Reversible disruption of pre-pulse inhibition in hypomorphic-inducible and reversible CB1-/- mice

Although several genes are implicated in the pathogenesis of schizophrenia, in animal models for such a severe mental illness only some aspects of the pathology can be represented (endophenotypes). Genetically modified mice are currently being used to obtain or characterize such endophenotypes. Since its cloning and characterization CB1 receptor has increasingly become of significant physiological, pharmacological and clinical interest. Recently, its involvement in schizophrenia has been reported. Among the different approaches employed, gene targeting permits to study the multiple roles of the endocannabinoid system using knockout ((-/-)) mice represent a powerful model but with some limitations due to compensation. To overcome such a limitation, we have generated an inducible and reversible tet-off dependent tissue-specific CB1(-/-) mice where the CB1R is re-expressed exclusively in the forebrain at a hypomorphic level due to a mutation (IRh-CB1(-/-)) only in absence of doxycycline (Dox). In such mice, under Dox(+) or vehicle, as well as in wild-type (WT) and CB1(-/-), two endophenotypes motor activity (increased in animal models of schizophrenia) and pre-pulse inhibition (PPI) of startle reflex (disrupted in schizophrenia) were analyzed. Both CB1(-/-) and IRh-CB1(-/-) showed increased motor activity when compared to WT animals. The PPI response, unaltered in WT and CB1(-/-) animals, was on the contrary highly and significantly disrupted only in Dox(+) IRh-CB1(-/-) mice. Such a response was easily reverted after either withdrawal from Dox or haloperidol treatment. This is the first Inducible and Reversible CB1(-/-) mice model to be described in the literature. It is noteworthy that the PPI disruption is not present either in classical full CB1(-/-) mice or following acute administration of rimonabant. Such a hypomorphic model may provide a new tool for additional in vivo and in vitro studies of the physiological and pathological roles of cannabinoid system in schizophrenia and in other psychiatric disorders.     

Adipokines,Ghrelin and Obesity‐Associated Insulin Resistance in Nondiabetic Patients with Acute Coronary Syndrome

Altered glucose metabolism negatively modulates outcome in acute coronary syndromes (ACS). Insulin resistance is commonly associated with increasing BMI in the general population and these associations may involve obesity‐related changes in circulating ghrelin and adipokines. We aimed at investigating interactions between BMI, insulin resistance and ACS and their associations with plasma ghrelin and adipokine concentrations. Homeostasis model assessment of insulin resistance (HOMA_IR)‐insulin resistance index, plasma adiponectin, leptin, total (T‐Ghrelin), acylated (Acyl‐Ghrelin), and desacylated ghrelin (Desacyl‐Ghrelin) were measured in 60 nondiabetic ACS patients and 44 subjects without ACS matched for age, sex, and BMI. Compared with non‐ACS, ACS patients had similar HOMA_IR and plasma adipokines, but lower T‐ and Desacyl‐Ghrelin and higher Acyl‐Ghrelin. Obesity (BMI > 30) was associated with higher HOMA_IR, lower adiponectin, and higher leptin (P < 0.05) similarly in ACS and non‐ACS subjects. In ACS (n = 60) HOMA_IR remained associated negatively with adiponectin and positively with leptin independently of BMI and c‐reactive protein (CRP) (P < 0.05). On the other hand, low T‐ and Desacyl‐Ghrelin with high Acyl‐Ghrelin characterized both obese and non‐obese ACS patients and were not associated with HOMA_IR. In conclusion, in ACS patients, obesity and obesity‐related changes in plasma leptin and adiponectin are associated with and likely contribute to negatively modulate insulin resistance. ACS per se does not however enhance the negative impact of obesity on insulin sensitivity. High acylated and low desacylated ghrelin characterize ACS patients independently of obesity, but are not associated with insulin sensitivity.     

B-RAF mutant alleles associated with Langerhans cell histiocytosis, a granulomatous pediatric disease

Background

Langerhans cell histiocytosis (LCH) features inflammatory granuloma characterised by the presence of CD1a+ dendritic cells or ‘LCH cells’. Badalian-Very et al. recently reported the presence of a canonical ^V600EB-RAF mutation in 57% of paraffin-embedded biopsies from LCH granuloma. Here we confirm their findings and report the identification of two novel B-RAF mutations detected in LCH patients.

