Molecular basis of ageing in chronic metabolic diseases.

AIM: Over the last decades, the shift in age distribution towards older ages and the progressive ageing which has occurred in most populations have been paralleled by a global epidemic of obesity and its related metabolic disorders, primarily, type 2 diabetes (T2D). Dysfunction of the adipose tissue (AT) is widely recognized as a significant hallmark of the ageing process that, in turn, results in systemic metabolic alterations. These include insulin resistance, accumulation of ectopic lipids and chronic inflammation, which are responsible for an elevated risk of obesity and T2D onset associated to ageing. On the other hand, obesity and T2D, the paradigms of AT dysfunction, share many physiological characteristics with the ageing process, such as an increased burden of senescent cells and epigenetic alterations. Thus, these chronic metabolic disorders may represent a state of accelerated ageing. MATERIALS AND METHODS: A more precise explanation of the fundamental ageing mechanisms that occur in AT and a deeper understanding of their role in the interplay between accelerated ageing and AT dysfunction can be a fundamental leap towards novel therapies that address the causes, not just the symptoms, of obesity and T2D, utilizing strategies that target either senescent cells or DNA methylation. RESULTS: In this review, we summarize the current knowledge of the pathways that lead to AT dysfunction in the chronological ageing process as well as the pathophysiology of obesity and T2D, emphasizing the critical role of cellular senescence and DNA methylation. CONCLUSION: Finally, we highlight the need for further research focused on targeting these mechanisms.

Can nature conservation and wood production be reconciled in managed forests? A review of driving factors for integrated forest management in Europe.

Integrated forest management (IFM) can help reconcile critical trade-offs between goals in forest management, such as nature conservation and biomass production. The challenge of IFM is dealing with these trade-offs at the level of practical forest management, such as striving for compromises between biomass extraction and habitat retention. This paper reviews some of the driving factors that influence the integration of nature conservation into forest management. The review was conducted in three steps - a literature review, an expert workshop and an expert-based cooperative analysis. Of 38 driving factors identified, three were prioritised by more of the participants than any of the others: two are socio-cultural factors, identity (how people identify with forest) as well as outreach and education, and one is economic - competitiveness in forest value chains. These driving factors correspond to what are considered in the literature as enablers for IFM. The results reveal that targeted, group-oriented, adaptive and innovative policy designs are needed to integrate nature conservation into forest management. Further, the results reveal that a "one-size-fits-all" governance approach would be ineffective, implying that policy instruments need to consider contextually specific driving factors. Understanding the main driving factors and their overall directions can help to better manage trade-offs between biodiversity conservation and biomass production in European forests.

Hoxa5 undergoes dynamic DNA methylation and transcriptional repression in the adipose tissue of mice exposed to high-fat diet.

BACKGROUND/OBJECTIVES: The genomic bases of the adipose tissue abnormalities induced by chronic positive calorie excess have been only partially elucidated. We adopted a genome-wide approach to directly test whether long-term high-fat diet (HFD) exposure affects the DNA methylation profile of the mouse adipose tissue and to identify the functional consequences of these changes. SUBJECTS/METHODS: We have used epididymal fat of mice fed either high-fat (HFD) or regular chow (STD) diet for 5 months and performed genome-wide DNA methylation analyses by methylated DNA immunoprecipitation sequencing (MeDIP-seq). Mouse Homeobox (Hox) Gene DNA Methylation PCR, RT-qPCR and bisulphite sequencing analyses were then performed. RESULTS: Mice fed the HFD progressively expanded their adipose mass accompanied by a significant decrease in glucose tolerance (P<0.001) and insulin sensitivity (P<0.05). MeDIP-seq data analysis revealed a uniform distribution of differentially methylated regions (DMR) through the entire adipocyte genome, with a higher number of hypermethylated regions in HFD mice (P<0.005). This different methylation profile was accompanied by increased expression of the Dnmt3a DNA methyltransferase (Dnmt; P<0.05) and the methyl-CpG-binding domain protein Mbd3 (P<0.05) genes in HFD mice. Gene ontology analysis revealed that, in the HFD-treated mice, the Hox family of development genes was highly enriched in differentially methylated genes (P=0.008). To validate this finding, Hoxa5, which is implicated in fat tissue differentiation and remodeling, has been selected and analyzed by bisulphite sequencing, confirming hypermethylation in the adipose tissue from the HFD mice. Hoxa5 hypermethylation was associated with downregulation of Hoxa5 mRNA and protein expression. Feeding animals previously exposed to the HFD with a standard chow diet for two further months improved the metabolic phenotype of the animals, accompanied by return of Hoxa5 methylation and expression levels (P<0.05) to values similar to those of the control mice maintained under standard chow. CONCLUSIONS: HFD induces adipose tissue abnormalities accompanied by epigenetic changes at the Hoxa5 adipose tissue remodeling gene.

