MicroRNA-Mediated Direct Reprogramming of Human Adult Fibroblasts Toward Cardiac Phenotype.

Modulation of microRNA expression holds the promise to achieve direct reprogramming of fibroblasts into cardiomyocyte-like cells as a new strategy for myocardial regeneration after ischemic heart disease. Previous reports have shown that murine fibroblasts can be directly reprogrammed into induced cardiomyocytes (iCMs) by transient transfection with four microRNA mimics (miR-1, 133, 208, and 499, termed "miRcombo"). Hence, study on the effect of miRcombo transfection on adult human cardiac fibroblasts (AHCFs) deserves attention in the perspective of a future clinical translation of the approach. In this brief report, we studied for the first time whether miRcombo transient transfection of AHCFs by non-viral vectors might trigger direct reprogramming of AHCFs into cardiomyocyte-like cells. Initially, efficient miRNA delivery to cells was demonstrated through the use of a commercially available transfection agent (DharmaFECT1). Transient transfection of AHCFs with miRcombo was found to upregulate early cardiac transcription factors after 7 days post-transfection and cardiomyocyte specific marker cTnT after 15 days post-transfection, and to downregulate the expression of fibroblast markers at 15 days post-transfection. The percentage of cTnT-positive cells after 15 days from miRcombo transfection was ∼11%, as evaluated by flow cytometry. Furthermore, a relevant percentage of miRcombo-transfected AHCFs (∼38%) displayed spontaneous calcium transients at 30 days post-transfection. Results evidenced the role of miRcombo transfection on triggering the trans differentiation of AHCFs into iCMs. Although further investigations are needed to achieve iCM maturation, early findings from this study pave the way toward new advanced therapies for human cardiac regeneration.

Temperature and pH sensors based on graphenic materials.

Point-of-care applications and patients' real-time monitoring outside a clinical setting would require disposable and durable sensors to provide better therapies and quality of life for patients. This paper describes the fabrication and performances of a temperature and a pH sensor on a biocompatible and wearable board for healthcare applications. The temperature sensor was based on a reduced graphene oxide (rGO) layer that changed its electrical resistivity with the temperature. When tested in a human serum sample between 25 and 43°C, the sensor had a sensitivity of 110±10Ω/°C and an error of 0.4±0.1°C compared with the reference value set in a thermostatic bath. The pH sensor, based on a graphene oxide (GO) sensitive layer, had a sensitivity of 40±4mV/pH in the pH range between 4 and 10. Five sensor prototypes were tested in a human serum sample over one week and the maximum deviation of the average response from reference values obtained by a glass electrode was 0.2pH units. For biological applications, the temperature and pH sensors were successfully tested for in vitro cytotoxicity with human fibroblast cells (MRC-5) over 24h.

A graphene oxide pH sensor for wound monitoring.

This article describes the fabrication and characterization of a pH sensor for monitoring the wound status. The pH sensitive layer consists of a graphene oxide (GO) layer obtained by drop-casting 5 µÎ of GO dispersion onto the working electrode of a screen-printed substrate. Sensitivity was 31.8 mV/pH with an accuracy of 0.3 unit of pH. Open-circuit potentiometry was carried out to measure pH in an exudate sample. The GO pH sensor proved to be reliable as the comparison with results obtained from a standard glass electrode pH-meter showed negligible differences (<; 0.09 pH units in the worst case) for measurements performed over a period of 4 days.

Metacercariae of Clinostomum complanatum (Trematoda: Digenea) in European newts Triturus carnifex and Lissotriton vulgaris (Caudata: Salamandridae).

Adults of Clinostomum spp. are digenetic trematodes found in fish-eating birds, reptiles and occasionally mammals, including humans. Freshwater snails serve as first intermediate hosts and many fish species and amphibians as second intermediate hosts. To date, amphibian hosts of Clinostomum metacercariae include members of urodele and anuran families in North America, but no data are available on infections of European amphibians, including newts. In this study, we characterize infections of Clinostomum complanatum metacercariae in four smooth (Lissotriton vulgaris) and 18 Italian crested newts (Triturus carnifex) from an artificial pond located in a protected area in Tuscany, Italy. Parasites were surgically removed from the infected newts and identified both morphologically and using sequences of a mitochondrial gene, cytochrome c oxidase I, and the ribosomal markers, internal transcribed spacers. This is the first record of C. complanatum in European newts and, more generally, in amphibians in Europe.

