovine EVC2, and was concordant with the recessive pattern of inheritance in affected and carrier animals. This loss of function mutation confirms the important role of EVC2 in bone development. Genetic testing can now be used to eliminate this form of chondrodysplastic dwarfism from Tyrolean Grey cattle.     

Diversity of Capsular Polysaccharide Gene Clusters in Kpc-Producing Klebsiella pneumoniae Clinical Isolates of Sequence Type 258 Involved in the Italian Epidemic

Strains of Klebsiella pneumoniae producing KPC-type beta-lactamases (KPC-Kp) are broadly disseminating worldwide and constitute a major healthcare threat given their extensively drug resistant phenotypes and ability to rapidly disseminate in healthcare settings. In this work we report on the characterization of two different capsular polysaccharide (CPS) gene clusters, named cps _BO-4 and cps _207-2, from two KPC-Kp clinical strains from Italy belonging in sequence type (ST) 258, which is one of the most successful ST of KPC-Kp spreading worldwide. While cps _BO-4 was different from known 78 K-types according to the recently proposed typing schemes based on the wzi or wzc gene sequences, cps _207-2 was classified as K41 by one of these methods. Bioinformatic analysis revealed that they were represented in the genomic sequences of KPC-Kp from strains of ST258 from different countries, and cps _BO-4 was also detected in a KPC-Kp strain of ST442 from Brazil. Investigation of a collection of 46 ST258 and ST512 (a single locus variant of ST258) clinical strains representative of the recent Italian epidemic of KPC-Kp by means of a multiplex PCR typing approach revealed that cps _BO-4 was the most prevalent type, being detected both in ST258 and ST512 strains with a countrywide distribution, while cps _207-2 was only detected in ST258 strains with a more restricted distribution.     

Exploiting the repertoire of CK2 inhibitors to target DYRK and PIM kinases

Advantage has been taken of the relative promiscuity of commonly used inhibitors of protein kinase CK2 to develop compounds that can be exploited for the selective inhibition of druggable kinases other than CK2 itself. Here we summarize data obtained by altering the scaffold of CK2 inhibitors to give rise to novel selective inhibitors of DYRK1A and to a powerful cell permeable dual inhibitor of PIM1 and CK2. In the former case one of the new compounds, C624 (naphto [1,2-b]benzofuran-5,9-diol) displays a potency comparable to that of the first-in-class DYRK1A inhibitor, harmine, lacking however the drawback of drastically inhibiting monoamine oxidase-A (MAO-A) as harmine does. On the other hand the promiscuous CK2 inhibitor 4,5,6,7-tetrabromo-1H-benzimidazole (TBI,TBBz) has been derivatized with a sugar moiety to generate a 1-(β-D-2′-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole (TDB) compound which inhibits PIM1 and CK2 with comparably high efficacy (IC₅₀ values < 100 nM) and remarkable selectivity. TDB, unlike other dual PIM1/CK2 inhibitors described in the literature is readily cell permeable and displays a cytotoxic effect on cancer cells consistent with concomitant inhibition of both its onco-kinase targets. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Essential‐Oil Composition of Helichrysum italicum (Roth) G.Don ssp. italicum from Elba Island (Tuscany,Italy)

The composition of 21 essential‐oil samples isolated from Helichrysum italicum collected in seven locations of Elba Island (Tuscany, Italy), characterized by different soil types, during three different periods (January, May, and October 2010) was determined by GC‐FID and GC/EI‐MS analyses. In total, 115 components were identified, representing 96.8–99.8% of the oil composition. The oils were characterized by a high content of oxygenated monoterpenes (38.6–62.7%), while monoterpene and sesquiterpene hydrocarbons accounted for 2.3–41.9 and 5.1–20.1% of the identified constituents, respectively. The main oxygenated derivatives were nerol (2.8–12.8%) and its ester derivative neryl acetate (5.6–45.9%). To compare the chemical variability of the species within Elba Island and between the island and other localities within the Mediterranean area, studied previously, multivariate statistical analysis was performed. The results obtained showed a difference in the composition of the essential oils of H. italicum from Elba Island, mainly due to the environment where the plant grows, and, in particular, to the soil type. These hypotheses were further confirmed by the comparison of these oils with essential oils obtained from H. italicum collected on other islands of the Tuscan archipelago.     