Epigenetics: spotlight on type 2 diabetes and obesity.

Type 2 diabetes (T2D) and obesity are the major public health problems. Substantial efforts have been made to define loci and variants contributing to the individual risk of these disorders. However, the overall risk explained by genetic variation is very modest. Epigenetics is one of the fastest growing research areas in biomedicine as changes in the epigenome are involved in many biological processes, impact on the risk for several complex diseases including diabetes and may explain susceptibility. In this review, we focus on the role of DNA methylation in contributing to the risk of T2D and obesity.

Salivary hormonal responses to resistance exercise in trained and untrained middle-aged men.

AIM: The aim of the present study was to investigate if there are differences in salivary hormonal responses to resistance exercise between long-term strength-trained and untrained men. METHODS: Twenty-eight subjects were recruited to this study, matched into a strength-trained group (SG, N=13) and an untrained group (UG, N=15). Upper and lower body absolute muscle strength was measured through the one-repetition maximum (1-RM) test. Saliva samples were collected at rest and after a resistance exercise protocol (REP) with intensity relative to 1-RM values. With these samples, testosterone (TES), dehydroepiandrosterone (DHEA) and cortisol (COR) were determined. RESULTS: SG subjects demonstrated significantly higher values in all muscle strength variables. While a significant increase in TES after REP was found in the SG (0.114 + or - 0.1 vs. 0.15 + or - 0.09 pg/mL, P<0.05), no differences were observed in the UG (0.144 + or - 0.1 vs. 0.17 + or - 0.1 pg/mL). In both groups, there were increases in salivary COR (SG: 1.4 + or - 0.6 vs. 2.06 + or - 1; UG: 1.5 + or - 0.8 vs. 2.3 + or - 1.2 ug/dL, P<0.05) and DHEA (SG: 0.6 + or - 0.3 vs. 0.9 + or - 0.6; UG: 0.65 + or - 0.3 vs. 0.97 + or - 0.7 ng/dL, P<0.05). CONCLUSIONS: These results suggest the possible presence of adaptation of TES responses to resistance exercise in long-term strength-trained men, with these subjects presenting higher responses to the same stimulus, compared with untrained subjects, while no such adaptation was seen at the adrenocortical level in these subjects as the responses observed were similar in both groups.

Acquisition of different carbapenem resistance mechanisms by an epidemic clonal lineage of Pseudomonas aeruginosa.

Multidrug-resistant isolates of a clonal lineage of Pseudomonas aeruginosa producing the VIM-2 metallo-beta-lactamase (MBL), involved in a large outbreak in an Italian hospital, were compared with MBL-negative strains that had caused outbreaks in two French hospitals. Although the isolates had different carbapenem MICs, the VIM-2-producing isolates from Italy carried identical, or very similar, allelic forms of the oprD gene, harboured a common class 1 integron, belonged to the same multilocus sequence type (ST111), and showed macrorestriction profiles that were related to those of the MBL-negative French strains. These results support the concept of independent acquisition of resistance determinants by members of a widespread clonal lineage of P. aeruginosa.

Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data.

Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing.

Neuroleptic-like effects of the l-isomer of fenfluramine on striatal dopamine release in freely moving rats.

l-Fenfluramine (l-F) was studied for its ability to release dopamine (DA) and its metabolites in freely moving rats through the trans-striatal dialysis technique. l-F's effect on striatal DA release was also studied in animals made tolerant to the effect of haloperidol by chronic treatment (1 mg/kg i.p. twice daily for 11 days and 48 hr wash-out) with the neuroleptic or pretreated with 300 mg/kg i.p. gamma-butyrolactone (GBL). Five and 10 mg/kg l-F dose-dependently increased the release of DA and its metabolites with a pattern of effects similar to that observed with neuroleptic drugs. The dose of 20 mg/kg l-F had the same effect as 10 mg/kg. Repeated haloperidol treatment reduced the basal release of DA and its metabolites and a much smaller amount of DA and metabolites was released by l-F (10 mg/kg i.p.) and haloperidol (0.1 mg/kg i.p.) in animals treated with haloperidol than in controls. GBL 300 mg/kg i.p. reduced basal DA release by about 50%. When 10 mg/kg l-F, 0.1 mg/kg haloperidol and 0.25 mg/kg d-amphetamine were injected i.p. 40 min after GBL, l-F and haloperidol did not significantly raise DA release in GBL-treated rats whereas a significant effect was observed at various times after d-amphetamine. The data show that l-F resembles haloperidol in its ability to release DA and its metabolites from the corpus striatum of freely moving rats. The cross-tolerance between haloperidol and l-F for their effect on DA release suggests that a common site is involved in the mechanism of these drugs.

