Foetal and adult cardiomyocyte progenitor cells have different developmental potential

In the past years, cardiovascular progenitor cells have been isolated from the human heart and characterized. Up to date, no studies have been reported in which the developmental potential of foetal and adult cardiovascular progenitors was tested simultaneously. However, intrinsic differences will likely affect interpretations regarding progenitor cell potential and application for regenerative medicine. Here we report a direct comparison between human foetal and adult heart‐derived cardiomyocyte progenitor cells (CMPCs). We show that foetal and adult CMPCs have distinct preferences to differentiate into mesodermal lineages. Under pro‐angiogenic conditions, foetal CMPCs form more endothelial but less smooth muscle cells than adult CMPCs. Foetal CMPCs can also develop towards adipocytes, whereas neither foetal nor adult CMPCs show significant osteogenic differentiation. Interestingly, although both cell types differentiate into heart muscle cells, adult CMPCs give rise to electrophysiologically more mature cardiomyocytes than foetal CMPCs. Taken together, foetal CMPCs are suitable for molecular cell biology and developmental studies. The potential of adult CMPCs to form mature cardiomyocytes and smooth muscle cells may be essential for cardiac repair after transplantation into the injured heart.     

The loss of phenol sulfotransferase 1 in hepatocellular carcinogenesis

Biomarkers for the detection of early hepatocellular carcinoma (HCC) are urgently needed. To identify biomarkers of HCC, we performed a comparative proteomics analysis, based on 2‐DE of HCC tissues and surrounding non‐tumor tissues. Six xenobiotic enzymes were significantly down‐regulated in the HCC tissue. Among these, phenol sulfotransferase (SULT1A1) was confirmed by Western blot analysis in 105 HCC patients. SULT1A1 showed a significant decrease in 98.1% of the HCC tissues, with 88.6% sensitivity and 66.7% specificity for the detection of HCC. Immunohistochemistry for SULT1A1 was performed and compared with glypican‐3, which is a well‐known marker of HCC. The results showed down‐regulation of SULT1A1 and up‐regulation of glypican‐3 in 52.6 and 71.9% of the HCCs, and the use of both markers improved the sensitivity up to 78.9%. Moreover, SULT1A1 was useful in differentiating early HCC from benign dysplastic nodules. Clinically, the down‐regulation of SULT1A1 was closely associated with an advanced International Union Against Cancer stage and high levels of serum α‐fetoprotein. In conclusion, the results of this study demonstrate that the loss of SULT1A1 appears to be a characteristic molecular signature of HCC. SULT1A1 might be a useful biomarker for the detection of early HCC and help predict the clinical outcome of patients with HCC.     

The Neandertal lower right deciduous second molar from Trou de l'Abîme at Couvin, Belgium

A human lower right deciduous second molar was discovered in 1984 at the entrance of Trou de l'Abîme at Couvin (Belgium). In subsequent years the interpretation of this fossil remained difficult for various reasons: (1) the lack of taxonomically diagnostic elements which would support its attribution to either Homo (sapiens) neanderthalensis or H. s. sapiens; (2) the absence of any reliable chronostratigraphic interpretation of the sedimentary sequence of the site; (3) the contradiction between archaeological interpretations, which attributed the lithic industry to a transitional facies between the Middle and Early Upper Palaeolithic, and the radiocarbon date of 46,820 ± 3,290 BP obtained from animal bone remains associated with the tooth and the flint tools.Thanks to recent progress regarding these three aspects, the tooth from Trou de l'Abîme may now be studied in detail. Analyses of the morphology and enamel thickness of the fossil yielded diagnostic characters consistent with an attribution to Neandertals. Re-examination of the lithic industry of Couvin shows that it corresponds to the late Middle Palaeolithic rather than a transitional facies. Furthermore, a new analysis of the site stratigraphy indicates that the unit situated above the archaeological layer in which the tooth was found is probably a palaeosol of brown soil type. Comparison with the regional cave sequences as well as with the reference sequence from the Belgian loess belt tends to show that the most recent palaeosol of this type is dated between 42,000 and 40,000 BP. This is consistent with both a recently obtained AMS result at 44,500 BP and the published conventional date.     

