Versatile and precise gene-targeting strategies for functional studies in mammalian cell lines.

The advent of programmable nucleases such as ZFNs, TALENs and CRISPR/Cas9 has brought the power of genetic manipulation to widely used model systems. In mammalian cells, nuclease-mediated DNA double strand break is mainly repaired through the error-prone non-homologous end-joining (NHEJ) repair pathway, eventually leading to accumulation of small deletions or insertions (indels) that can inactivate gene function. However, due to the variable size of the indels and the polyploid status of many cell lines (e.g., cancer-derived cells), obtaining a knockout usually requires lengthy screening and characterization procedures. Given the more precise type of modifications that can be introduced upon homology-directed repair (HDR), we have developed HDR-based gene-targeting strategies that greatly facilitate the process of knockout generation in cell lines. To generate reversible knockouts (R-KO), a selectable promoter-less STOP cassette is inserted in an intron, interrupting transcription. Loss-of-function can be validated by RT-qPCR and is removable, enabling subsequent restoration of gene function. A variant of the R-KO procedure can be used to introduce point mutations. To generate constitutive knockouts (C-KO), an exon is targeted, which makes use of HDR-based gene disruption together with NHEJ-induced indels on non-HDR targeted allele(s). Hence the C-KO procedure greatly facilitates simultaneous inactivation of multiple alleles. Overall these genome-editing tools offer superior precision and efficiency for functional genetic approaches. We provide detailed protocols guiding in the design of targeting vectors and in the analysis and validation of gene targeting experiments.

Physical activity and cancer prevention: a review of current evidence and biological mechanisms.

OBJECTIVE: The main aim of this paper is to review the evidence available from the date of PubMed's inception to May 2011 for a link between cancer and physical activity (PA) in both animal models and humans. METHODS: We decided to select studies that comply with the scheme proposed by the American College of Sports Medicine/American Heart Association (ACSM/AHA) that distinguish occupational physical activity (OPA) and leisure-time physical activity (LT-PA), further classified in three levels of intensity (low, moderate and heavy) based on the Metabolic Equivalent of Task (MET) index. RESULTS: Considering animal models, there was strong evidence for an inverse association between voluntary wheel exercise and the risk of colon and breast cancer. Regarding human studies, we identified the following main results: 1) colorectum: LT-PA provided an overall colon risk reduction of 13-14%; 2) breast: significant reduction in the frequency of post-menopausal (PMP) cancers in women that practiced heavy and moderate LT-PA; 3) prostate: heavy OPA and LT-PA seemed to reduce the risk of advanced prostate cancers; 4) endometrium: strong protective effect of heavy/moderate LT-PA among overweight/ obese women; 5) lung: inverse relationship between heavy LT-PA and lung cancer in former or current smokers across all histologies. CONCLUSION: Increased LT-PA is associated with cancer prevention in several organs, but strong biases, such as body mass index (BMI), gender and age, make it difficult to assess which aspects of PA contribute most strongly to the reduced risk. Furthermore, we found few studies that indicated a protective role for OPA in cancer prevention when compared with LT-PA.

[Healthy design for sustainable communities]. / Sistema multicriteriale di valutazione degli aspetti igienico- sanitari di piani di governo del territorio e piani attuativi.

Health, quality of life and sustainable development are strongly interconnected. The quality of living is a complex concept that includes different meanings. The quality of life issue has been studied for a long time, even if its measurement is a more recent matter. It's possible to distinguish two main approaches: the first one, depending on which the quality of life corresponds to the social wellbeing and it can be measured objectively; the second one, that emphasizes the perceptive dimension of quality of life, such as needs, feelings and aspirations. According to the WHO's wide definition of wellbeing, this paper suggests an approach focused on the effects that urban planning and designing can have on the health of citizens. Actually many of the problems of the cities like pollution, inequity, lack of services and accessibility depends on decisions about the development of land and buildings. To have more attractive cities in the future it is important that professionals involved in planning and local authorities focus on the major determinants of health: the physical and social environment in which people live and the nature of their lifestyles. The experience explained in this paper shows as local authorities can support professionals in designing process, producing quick and effective benchmark in order to improve the quality of urban spaces and architecture. More in deep the tool works by a set of performance indicators developed with the purpose to assess the degree of sustainability of building and urban space proposals at the planning stage (and at later stages), against a range of criteria. This evaluation procedure can be considered as a common platform from which different stakeholders can agree goals and work together contributing to increase the benefits of a well-designed built environment.

Gene-environment interactions in ocular diseases.