Using multiple sources of characters to delimit species in the genus Crataegus (Rosaceae): the case of the Crataegus rosei complex

The aim of this study was to delimit the taxa of the Crataegus rosei complex using an integrative approach that incorporates a suite of molecular (cpDNA and nuclear microsatellite markers), morphological, and geometric morphometric characters. One hundred and ten plants from 19 populations that encompass the entire distribution range of the species complex were collected and examined along with herbarium specimens. Parsimony and Bayesian inference analyses were run using morphological, molecular, and both the morphological and molecular data sets combined. Analyses to determine genetic structure based on microsatellite data and multivariate analyses incorporating geometric morphometrics were also done to identify differences in leaf shape. The results supported the recognition of two taxa: C. rosei with high levels of gene flow among its populations, remarkable morphological variation and a wide distribution range and C. rosei var. amoena, composed of a few isolated populations in the high elevation location of Cerro Potosí; a new specific epithet will be decided for the latter in accordance with the International Code of Nomenclature for algae, fungi, and plants.     

Tamoxifen in breast cancer ipse dixit in uterine malignant mixed Müllerian tumor and sarcoma—A report of 8 cases and review of the literature

Aim

Report the outcome of 8 patients (pts) with breast cancer (BC) treated with Tamoxifen (TAM) that developed malignant mixed Müllerian tumor (MMMT) and rare uterine sarcoma (RUS).

Patients and methods

Retrospective study based on data collected from the department medical records between April 1999 and September 2010 among 583 pts with endometrial cancer, 36 pts with MMMT and RUS histopathology. Among them, 8 pts underwent TAM between 4 and 10 years due to a previous diagnosis of BC; all pts were post-menopausal with regular gynecological surveillance; 6 pts (75%) with abnormal uterine bleeding. The diagnosis of 6 pts (MMMT) and 2 pts (RUS) occurred at median interval of 8 years (range 4–12) after initial BC treatment. Pts underwent surgical treatment and were staged as stage I (3pts), IIIA (3pts) and IIIC (2 pts) (FIGO 1988); followed by whole pelvis irradiation (50 Gy) and intracavitary HDR brachytherapy boost (24 Gy). Two pts underwent chemotherapy (CT). Overall and disease free survival was calculated by Kaplan Meier method.

Results

With a median follow-up of 47 months (range 17–130), 3 pts remain alive recurrence-free of BC and RUS. Four pts died with distant metastasis within the first follow-up year, without BC. One pt died from non-related cancer cause. No evidence of local recurrence was found in the whole group of pts. At two years, DFS and OS were 40% and 80%, respectively.

Conclusion

As reported in the literature, TAM administration and causal effect on MMMT and RUS in BC pts is still unknown. No reports about outcome from these specific pts were found.     

Chirally-Modifiedoligonucleotides and the Control of Gene Expression. The Case of L-DNAS And-RNAS

Abstract

The affinity of L-DNAs, L-RNAs and L/D-DNAs for homopurine homopyrimidine d.s. D-DNA and s.s. D-RNA was probed by gel electrophoresis and CD spectroscopy. It was found that the L-modified oligomers do not bind to d.s. DNA and to natural RNA that contains all four natural bases. Thus they cannot be used, in general, for the control of gene expression according to the antigene and antisense methodologies. Heterochiral complexes with 1:1 stoichiometry and low thermal stability are formed, instead, by homopurinic L-RNA or L/D-DNA and homopyrimidinic L-RNA with the W/C complementary natural RNA sequences.     

Occupational exposure to anaesthetic gases and urinary excretion of D-glucaric acid

In order to ascertain whether the urinary excretion of D-glucaric acid (DGA) might be a suitable biomarker of effect in monitoring workers exposed to anaesthetic gases, we measured DGA before and after an operating session (and, in some workers, before and after a 2-week vacation) in 229 workers of surgical units and in 229 controls. In the former, we also measured urinary levels of nitrous oxide (N₂O) and isofiurane after at least 4 h of exposure. For all subjects, information on age, smoking habits, daily intake of alcohol, coffee, and drugs, history of liver or kidney disease was collected. Study subjects were ranked according to: exposure (class 0: subjects not exposed; class 1: N₂