Circulating tumour cells: insights into tumour heterogeneity.

Tumour heterogeneity is a major barrier to cure breast cancer. It can exist between patients with different intrinsic subtypes of breast cancer or within an individual patient with breast cancer. In the latter case, heterogeneity has been observed between different metastatic sites, between metastatic sites and the original primary tumour, and even within a single tumour at either a metastatic or a primary site. Tumour heterogeneity is a function of two separate, although linked, processes. First, genetic instability is a hallmark of malignancy, and results in 'fixed' genetic changes that are almost certainly carried forward through progression of the cancer over time, with increasingly complex additional genetic changes in new metastases as they arise. The second type of heterogeneity is due to differential but 'plastic' expression of various genes important in the biology and response to various therapies. Together, these processes result in highly variable cancers with differential response, and resistance, to both targeted (e.g. endocrine or anti-human epithelial growth receptor type 2 (HER2) agents) and nontargeted therapies (e.g. chemotherapy). Ideally, tumour heterogeneity would be monitored over time, especially in relation to therapeutic strategies. However, biopsies of metastases require invasive and costly procedures, and biopsies of multiple metastases, or serially over time, are impractical. Circulating tumour cells (CTCs) represent a potential surrogate for tissue-based cancer and therefore might provide the opportunity to monitor serial changes in tumour biology. Recent advances have enabled accurate and reliable quantification and molecular characterization of CTCs with regard to a number of important biomarkers including oestrogen receptor alpha and HER2. Preliminary data have demonstrated that expression of these markers between CTCs in individual patients with metastatic breast cancer reflects the heterogeneity of the underlying tumours. Future studies are designed to determine the clinical utility of these novel technologies in either research or routine clinical settings.

Molten carbonate fuel cells fed with biogas: combating H(2)S.

The use of biomass and waste to produce alternative fuels, due to environmental and energy security reasons, is a high-quality solution especially when integrated with high efficiency fuel cell applications. In this article we look into the coupling of an anaerobic digestion process of organic residues to electrochemical conversion to electricity and heat through a molten carbonate fuel cell (MCFC). In particular the pathway of the exceedingly harmful compound hydrogen sulphide (H(2)S) in these phases is analysed. Hydrogen sulphide production in the biogas is strongly interrelated with methane and/or hydrogen yield, as well as with operating conditions like temperature and pH. When present in the produced biogas, this compound has multiple negative effects on the performance and durability of an MCFC. Therefore, there are important issues of integration to be solved. Three general approaches to solve the sulphur problem in the MCFC are possible. The first is to prevent the formation of hydrogen sulphide at the source: favouring conditions that inhibit its production during fermentation. Secondly, to identify the sulphur tolerance levels of the fuel cell components currently in use and develop sulphur-tolerant components that show long-term electrochemical performance and corrosion stability. The third approach is to remove the generated sulphur species to very low levels before the gas enters the fuel cell.

Lactobacillus rhamnosus GG inhibits invasion of cultured human respiratory cells by prtF1-positive macrolide-resistant group A streptococci.

AIMS: This study was designed to determine whether the probiotic strain Lactobacillus GG, which is extensively used in the treatment and prevention of intestinal disorders, is able to inhibit invasion of cultured human respiratory cells by macrolide-resistant group A streptococci (GAS) carrying the prtF1 gene, which encodes the fibronectin (Fn)-binding invasin F1. METHODS AND RESULTS: Eight prtF1-positive erythromycin-resistant GAS strains were used to infect A549 monolayers in competition and displacement assays with Lactobacillus GG. Live (L-LGG) and heat-killed (HK-LGG) lactobacilli and their spent culture supernatant (SCS) significantly reduced (P < 0.001) GAS invasion efficiency in both assays. No antibacterial activity of Lactobacillus GG against GAS was detected. Both L-LGG and HK-LGG and all prtF1-positive GAS induced a strong agglutination reaction using Fn-coated particles. CONCLUSIONS: Lactobacillus GG exerts an antagonistic action against GAS by inhibiting cell invasion. Competitive binding of Lactobacillus GG and GAS to Fn might be involved in the inhibition process. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding that Lactobacillus GG can prevent in vitro invasion of respiratory cells by GAS suggests new applications for this probiotic strain and warrants further studies of its capacity to prevent GAS throat infections.