Degenerative ocular diseases are widespread in the population and represent a major cause of reversible and irreversible blindness. Scientific evidences have been accumulating supporting the role of genotoxic damage and gene environment interactions in the pathogenesis of these diseases mainly including glaucoma, age-related macular degeneration, and cataract. Glaucoma, in its degenerative form, is characterized by the degeneration of the trabecular meshwork, the tissue of the anterior chamber of the eye devoted to aqueous-humour outflow. Such a degenerative process results in intra-ocular pressure increase and progressive damage of optic nerve head. Oxidative stress and DNA damage play an important role in inducing the degeneration of these well differentiated target tissues in which DNA damage results in a progressive cell loss. Macular degeneration is a common age-related disease affecting the central regions of the retina inducing progressive accumulation of oxidized lipoproteins and neovascularization. Environmental genotoxic risk factors include diet, light, and cigarette smoke paralleled by individual susceptibility as determined by adverse genetic assets. Cataract is a progressive opacity of the crystalline lens resulting from molecular damages induced by various risk factors including UV-containing light. This disease has been related to a failure in antioxidant defences. Experimental study provides evidence that cataract patients possess higher basal level of DNA damage, as evaluated by Comet test, in lymphocytes than controls. This finding is paralleled by the higher susceptibility to oxidative stress observed in the same patients. These novel experimental data further support the role of DNA damage as a main factor contributing to cataract onset. In conclusion, the examined degenerative ocular diseases recognise environmental risk factors often displaying genotoxic attitudes. Whenever these factors target individuals who are susceptible due their genetic assets the results is the onset of a specific eye disease depending on the affected ocular tissue.

A Clostridium difficile outbreak in an Italian hospital: the efficacy of the multi-disciplinary and multifaceted approach.

INTRODUCTION: We described an outbreak of C. difficile that occurred in the Internal Medicine department of an Italian hospital and assessed the efficacy of the measures adopted to manage the outbreak. METHODS: The outbreak involved 15 patients and was identified by means of continuous integrated microbiological surveillance, starting with laboratory data (alert organism surveillance). Diarrheal fecal samples from patients with suspected infection by C. difficile underwent rapid membrane immuno-enzymatic testing, which detects both the presence of the glutamate dehydrogenase antigen and the presence of the A and B toxins. Extensive microbiological sampling was carried out both before and after sanitation of the environment, in order to assess the efficacy of the sanitation procedure. RESULTS: The outbreak lasted one and a half month, during which time the Committee for the Prevention of Hospital Infections ordered the implementation of multiple interventions, which enabled the outbreak to be controlled and the occurrence of new cases to be progressively prevented. The strategies adopted mainly involved patient isolation, reinforcement of proper hand hygiene techniques, antimicrobial stewardship and environmental decontamination by means of chlorine-based products. Moreover, the multifaceted management of the outbreak involved numerous sessions of instruction/training for nursing staff and socio-sanitary operatives during the outbreak. Sampling of environmental surfaces enabled two sites contaminated by C. difficile to be identified. CONCLUSIONS: Joint planning of multiple infection control practices, together with effective communication and collaboration between the Hospital Infections Committee and the ward involved proved to be successful in controlling the outbreak.

Temporal sequence of changes in central noradrenergic system of rat after prolonged antidepressant treatment: receptor desensitization and neurotransmitter interactions.

It has been shown that different receptor components may be involved in the adaptive changes occurring in noradrenergic (NE) neurones after prolonged periods of exposure to antidepressant drugs. In this report the desensitization of NE-coupled adenylate cyclase (NE-AC). beta-adrenergic receptors and [3H]imipramine ([3H]-IMI) or [3H]desipramine ([3H]-DMI) binding sites have been temporally correlated with in vivo changes of NE utilization. Normetanephrine (NMN) was measured as indicator of NE synaptic events involved in antidepressant action. Concentrations of normetanephrine were increased after acute desipramine (DMI), viloxazine and mianserin administration. Following 3 days of treatment, the antidepressant-induced increase of normetanephrine became tolerant and NE neurones were resistant to the antidepressant effect until the 15th day of treatment. After two weeks, DMI elicited a significant decrease in the content of normetanephrine. A different pattern of changes has been found in the temporal modification of [3H]-IMI recognition sites, beta-adrenoceptors and NE-AC activity after chronic DMI treatment. Binding sites and receptors were down regulated after 10 days of treatment preceding the decrease in normetanephrine content. No down-regulation was observed in [3H]-DMI binding sites. Studies on the effects of antidepressants during brain maturation revealed that the mechanisms which cause desensitization of beta-receptors and [3H]-IMI binding sites appear in the early stages of postnatal life. Since [3H]-IMI and [3H]-DMI recognition sites have been shown to be located on serotonergic (5-HT) and noradrenergic neurones respectively, the interactions between NE and 5-HT neurones could represent possible mechanisms implicated in receptor desensitization. The experiments presented involving lesions of 5-HT neurones have clearly demonstrated that NE release in rat cerebral cortex is under a tonic serotonergic influence. Alterations in the chemico-physical properties of the synaptic membranes might be also taken in consideration for the mechanisms underlying receptor modulation. In fact, evidence is provided that in neural tissue phospholipid methylation can be affected. In conclusion, the temporal sequence of changes in cortical noradrenergic neurones, after chronic antidepressant treatment, has demonstrated that integrated mechanisms are operative for the function of the overall system.