Binocular probability summation in a choice reaction-time task in pigeons.

We present evidence for binocular summation in pigeons in a choice reaction time paradigm. Considering binocular vision as the result of a race between the equivalent eyes, pigeons can adopt both a 'subject by subject' and a 'trial by trial' race model, and binocular advantage can be attributed merely to a probability summation. Pigeons were tested on a visual discrimination task in which the time for detection was gradually reduced and a quick response was required. The time presentation of a small black spot could vary from 2000 ms to the shortest value (157-226 ms) allowing maintenance of a satisfactory performance criterion.

Cell-mediated immunity in breast cancer patients.

In clinical practice, reports have been made on immunosuppression after surgical excision of primary tumor or at relapse. However, the relationship between undefined or overt metastases and the host immune system has not been sufficiently examined over a prolonged period. These aspects were investigated in 160 breast cancer patients followed up post-operatively with serial controls over a long period. One hundred and thirty-four cases (91 node negative, (N-), 43 node positive (N+)) were disease-free and 26 relapsed. In all patients, serum T cell populations, serum B lymphocytes and skin reaction of delayed hypersensitivity (SRDH) were serially determined for 39 +/- 12 months (m +/- SD). The reference values for these parameters were assessed as follows: T populations were evaluated in 24 healthy donors and SRDH in 95 healthy females. In non-relapsed patients, constant CD8+ T cell decrease and T4/T8 ratio increase were observed; the T4/T8 ratio was significantly higher (ranging from P < 0.05 - P < 0.001) than in the control group. The mean values of NK cells and B lymphocytes, the former parameter being highly significant (P < 0.001), were higher than in controls. In the 26 metastatic patients, the T4/T8 ratio from 20 months before to 30 months after the first sign of relapse decreased from 3.2 to about 1 (r = -0.256, P < 0.05) and from 30 to 92 months after relapse progressively increased to 2. Similarly, in the former subinterval a progressive decrease in the number of positive antigens and score was found (from 2.4 to 0 and from 10 to zero respectively). A significant inverse correlation between these two parameters and observation time occurred (P < 0.01 and P < 0.001 respectively). From 30 to 86 months after relapse, a progressive increase in the number of positive antigens and scores up to 2 and 12 were observed. A significant direct correlation (P < 0.05) was noted. In conclusion, these data indicate significant changes in T populations during the disease-free interval in breast cancer patients. The decrease in circulating CD8+ T cells is compatible with the hypothesis of CD8+ T cell localization at the site of the micrometastases. The increase in circulating B lymphocytes and NK cells suggests activation of aspecific humoral immunity and NK function. In addition, they show that progressive deficiency in cell-mediated immunity appears many months before and that recovery continues for a long time after overt metastatic disease.

Reduced expression of the chemokine receptor CCR1 in human macrophages and U-937 cells in vitro infected with Leishmania infantum.

Chemokines exert their actions through G-proteinlinked receptors, which are expressed to variable extents by different cell types. In accordance with the chemokine classification, these receptors are designated as CXC, CC, XC, and CX(3)C, followed by R and a number. The purpose of this investigation was to evaluate CCR1 expression in human peripheral blood-derived macrophages and the human monocytic U-937 cell line. Cells in vitro were infected with live Leishmania infantum promastigotes (zymodeme MON1); cell lysates were then subjected to SDS-PAGE and immunoblotting, by using an anti-CCR1 affinity purified polyclonal antibody. The expression of the CCR1 gene was analyzed by RT-PCR, using specific human primers. The results of both immunoblotting and RT-PCR showed that CCR1 expression in Leishmania-infected cells was lower than in uninfected control cells. These results indicate that Leishmania infantum infection causes a down-regulation of the CCR1 gene and protein expression, suggesting that reduced phagocyte recruitment at the inflammation sites could favor parasite progression and the spread of Leishmania infection.

Macrophage chemotactic protein-1 and macrophage inflammatory protein-1 alpha induce nitric oxide release and enhance parasite killing in Leishmania infantum-infected human macrophages.

Chemokines are a group of structurally defined small proteins that act as chemoattractants for leukocytes and are involved in many different biological activities, including leukocyte activation for antimicrobial mechanisms. We studied the effect of the chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1 alpha on nitric oxide release and parasitocidal ability of peripheral blood-derived human macrophages in vitro infected with Leishmania infantum, zymodeme MON1. In infected human macrophages, treatment with MCP-1 or MIP-1 alpha significantly enhanced nitric oxide production and leishmanicidal ability, compared with untreated cells, to the same levels induced by interferon-gamma. Both nitric oxide release and parasitocidal ability of macrophages were significantly reduced by addition of L- N(G)monomethylarginine ( L-NMMA), which is a competitive inhibitor of the L-arginine nitric oxide pathway. These data suggest that MCP-1 and MIP-1 alpha mediate macrophage activation for nitric oxide release and subsequent parasite clearance, and thus may play a role in the containment of Leishmania infection.