Methods and Results

Mutations of B-RAF were observed in granuloma samples from 11 out of 16 patients using ‘next generation’ pyrosequencing. In 9 cases the mutation identified was ^V600EB-RAF. In 2 cases novel polymorphisms were identified. A somatic ^600DLATB-RAF insertion mimicked the structural and functional consequences of the ^V600EB-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The ^600DLATB-RAF and ^V600EB-RAF mutations were found enriched in DNA and mRNA from the CD1a+ fraction of granuloma. They were absent from the blood and monocytes of 58 LCH patients, with a lower threshold of sequencing sensitivity of 1%–2% relative mutation abundance. A novel germ line ^T599AB-RAF mutant allele was detected in one patient, at a relative mutation abundance close to 50% in the LCH granuloma, blood monocytes and lymphocytes. However, ^T599AB-RAF did not destabilize the inactive conformation of the B-RAF kinase, and did not induce increased ERK phosphorylation or C-RAF transactivation.

Conclusions

Our data confirmed presence of the ^V600EB-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that ^V600EB-RAF and ^600DLATB-RAF mutations are somatic mutants enriched in LCH CD1a⁺ cells and absent from the patient blood. Further studies are needed to assess the functional consequences of the germ-line ^T599AB-RAF allele.     

hERG1 Channels and Glut-1 as Independent Prognostic Indicators of Worse Outcome in Stage I and II Colorectal Cancer: A Pilot Study

BACKGROUND: There is a need to identify new markers to assess recurrence risk in early-stage colorectal cancer (CRC) patients. We explored the prognostic impact of ether-a-gò-gò-related gene 1 channels and some hypoxia markers, in patients with nonmetastatic (stage I, II, and III) CRC. METHODS: The expression of hERG1, vascular endothelial growth factor A (VEGF-A), glucose transporter 1, carbonic anhydrase IX (CA-IX), epidermal growth factor receptor (EGF-R), and p53 was tested by immunohistochemistry in 135 patients. The median follow-up was 35 months. Clinicopathologic parameters and overall survival were evaluated. RESULTS: hERG1 displayed a statistically significant association with Glut-1, VEGF-A, CA-IX, and EGF-R; p53 with VEGF-A and CA-IX; Glut-1 with the age of the patients; and EGF-R with TNM and mucin content. TNM and CA-IX were prognostic factors at the univariate analysis; TNM, hERG1, and Glut-1, at the multivariate analysis. Risk scores calculated from the final multivariate model allowed to stratify patients into four different risk groups: A) stage I-II, Glut-1 positivity, any hERG1; B) stage I-II, Glut-1 and hERG1 negativity; C) stage I-II, Glut-1 negativity, hERG1 positivity; D) stage III, any Glut-1 and any hERG1. CONCLUSIONS: hERG1 positivity with Glut-1 negativity identifies a patient group with poor prognosis within stage I-II CRC. The possibility that these patients might benefit from adjuvant therapy, independently from the TNM stage, is discussed. IMPACT: More robust prognostic and predictive markers, supplementing standard clinical and pathologic staging, are needed for node-negative patients.     

Expression, purification and characterisation of Haemophilus influenzae 3-isopropylmalate dehydrogenase (LeuB)

3-isopropylmalate dehydrogenase (3IPMDH) is the third enzyme in leucine biosynthesis and a promising target for the development of broad-spectrum antibacterial agents. We report here the expression, purification and biochemical characterisation of Haemophilus influenzae 3-isopropylmalate dehydrogenase. The observed enzyme inhibition by the reaction product NADH could represent a regulatory mechanism for 3IPMDH.     