Apomorphine-induced inhibition of histamine release in rat peritoneal mast cells.

The apomorphine-induced inhibition of histamine release in rat peritoneal mast cells was studied by means of secretagogues stimulating different pathways of mast cell activation. Apomorphine inhibited the mast cell response to all releasing agents (lysophosphatidylserine plus nerve growth factor, compound 48/80, substance P, ATP, tetradecanoylphorbolacetate, melittin). The IC50 ranged from 4 microM to 24 microM at concentrations of secretagogues releasing 30-50% of mast cell histamine. However, the potency of the drug decreased at higher secretagogue concentrations. Mast cells, pretreated with apomorphine and washed, released little histamine upon stimulation. The secretory response could be partially restored on increasing the concentration of secretagogues. The results suggest that apomorphine affects a regulatory step controlling the terminal sequence of mast cell secretory activity. As indicated by the reduced potency of the drug, the control by the apomorphine-sensitive reaction loses efficiency under conditions of massive histamine release.

Age dependent changes in the methylation of rat brain phospholipids.

Phospholipid methyltransferase I and II enzymes methylate phosphatidylethanolamine three times to form phosphatidylcholine. The activity of these two enzymes was determined in synaptosome-enriched fractions from rats 1, 3, 7, 15 and 21 months old. The activity of phospholipid methyltransferase I was significantly greater in 7-, 15- and 21-month-old rats than in 1- and 3-month-old rats. In contrast, the activity of phospholipid methyltransferase II did not change with age. These changes in methyltransferase activity with increasing age may be related to changes in beta-receptor function with increasing age.

Synergism between lysophosphatidylserine and the phorbol ester tetradecanoylphorbolacetate in rat mast cells.

In rat peritoneal mast cells tetradecanoylphorbolacetate (TPA) induced a non cytotoxic histamine release in the absence of extracellular calcium. The addition of calcium prevented the TPA effect but micromolar concentrations of lysophosphatidylserine (lysoPS) converted the calcium-induced inhibition into a stimulation. Other lysophospholipids were inactive. In agreement with a mutual influence between lysoPS and TPA, minimal TPA concentrations enhanced the calcium-dependent histamine release induced by lysoPS in the presence of nerve-growth factor. It is proposed that the calcium-dependent pathway promoted by lysoPS and the activation of protein kinase C by TPA act synergically to induce histamine release from mast cells.

Spontaneous partial regression of low-grade glioma in children with neurofibromatosis-1: a real possibility.

At the age of 41 and 31 months, respectively, a boy and a girl affected by neurofibromatosis-1 were diagnosed with a visual pathway glioma during surveillance contrast-enhanced head magnetic resonance imaging (MRI). In the first child, the initial MRI showed that the entire optic chiasm, the intracranial tract of the left optic nerve, and hypothalamus were grossly enlarged and enhanced in the post-gadolinium T1-weighted images. Ten months later, the hypothalamic component of the lesion had regressed markedly and there were no more areas of contrast enhancement. In the second child, the initial MRI showed that the optic chiasm, the right optic tract, and geniculate body were enlarged and enhanced after gadolinium injection. At 6-month follow-up, the MRI showed that the right optic tract and the anterior aspect of the optic chiasm decreased in size and the contrast enhancement of the entire lesion was reduced dramatically. These findings, as indicated by other similar reports, confirm that spontaneous regression of visual pathway glioma is a rare but real possibility in children with neurofibromatosis-1. Therefore, clinicians need to be aware of visual pathway glioma's erratic behavior in children with neurofibromatosis-1 with special attention given to the importance of a very conservative attitude toward any type of treatment for such patients.