Perforation of the ileum due to foreign body treated laparoscopically.

Laparoscopy has gained widespread acceptance in the setting of acute abdominal pain. We report the case of a patient with acute abdominal pain in the right lower quadrant that proved to be due to a fish bone perforation of the ileum at laparoscopy. The fish bone was retrieved and the perforation closed laparoscopically. Laparoscopy is a powerful diagnostic and therapeutic tool and should be used routinely in patients with acute abdominal affections.

[Primary neuroendocrine tumor in the intrahepatic bile ducts: a rare finding in an asymptomatic patient]. / Tumeur neuro-endocrine primitive des voies biliaires intrahépatiques: une découverte exceptionnelle chez un malade asymptomatique.

Neuroendocrine tumors of the biliary tree are rare. In all cases except one, diagnosis was made in symptomatic patients. We report a case of an asymptomatic intrahepatic bile duct neuroendocrine tumor in a 74-year old man. To our knowledge, this is the second reported case of an asymptomatic intrahepatic bile duct neuroendocrine tumor. Diagnosis was only made by anatomopathological examination of the tumor after resection. Systemic and immunohistochemical hormonal screening was negative. Twenty months after surgery, the patient was asymptomatic and there was no recurrence.

Voluminous frontoethmoidal mucocele with epidural involvement. Surgical treatment by coronal approach.

The case is described of mucocele of the right frontoethmoidal sinus with bilateral maxillary sinusitis and a large polyp in the right nasal cavity. The mucocele had determined erosion of the anterior and posterior walls of the frontal sinus and superomedial wall of the orbit. The patient was operated upon by a surgical team comprising ENT and maxillofacial specialists. Right maxillary sinusotomy (Caldwell-Luc procedure) was performed, and an osteoplastic flap was prepared, repositioned in the canine fossa and fixed with a titanium plate. Debris was removed from the left osteomeatal complex during endoscopy. To reach the mucocele, an external surgical approach was used, through a bitemporal coronal cutaneous incision, according to Unterberger. This approach was used in order to gain better access to the area of the lesion and in order to make reconstruction easier, with a view to achieving good functional results without untoward scarring. The scalp was detached down to the root of the nose to allow optimal visualisation of the anterior area of erosion determined by the mucocele, and, after excision and removal of the latter from the bony walls, of the posterior bony breach and underlying dura mater. Another bony breach involved the medial and superior walls of the orbit. The nasofrontal canal was obliterated with bone fragments and Tissucol; the posterior breach, with Surgical and Tissucol. The orbit wall was repaired with high-density porous polyethylene sheeting; the frontal sinus was filled with fat. The anterior wall of the frontal sinus was repaired with two split of calvarial bone grafts harvested from the parietal bone and fixed with a titanium microplate. The morphological outcome of reconstruction was satisfactory, with no recurrences, as confirmed at post-operative follow-up, including computed tomography scan, at 5 months. Ocular motility and patency of the tear drainage system were also normal. No diplopia, or inflammation occurred.

DNA copy-number abnormalities do not occur in infant ALL with t(4;11)/MLL-AF4.

The pathogenesis of infant acute lymphoblastic leukemia (ALL) is still not well defined. Short latency to leukemia and very high concordance rate for ALL in Mixed-Lineage Leukemia (MLL)-positive infant twins suggest that the MLL rearrangement itself could be sufficient for overt leukemia. Attempts to generate a suitable mouse model for MLL-AF4-positive ALL did not thoroughly resolve the issue of whether cooperating mutations are required to reduce latency and to generate overt leukemia in vivo. In this study, we applied single-nucleotide polymorphism array technology to perform genomic profiling of 28 infant ALL cases carrying t(4;11) to detect MLL-cooperating aberrations hidden to conventional techniques and to gain new insights into infant ALL pathogenesis. In contrast to pediatric, adolescent and adult ALL cases, the MLL rearrangement in infant ALL is associated with an exceptionally low frequency of copy-number abnormalities, thus confirming the unique nature of this disease. By contrast, additional genetic aberrations are acquired at disease relapse. Small-segmental uniparental disomy traits were frequently detected, mostly constitutional, and widely distributed throughout the genome. It can be argued that the MLL rearrangement as a first hit, rather than inducing the acquisition of additional genetic lesions, has a major role to drive and hasten the onset of leukemia.