Knock-down of the COX3 and COX17 gene expression of cytochrome c oxidase in the unicellular green alga Chlamydomonas reinhardtii

The COX3 gene encodes a core subunit of mitochondrial cytochrome c oxidase (complex IV) whereas the COX17 gene encodes a chaperone delivering copper to the enzyme. Mutants of these two genes were isolated by RNA interference in the microalga Chlamydomonas. The COX3 mRNA was completely lacking in the cox3-RNAi mutant and no activity and assembly of complex IV were detected. The cox17-RNAi mutant presented a reduced level of COX17 mRNA, a reduced activity of the cytochrome c oxidase but no modification of its amount. The cox3-RNAi mutant had only 40% of the wild-type rate of dark respiration which was cyanide-insensitive. The mutant presented a 60% decrease of H₂O₂ production in the dark compared to wild type, which probably accounts for a reduced electron leakage by respiratory complexes III and IV. In contrast, the cox17-RNAi mutant showed no modification of respiration and of H₂O₂ production in the dark but a two to threefold increase of H₂O₂ in the light compared to wild type and the cox3-RNAi mutant. The cox17-RNAi mutant was more sensitive to cadmium than the wild-type and cox3-RNAi strains. This suggested that besides its role in complex IV assembly, Cox17 could have additional functions in the cell such as metal detoxification or Reactive Oxygen Species protection or signaling. Concerning Cox3, its role in Chlamydomonas complex IV is similar to that of other eukaryotes although this subunit is encoded in the nuclear genome in the alga contrary to the situation found in all other organisms.     

Detection by whole genome microarrays of a spontaneous 126-gene deletion during construction of a ytfE mutant: confirmation that a ytfE mutation results in loss of repair of iron-sulfur centres in proteins damaged by oxidative or nitrosative stress

We show that genomic hybridization allows detection of a spontaneous secondary deletion of 126 genes that occurred during construction of an Escherichia coli ytfE mutant, LMS4209, explaining some of its unexpected growth defects. We confirm that YtfE is required to repair damage to iron-sulfur centres and for hydrogen peroxide resistance.     

Use of Purified Clostridium difficile Spores To Facilitate Evaluation of Health Care Disinfection Regimens

Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 10⁶ pure C. difficile spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile spores (17 to 29 spores/cm²) and block host transmission. Thus, pure C. difficile spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.Clostridium difficile is a Gram-positive, spore-forming, anaerobic bacterium that is a major cause of health care-acquired infections and antibiotic-associated diarrhea (2). In recent years, several genetic variants of C. difficile have emerged as important health care pathogens (6). Perhaps most notable is the “hypervirulent” variant, commonly referred to as PCR ribotype 027/restriction endonuclease analysis (REA) group BI, that produces elevated levels of toxins TcdA and TcdB (17, 19). Other virulent ribotypes that display extensive heterogeneity among their toxin protein sequences (26) and gene activities (8) have emerged. Using whole-genome sequencing, we demonstrated that there are broad genetic differences between the entire genomes of several common variants, including ribotype/REA group variants 012/R, 017/CF, and 027/BI used in this study (12, 27, 31). In contrast, phylogeographic analysis of 027/BI isolates from Europe and the United States demonstrates that this clade is extremely clonal and implies recent transcontinental spread of hypervirulent C. difficile (12).C. difficile is distinct from many other health care pathogens because it produces highly infectious spores that are shed into the environment (25, 28). C. difficile spores can resist disinfection regimens that normally inactivate other health care pathogens, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, therefore challenging current infection control measures (2). A multifaceted approach is normally used to control C. difficile in health care facilities (32). Interventions include antimicrobial stewardship, increased clinical awareness, patient isolation (11), and enhanced environmental disinfection regimens based on hydrogen peroxide (H₂O₂) vapor (HPV) (4). While attempts to break the spore-mediated infection cycle and interrupt these efficient routes of transmission are important for infection control measures, there is little quantitative evidence indicating which interventions are most effective (7). Here we describe the exploitation of pure C. difficile spores (16) and a murine transmission model (15) in simple, practical methods to quantitatively monitor the impact of health care disinfection regimens on C. difficile viability. These methods can be used to optimize disinfection regimens targeted at C. difficile.     

Beneficial Effect of Verminephrobacter Nephridial Symbionts on the Fitness of the Earthworm Aporrectodea tuberculata