VanA-type enterococci from humans, animals, and food: species distribution, population structure, Tn1546 typing and location, and virulence determinants.

VanA-type human (n=69), animal (n=49), and food (n=36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n=4) and M49 (n=13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P>0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P<0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.

Oxaliplatin, 5-fluorouracil/leucovorin and epirubicin as first-line treatment in advanced gastric carcinoma: a phase II study.

The association between oxaliplatin and 5-fluorouracil (5-FU) has been extensively reported to improve prognosis of gastric cancer patients. The present study is aimed at evaluating response rate and the toxicity profile of the association with oxaliplatin, 5-FU/lecovorin and epirubicin in gastric cancer patients with locally advanced or metastatic disease. Thirty-six patients have been enrolled and 35 evaluated. The treatment schedule was oxaliplatin (100 mg m(-2)), 5-FU (400 mg m(-2)), leucovorin (40 mg m(-2)) and epirubicin (60 mg m(-2)) intravenously. administered every 3 weeks for 6 months, for a total of 185 therapy cycles . Response rate and toxicity were assessed according to the international WHO criteria. Every patient received a mean of 5.3 therapy cycles in a day-hospital setting. Sixteen of 35 patients (46%) showed an objective response, two complete response and 14 partial response. Median time to progression was 33 weeks with an overall median survival of 49 weeks. During the study, anaemia grade 3 and neutropenia grade 3 were observed in 9 and 11% of patients respectively. A grade 3 periferic sensorial neuropathy was observed in 6% of patients. No life threatening or cardiac toxicity was recorded. The regimen used showed anticancer activity against gastric carcinoma, a tolerable toxicity profile and excellent patient compliance.

Long-term survival in metastatic melanoma patients treated with sequential biochemotherapy: report of a Phase II study.

The overall survival for patients with metastatic melanoma is very poor, with a median survival of 8.5 months. In this Phase II trial, we assessed the efficacy, safety, and tolerability of a sequential biochemotherapy schedule, using dacarbazine as antiblastic agent and immunomodulant doses of interleukin-2 and interferon-alfa. Thirty-one eligible patients with metastatic melanoma received dacarbazine IV as antiblastic therapy and interluekin-2, plus interferon-alfa SC as sequential immunotherapy, for 6 months. Responding and nonprogressing patients were subsequently maintained on immunotherapy treatment for further 6 months. Twenty-nine patients had an adequate trial, and were assessable for both response and toxicities, with a median follow-up of 49 months. The overall response rate was 52 percent (3 CR and 12 PR), SD was 8 (27 percent) and PD were achieved in 6 patients (21 percent). The median survival duration of responders was 28 months, significantly longer (p < 0.001) than the 16 months of nonresponders. Therapy was well tolerated and produced a significant improvement in progressive-free survival. Further studies, thus, are recommended for larger groups of patients not only to confirm these results, but also to apply this biochemotherapy regimen as adjuvant postsurgical treatment in early stages of malignant melanoma.

Risk of melanoma following adulthood cancer: a case-control study.

Melanoma is a severe skin cancer related to sun exposure. Whether this malignancy is linked to exposure to ionising radiation during adulthood is still controversial. This case-control study examined the risk of melanoma following treatment for an adulthood first malignant neoplasm (FMN). Cases were patients who presented with cutaneous melanoma after a first cancer in adulthood. Controls (3 per case) were patients free of melanoma, matched for age, duration of follow-up since the FMN, type of FMN, and followed in the same institution. A total of 57 cases and 171 controls were included. In the final multivariate analysis, no risk of melanoma was associated with radiotherapy (odds ratio (OR) for 1 Gy = 1.01, 95% confidence interval (95%CI) 0.96-1.07) nor hormonotherapy, whereas chemotherapy use (OR = 2.3, 95%CI 0.93-5.6) and having a history of familial cancer (OR = 2.8, 95%CI 1.3-5.9) exhibited a nearly significant risk. In conclusion, unlike the evidence for risk of exposure to ionising radiation during childhood, we did not substantiate a risk for association of melanoma with exposure to ionising radiation during adulthood. The risk associated with chemotherapy should justify the implementation of skin surveillance for early detection of melanoma in these patients.