Characterization of the Topography and Wettability of English Weed Leaves and Biomimetic Replicas

The topography and wettability of the underside of English weed （Oxalis pes-caprae） leaves and of their biomimetic replicas are investigated. Polyvinyl siloxane molds were cast from the leaves and then filled with an epoxy pre-polymer to produce replicas. The particular topographical structures of leaves and replicas were evaluated by optical microscopy and Scanning Electron Microscopy （SEM） analysis. The static wettability of leaves and replicas was assessed by contact angle measurements, while the dynamic wettability was characterized by estimating contact angle hysteresis and studying the dynamic behavior of impacting water droplets. A smooth glass slip and its replica were used as control surfaces. The replica moulding method used was able to transfer the characteristic pattern of irregular 100 μm - 200 μm × 60 μm convex papillae interspersed with stomata of the original leaf to the epoxy replicas. The static contact angle of 143°± 3° and the contact angle hysteresis of 2~ indicate that the underside of the English weed leaf is close to superhydrophobic. The lower contact angles （130° ± 4°） and higher hysteresis （31°） observed for the replica when compared with the original leaves were associated to an inaccurate replication of the chemistry and structures of the three-dimensional wax projections covering the plant surface. Also, trichomes in the original leaves could not be accurately reproduced due to their flexibility and fragility. Differences in wetting behavior were also evident from droplet impact experiments, with rebound regimes prevailing in the original leaves and regimes characterized by higher adhesion and larger dissipation predominating in the replicas. Nevertheless, the morphological features of the leaf transferred to the replica were sufficient to promote a clear hydrophobic behavior of the replica when compared with the smooth epoxy reference surface.     

Microbe removal in secondary effluent by filtration

A study was carried out to assess the efficiency of filtration in reducing microbial contamination in municipal secondary effluent. After primary and secondary treatments, the wastewater underwent filtration through sand/hydroanthracite filters. A total of 20 samplings were made, each consisting of two instant samples (secondary effluent and filtered effluent). Each of the 40 samples was tested for: total and faecal coliforms,Escherichia coli, enterococci and somatic coliphages. The mean concentrations detected in the secondary effluent were in the order of 5 log for the total and faecal coliforms, 4 log for enterococci andEscherichia coli, and approx. 3 log for the coliphages. The filtration showed a higher efficacy in the reduction of feacal coliforms,Escherichia coli and in particular total coliforms. The results obtained for enterococci and coliphages were significanty lower. Filtration alone was not enough to reduce the bacterial indicators to within Italian legal limits, and showed a poor capacity to abate coliphages. However, by performing the filby-products and a consequent reduction in the chemical risk for the general population.     

Synthesis and biological evaluation of tetracyclic fluoroquinolones as antibacterial and anticancer agents

A simple and efficient synthesis of 6-fluoro-4-oxopyrido[2,3-a]carbazole-3-carboxylic acids (13a–e) and a structurally related 6-fluoro-4-oxothieno[2′,3′:4,5]pyrrolo[3,2-h]quinoline (13f) was achieved via Stille arylation of 7-chloro-6-fluoro-8-nitro-4-oxoquinoline-3-carboxylate and a subsequent microwave-assisted phosphite-mediated Cadogan reaction. The new compounds were tested for their in vitro antimicrobial and antiproliferative activity. The ability of 13a–f to inhibit the activity of DNA gyrase and topoisomerase IV was also investigated. The thieno isostere (13f) emerged as the most active antibacterial, while the 9-fluoro derivative (13e) was the most potent against multidrug-resistant staphylococci. Compounds 13a, 13c–f displayed growth inhibition against MCF-7 breast tumor and A549 non-small cell lung cancer cells coupled with an absence of cytotoxicity toward normal human-derm fibroblasts (HuDe). Compound 13e was the most active anticancer against MCF-7 cells, with greater potency than ellipticine (IC₅₀ 0.8 and 1.6 μM, respectively). The most active compounds in this series show promise as dual acting anticancer and antibacterial chemotherapeutics.     

A Genetic Insight Into Peptide and Amino-Acid Utilization by Propionibacterium freudenreichii LMG 16415

In this note the genetic characterization of the peptide degrading system of Propionibacterium freudenreichii was addressed. Genomic fragments of P. freudenreichii subsp. freudenreichii LMG 16415 were cloned in Escherichia coli XL1 Blue, and those leading to an increase in peptidase-like activity using chromogenic substrates aminoacyl-β-naphtylamides (aminoacyl-βNA) were isolated and sequenced. This strategy allowed the identification of partial gene regions of P. freudenreichii LMG 16415 with significant similarity to proteins directly or indirectly involved in peptide and amino acid metabolism, i.e., an oligopeptide transporter, a D-amino acid oxidase, a muropeptidase, and an ABC transporter involved in osmoregulation similar to glycine betaine transporters.