Almost all lumbricid earthworms (Oligochaeta: Lumbricidae) harbor species-specific Verminephrobacter (Betaproteobacteria) symbionts in their nephridia (excretory organs). The function of the symbiosis, and whether the symbionts have a beneficial effect on their earthworm host, is unknown; however, the symbionts have been hypothesized to enhance nitrogen retention in earthworms. The effect of Verminephrobacter on the life history traits of the earthworm Aporrectodea tuberculata (Eisen) was investigated by comparing the growth, development, and fecundity of worms with and without symbionts given high (cow dung)- and low (straw)-nutrient diets. There were no differences in worm growth or the number of cocoons produced by symbiotic and aposymbiotic worms. Worms with Verminephrobacter symbionts reached sexual maturity earlier and had higher cocoon hatching success than worms cured of their symbionts when grown on the low-nutrient diet. Thus, Verminephrobacter nephridial symbionts do have a beneficial effect on their earthworm host. Cocoons with and without symbionts did not significantly differ in total organic carbon, total nitrogen, or total hydrolyzable amino acid content, which strongly questions the hypothesized role of the symbionts in nitrogen recycling for the host.Symbiosis has long been recognized as a source of evolutionary innovation (24), and the acquisition of symbionts can enable animal hosts to exploit previously inaccessible niches (3). The phylum Annelida is no exception to this; chemosynthetic symbionts in marine annelids (e.g., the giant tubeworm Riftia sp. and other gutless marine oligochaetes [9]) gain energy from the oxidation of reduced sulfur compounds and fix CO₂ and supply their animal host with fixed carbon. A more obscure partnership is known from the bone-eating annelid Osedax sp., where endosymbionts help degrade the bones of whale carcasses, the only known habitat of the worms (33). The medicinal leech Hirudo sp., like other blood-feeding animals (3, 7), has symbionts that are thought to produce essential vitamins missing from a blood meal (13). In addition, leeches have a number of symbionts of unknown function in their nephridia (excretory organs) (18). Earthworms (Oligochaeta: Lumbricidae) have also long been known to harbor symbiotic bacteria in their nephridia (19, 36). The function of this symbiosis, however, is still not known, but the stability of the symbiosis over evolutionary time (23) suggests that the symbionts benefit the host.The earthworm symbionts reside in the nephridia and have therefore been proposed to be involved in internal recycling of nitrogen in the host (29). The earthworm nephridia play an important role in both nitrogenous waste excretion and osmoregulation (20). The nephridia are found in pairs in each segment of the worm and consist of a coiled tube leading from the coelom to the exterior (Fig. ​(Fig.1).1). The tube forms three loops, and the symbiotic bacteria are situated in the ampulla in the second loop, where they form a dense population lining the lumen wall (19, 36).Open in a separate windowFIG. 1.The nephridia are found as paired organs in each segment of the worm. The nephridostome (the inlet to the nephridia) protrudes into the previous segment. The nephridial tube forms three loops and finally empties out through the body wall via the nephridopore. The symbionts (black) reside in the ampulla in the second loop. (Modified from reference 23 with permission.)The symbionts form the monophyletic genus Verminephrobacter (Betaproteobacteria) (30, 36); they are species specific and present in almost all lumbricid earthworms (23). The Verminephrobacter symbionts are transmitted vertically via the cocoon, where they are deposited along with eggs and sperm (5). During embryogenesis, the symbionts migrate into the developing nephridia, and after the worm hatches, the symbionts can no longer infect (5, 6). By taking advantage of the vertical transmission mode, it has been possible to establish symbiont-free earthworm cultures in the laboratory through controlled antibiotic treatment of newly deposited cocoons (5; this study). Separation of the symbiotic partners allows studies of the effect of the symbionts on their earthworm hosts.Pandazis (29) hypothesized that the symbionts enhance earthworm nitrogen retention by excreting proteolytic enzymes that will degrade peptides and proteins lost in urine; this would allow the earthworm to reabsorb the resulting amino acids. As a consequence, earthworms cured of their symbionts should have a lower fitness level than control worms when grown under nitrogen-limiting conditions. To test this hypothesis, growth, development, fecundity, and cocoon hatching success were compared for symbiotic and aposymbiotic earthworms of the species Aporrectodea tuberculata (Eisen) under high and low nutrient availability conditions.     

Development and Validation of a Continuous In Vitro System Reproducing Some Biotic and Abiotic Factors of the Veal Calf Intestine