Estimation of the familial relative risk of cancer by site from a French population based family study on colorectal cancer (CCREF study).

BACKGROUND: Colorectal cancer (CRC) is the second most common cause of death from cancer in France. A family history of CRC increases an individual's risk of developing CRC. Family history has been suggested to have a greater impact on proximal than distal tumours. AIM: We estimated the familial risk of CRC and other cancers, and examined how risk varies according to localisation of the tumour in the colorectal tract. SUBJECTS: We recorded all cases of CRC diagnosed between 1993 and 1998 in the region served by the Calvados Cancer Registry. A trained interviewer asked all participants about their family history of cancer. STATISTICAL METHODS: Familial risk was estimated from a cohort analysis of the relatives of the CRC cases. The expected numbers of cancers were calculated from Calvados incidence rates. Familial relative risks were calculated using standardised incidence ratios. RESULTS: Our findings showed that colon cancer had a stronger familial/genetic component (relative risk (RR) 1.47) than rectal cancer (RR 0.98). The familial/genetic component appeared stronger for proximal colon cancer than for distal colon cancer only among women (RR 2.24 v RR 1.45). CRC appeared to be positively associated with leukaemia (RR 1.77), stomach cancer (RR 1.32), and testicular cancer (RR 3.13), and negatively associated with urinary bladder cancer (RR 0.57) within families. The cancer spectrum associated with CRC among younger participants included prostate (RR 1.93), uterus (RR 2.49), and thyroid (RR 3.85) cancers. CONCLUSION: If our results are confirmed, follow up guidelines for patients with a family history of CRC should depend on the sex and tumour site of affected relatives to avoid needless invasive screening.

Familial relative risk of colorectal cancer: a population-based study.

This study aimed to assess the familial relative risk for colorectal cancer (CRC) and its variation according to age and gender. A population-based family study was carried out in France, from 1993 to 1998, including 761 families. Familial CRC risks were estimated from a cohort analysis of the relatives. No obvious decrease in CRC risk was found with increasing age, except when either the proband, or the relative, were in the youngest age class. The effect of the relatives' and probands' ages on the CRC risk differed according to their gender. The cumulative risk of CRC increased at an earlier age in male relatives of probands younger than 60 years of age, than in female relatives. This result suggests that mechanisms specific to females, possibly interacting with genetic factors, explain the difference in the cumulative risks between families with male and female probands.

Smoking cessation rates 4 years after treatment by nicotine gum and acupuncture.

BACKGROUND: This study was done to estimate the smoking cessation rates 4 years after treatment with acupuncture and nicotine gum. METHODS: Participants were randomized in a 2 x 2 factorial design to four groups: double active treatments (nicotine gum and acupuncture), double placebo, and the combination of one active treatment and placebo. RESULTS: The success rates were quite similar in the four groups at the different points of follow-up. They sharply decreased between 1 month (around 23%) and 1 year (around 10%). The decrease slowed down thereafter to around 6% at 4 years. CONCLUSIONS: Results from our study suggest that the two treatments did not offer any long-term improvement over placebo. Additional investigations are necessary to estimate the magnitude of their long-term success rate.

Effects of CYP2D6 activity and tobacco on larynx cancer risk.

The genetically determined capacity of the cytochrome P450 CYP2D6 is suspected to be involved in the activation of tobacco carcinogens. From a multicentric case-control study carried out to analyze the interaction between host and environmental factors on tobacco-related cancers, we reported recently that the effect of tobacco on lung cancer risk rose with increasing CYP2D6 activity, and the effect of CYP2D6 activity rose with increasing tobacco consumption. The aim of the present report was to investigate whether results on lung cancer could be observed for larynx cancer, from a study on 140 cases and 157 controls. A weak interaction between increasing levels of both CYP2D6 activity and average daily consumption of tobacco was found (P = 0.12). The only significant interaction between these two factors was observed when CYP2D6 activity was considered with the two conventional phenotypes (P < 0.05). A dose-response effect of tobacco on larynx cancer risk was found only among one-third of the smokers with the highest level of CYP2D6 activity, and CYP2D6 was a risk factor only among heavy smokers. The highest risk for larynx cancer was then observed among smokers having both the highest levels of CYP2D6 activity and daily consumption of tobacco. The interaction between CYP2D6 activity and tobacco was weaker for larynx cancer than that reported previously for lung cancer. However, the similarities in the results found for these two cancers, i.e., a greater effect of tobacco among smokers with the highest CYP2D6 activity, reinforce the hypothesis that this activity could modify the effect of tobacco on cancer risk.