Modulation of lysophosphatidylserine-dependent histamine release.

Apomorphine (2-30 microM) inhibits lysophosphatidyl-serine-dependent histamine release in rat and mouse peritoneal mast cells. The drug-induced inhibition is influenced by the concentration of lysophosphatidylserine. Log concentration-response curves show a surmountable type of antagonism between the two compounds.

Inhibition of protein kinase activity by apomorphine.

The effect of apomorphine (1-20 microM) on protein kinase activity was studied in extracts from rat peritoneal mast cells and brain tissue. Apomorphine inhibited the cyclic AMP-dependent and the calcium-plus phosphatidylserine-dependent protein kinase activity with an IC50 between 1 and 6 microM, depending on the tissue and on the protein kinase involved. This effect might explain previous results on the apomorphine-induced inhibition of histamine release in rat peritoneal mast cells.

Biochemical changes of rat brain membranes with aging.

Modification of membrane composition and enzymatic activities both in total brain homogenate and purified synaptic plasma membrane of 3 and 24 month old rats has been investigated. Protein, cholesterol and phospholipid content and (Na+, K+)ATPase and 2',3' cyclic nucleotide phosphohydrolase activities were determined. The major changes occurred in the whole homogenate where a general increase in total protein and cholesterol content with age and a significant increase of the cholesterol/phospholipids molar ratio has been detected. In S.P.M. aging process induced a decrease of protein, cholesterol and phospholipids content associated with an increased membrane viscosity and a decrease of delta E. These data are consistent with a change in the structural organization and in the distribution pattern of different cell population in the aging brain. A possible artifactual effect of freezing on the reported parameter is also discussed.

Serine phospholipids as endocoids.

Unusual phospholipid effects may occur when their distribution in the membrane is altered or when uncontrolled metabolic reactions yield elevated concentrations of their short lived derivatives. Serine phospholipids are normally buried in the internal side of plasma membrane. Upon exposure to the extracellular environment they elicit a response from selected cell populations. The interaction between these phospholipids and neuroactive compounds in rat peritoneal mast cells may indicate that serine phospholipids have a role in the nervous system during development.

Lysophosphatidylserine as histamine releaser in mice and rats.

Lysophosphatidylserine is a specific inducer of histamine release in isolated mast cells. To determine whether a similar effect is manifest in vivo, the phospholipid was injected (1-5 mg/kg i.v.) into mice and rats. A dose-dependent rise in blood histamine was observed in both animals. The several-fold increase in blood histamine occurred in the first minutes and was followed by a slower decline toward normal values. A second dose of lysophosphatidylserine was without effect. Systemic manifestations (depression, hypothermia, hypotension) were associated with the increased blood histamine level. When the tissue histamine stores accessible to lysophosphatidylserine were previously decreased by repeated phospholipid injections, no systemic symptoms occurred. Mobilization of carbohydrate reserves was also manifest during the action of lysophosphatidylserine. Prior treatment with compound 48/80 induced sustained refractoriness to lysophosphatidylserine. Structure-activity relationship demonstrated that the property to induce histamine release was linked to the structure of serine head group. Thus, other natural phospholipids or lysophospholipids were inactive. It is concluded that in analogy with the effect seen in vitro lysophosphatidylserine produces in vivo release of mast cell histamine.

Phospholipid methylation increases [3H]diazepam and [3H]GABA binding in membrane preparations of rat cerebellum.

The effect of phospholipid methylation on both [3H]diazepam and [3H]GABA ( [3H]gamma-aminobutyric acid) binding to crude synaptic plasma membrane from rat cerebellum has been studied. S-Adenosylmethionine (SAM) stimulates [3H]methyl group incorporation into membrane phospholipids and enhances [3H]diazepam binding by increasing the apparent Bmax. Conversely, inhibition of [3H]methyl group transfer from [3H]SAM to phospholipids by preincubation with SAM at 0 degrees C or with SAH abolishes the increase of binding. After preincubation with SAM, analysis of the GABA binding reveals the presence of binding sites with high affinity, a property absent in control membranes preincubated without SAM. Among the neurotransmitter bindings tested, only those of GABA and benzodiazepine in the cerebellum and beta-adrenergic ligands in the cerebral cortex are enhanced upon stimulation of phospholipid methyltransferase activity. [3H]Dihydromorphine, [3H]dihydro-alpha-ergokryptine and [3H]spiroperidol bindings are not affected by SAM. The present data suggest an involvement of phospholipid methylation in regulation of both [3H]GABA and [3H]-diazepam binding.