Following the January 2006 European ban of antibiotics used as growth promoters in the veal calf industry, new feed additives are needed in order to maintain animal health and growth performance. As an alternative to in vivo experiments in the testing of such additives, an in vitro system modeling the intestinal ecosystem of the veal calf was developed. Stabilization of the main cultured microbial groups and their metabolic activity were tracked in an in vitro continuous fermentor operated under anaerobiosis, at pH 6.5, and at a temperature of 38.5°C and supplied with one of three different nutritive media (M1, M2, or M3). These media mainly differed in their concentrations of simple and complex carbohydrates and in their lipid sources. In vitro microbial levels and fermentative metabolite concentrations were compared to in vivo data, and the biochemical composition of the nutritive media was compared to that of the veal calf intestinal content. All three nutritive media were able to stabilize anaerobic and facultative anaerobic microflora, lactate-utilizing bacteria, bifidobacteria, lactobacilli, enterococci, and Bacteroides fragilis group bacteria at levels close to in vivo values. The microbiota was metabolically active, with high concentrations of lactate, ammonia, and short-chain fatty acids found in the fermentative medium. Comparison with in vivo data indicated that M3 outperformed M1 and M2 in simulating the conditions encountered in the veal calf intestine. This in vitro system would be useful in the prescreening of new feed additives by studying their effect on the intestinal microbiota levels and fermentative metabolite production.European regulations introduced in January 2006 banned the use of antibiotics as growth promoters (AGP) at subtherapeutic levels in animal feed (regulation EC 1831/2003), particularly for veal calves. AGP generated significantly enhanced growth performance via complex processes. The mechanism of growth promotion is still speculative, but many studies suggest the involvement of the intestinal microbiota (7, 9). First of all, AGP did not promote the growth of germfree animals (6). Moreover, they strongly inhibited the bacterial catabolism of urea and amino acids and the fermentation of carbohydrates both in vitro and in vivo (10, 28, 35). AGP treatment thus provided the animal with higher nutrient availability and led to a decrease in the toxic metabolites produced by bacteria, like ammonia or amines, limiting the energy needed by the animal to detoxify the organism. Some authors also argue that another beneficial effect of AGP results from improved control of intestinal pathologies, such as necrotic enteritis in poultry (12). The January 2006 ban is thus expected to have an impact on veal calf health by leading to more frequent digestive disorders, as previously observed in pigs and poultry in the Nordic countries (36), where AGP have been totally prohibited since the 1990s. Even though no scientific study has yet been done on calves, there have already been reports of higher death rates on experimental commercial farms subsequent to the withdrawal of AGP. The main digestive diseases leading to veal calf deaths are enteritis and enterotoxemia, which are mainly triggered by pathogenic strains of Escherichia coli and Clostridium perfringens (22, 30).Veal calf producers are looking for new feed additives to allay the consequences of the AGP ban. Alternative approaches include the use of prebiotics, probiotics, or plant extracts. Several studies have reported both consistent improvements in weight gain and feed conversion and a reduction of the incidence of diarrhea with the addition of such additives to the veal calf diet (1, 11, 14). One of the hypotheses used to explain these beneficial effects involves the modulation of the intestinal microbiota. In particular, oligosaccharides containing mannose or fructose are known to selectively increase the growth of beneficial intestinal bacteria, including lactobacilli and bifidobacteria (21). Timmerman et al. (33) showed that a calf-specific probiotic containing six Lactobacillus species reduced the fecal counts of E. coli. Green tea extracts also improved the intestinal microbial balance by maintaining high fecal levels of Bifidobacterium and Lactobacillus spp. and decreasing those of C. perfringens (16).As indicated above, it is important to assess the action of newly developed feed additives on the veal calf intestinal microbiota. High interindividual variability makes it difficult and expensive to carry out in vivo studies. Alternatively, experiments can be conducted via in vitro systems modeling the intestinal environment of the animals, provided the model has been checked as pertinent. This approach should allow an economical and ethical way to prescreen feed additives by studying their effects on the intestinal microbiota cultured in the in vitro system and its metabolic activity. With this objective in mind, a necessary requirement is knowledge of the veal calf intestinal ecosystem. Thus, the bacterial and biochemical composition of the jejunoileal chyme of calves was previously characterized (13).The aims of the present study were (i) to set up an in vitro system where the main cultured microbial groups identified in the veal calf intestinal chyme are reproducibly stabilized and metabolically active and (ii) to validate our model by comparing the in vitro and in vivo levels of selected biotic and abiotic variables.     

Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates

Fungi belonging to the Fusarium solani Species Complex (FSSC) are well known plant pathogens. In addition to being the causative agent of some superficial infections, FSSC has recently emerged as a group of common opportunistic moulds, mainly in patients with haematological malignancies. Molecular typing methods are essential in order to better understand the epidemiology of such opportunistic agents with the final goal of preventing contamination. A three-locus typing scheme has thus been developed for FSSC; based on polymorphisms in the domains of the ITS, EF-1 alpha, and RPB2 genes. This method is now considered to be a useful reference for phylogenetic and taxonomic studies. In other significant clinical fungi (e.g., Candida sp., Cryptococcus neoformans, and Aspergillus fumigatus), genes coding for metabolic enzymes have been widely used and proven to be very informative for diagnosis and epidemiology. The contribution of these genes has never been evaluated for Fusarium sp. and more specifically for F. solani Species Complex.Here, we have evaluated the contribution of 25 genes for diagnosis and epidemiological purposes. We then report a new five-locus MLST scheme useful for diagnosis and typing of clinical FSSC isolates. The method has been validated on 51 epidemiologically unrelated strains of FSSC and presents a high power of discrimination calculated at 0.991.