Occupational risk factors for renal cell carcinoma: a case-control study.

A case-control study of 196 histologically proved cases of renal cell carcinoma and 347 controls matched for age at interview, sex, hospital, and interviewer was conducted in France between 1987 and 1991. A complete occupational history was recorded for each patient and occupations were coded blindly according to the International Standard Classification of Occupations. In women, none of the risks were significant. Among men, after adjustment for the educational level, cigarette smoking, and Quetelet index before diagnosis, significantly increased matched odds ratios (ORs) were found for sales workers (OR = 2.1, 95% confidence interval (95% CI) 1.2-4.0), managers (OR = 3.3, 95% CI 1.2-8.9), and textile workers and tailors (OR = 6.2, 95% CI 1.1-33.7). For this last occupational group, an increase in risk was found with an increased duration of exposure.

[In vitro stimulation by ellipticine derivatives of DNA cleavage induced by DNA topoisomerase II: a structure-activity relationship]. / Stimulation in vitro par des dérivés de l'ellipticine des coupures de l'ADN induites par l'ADN topo-isomérase II: relation structure-activité.

Ellipticines are intercalating planar polycyclic aromatic molecules that display antitumor activity. The cytotoxicity of these compounds is related to the presence of an hydroxy group at position 9 of the pyridocarbazole ring system and to their interaction with DNA topoisomerase II. The ability of 13 ellipticine derivatives to stabilize the topoisomerase II-DNA covalent complex in vitro is reported. The following observations emerge from our structure-activity relationship study: i) the hydroxy group at position 9 is essential for stabilizing the covalent complex, ii) the replacement of the methyl group at position 5 by an ethyl group (EPC) enhances the complex stabilization. The interaction of EPC and three other ellipticine analogues with DNA shows that the covalent complexes which are most stable have the lowest drug-DNA binding constants. In addition our study suggests that ellipticines induce covalent complex stabilization by a cooperative mechanism. A model is proposed to explain this stabilization by ellipticines. This study supports the idea that topoisomerase II is the primary target involved in the mechanisms of action of ellipticines.

The localization of topoisomerase II cleavage sites on DNA in the presence of antitumor drugs.

Type II topoisomerase are enzymes that break and religate DNA phosphodiester bonds while crossing over DNA strands and altering DNA topology. They also are structural proteins that play a role in the spatial organization of chromatin and are involved in several crucial biological functions, such as DNA replication and transcription, chromosome segregation and recombination. Many drugs interfere with type II topoisomerases and can be assigned to two groups. Coumarin derivatives and synthetic quinolones act at the level of ATP binding or hydrolysis and are used for controlling bacterial infections. Drugs belonging to the second group produce DNA lesions by trapping a "cleavable complex" consisting of the normal transient topoisomerase II-DNA reaction intermediate in which the enzyme and the DNA are joined by two covalent bonds. There are four main categories of antitumour drugs that form cleavable complexes in eukaryotes: acridines, anthracyclines, ellipticines and epipodophyllotoxins. These drugs are cytotoxic and many--but not all--are endowed with antitumoral properties. The mechanisms of this pharmacological activity are not understood. Topoisomerase II-induced DNA breaks generated from cleavable complexes display different levels of cytotoxicity depending on their localization on DNA. The primary structure of DNA is not the only parameter that determines this localization. The spatial organization of the enzyme-DNA complex and both the topology and the structure of the underlying chromatin fiber constitute additional critical factors. It, therefore, may be unrealistic to expect that the actual pharmacological potency of antitumor drugs that act on type II topoisomerases can be accurately predicted solely on the basis of simple in vitro test tube experiments carried out using pure enzymes and naked DNA.