Cetobacterium somerae sp nov from human feces and emended description of the genus Cetobacterium

Phenotypic and phylogenetic studies were performed on four isolates of an unidentified gram-negative, microaerotolerant, non-spore-forming, rod-shaped bacterium isolated from the feces of children. The unknown organism was bile resistant and produced acetic acid as the major end product of metabolism of peptides and carbohydrates. It possessed a low DNA G + C content of 31 mol %. Comparative 16S rRNA gene sequencing demonstrated that the four isolates were phylogenetically identical (100% 16S rRNA sequence similarity) and represent a hitherto unknown sub-line within the genus Cetobacterium. The novel bacterium displayed approximately 5% sequence divergence with Cetobacterium ceti, and can be readily distinguished from the latter by physiological and biochemical criteria. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown fecal bacterium be classified in the genus Cetobacterium, as Cetobacterium somerae sp. nov. The proposed type strain of Cetobacterium somerae is WAL 14325(T) (ATCC BAA-474(T) = CCUG 46254T).

Anaerofustis stercorihominis gen. nov., sp nov., from human faeces

Phenotypic and phylogenetic studies were performed on an unidentified Gram-positive, strictly anaerobic, non-spore-forming, rod-shaped bacterium isolated from human feces. The organism was catalase-negative, resistant to 20% bile, produced acetic and butyric acids as end products of glucose metabolism, and possessed a G + C content of approximately 70 mol %. Comparative 16S rRNA gene sequencing demonstrated that the unidentified bacterium was a member of the Clostridium sub-phylum of the Gram-positive bacteria, and formed a loose association with rRNA cluster XV. Sequence divergence values of 12% or greater were observed between the unidentified bacterium and all other recognized species within this and related rRNA clusters. Treeing analysis showed the unknown anaerobe formed a deep line branching near to the base of rRNA cluster XV and phylogenetically represents a hitherto unknown taxon, distinct from Acetobacterium, Eubacterium sensu stricto, Pseudoramibacter and other related organisms. Based on both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium from feces be classified in a new genus Anaerofustis, as Anaerofustis stercorihominis sp. nov. The type strain of Anaerofustis stercorihominis is ATCC BAA-858(T) = CCUG 47767(T). (C) 2003 Elsevier Ltd. All rights reserved.

Fastidiosipila sanguinis gen. nov., sp nov., a new Gram-positive, coccus-shaped organism from human blood

Phenotypic and phylogenetic studies were performed on two strains of an unidentified Gram-positive, fastidious, non-spore-forming, coccus-shaped bacterium recovered from human blood. The organism was catalase-negative and grew under strictly anaerobic conditions and in the presence of 2 and 6% O-2. Comparative 16S rRNA gene sequencing demonstrated that the unidentified bacterium was, phylogenetically, far removed from peptostreptococci and related Gram-positive coccus-shaped organisms, but exhibited a phylogenetic association with Clostridium rRNA cluster III [as defined by Collins et al, Int J Syst Bacteriol 44 (1994), 812-826]. Sequence divergence values of 15% or more were observed between the unidentified bacterium and all other recognized species within this and related rRINIA clostridial clusters. Treeing analysis showed that the unknown bacterium formed a deep line branching at the periphery of rRNA cluster III and represents a hitherto unknown genus within this supra-generic grouping. On the basis of both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium from blood be classified in a new genus, Fastidiosipila gen. nov., as Fastidiosipila sanguinis sp, nov. The type strain of Fastidiosipila sanguinis is CCUG 47711(T) (= CIP 108292(T)).

Differential induction of apoptosis in human colonic carcinoma cells (Caco-2) by Atopobium, and commensal, probiotic and enteropathogenic bacteria: mediation by the mitochondrial pathway

The induction of apoptosis in mammalian cells by bacteria is well reported. This process may assist infection by pathogens whereas for non-pathogens apoptosis induction within carcinoma cells protects against colon cancer. Here, apoptosis induction by a major new gut bacterium, Atopobium minutum, was compared with induction by commensal (Escherichia coli K-12 strains), probiotic (Lactobacillus rhamnosus, Bifidobacterium latis) and pathogenic (E. coli: EPEC and VTEC) gut bacteria within the colon cancer cell line, Caco-2. The results show a major apoptotic effect for the pathogens, mild effects for the probiotic strains and A. minutum, but no effect for commensal E. coli. The mild apoptotic effects observed are consistent with the beneficial roles of probotics in protection against colon cancer and suggest, for the first time, that A. minutum possesses similar advantageous, anti-cancerous activity. Although bacterial infection increased Caco-2 membrane FAS levels, caspase-8 was not activated indicating that apoptosis is FAS independent. Instead, in all cases, apoptosis was induced through the mitochondrial pathway as indicated by BAX translocation, cytorchrome c release, and caspase-9 and -3 cleavage. This suggests that an intracellular stimulus initiates the observed apoptosis responses.

An automated calibration method for non-see-through head mounted displays

Accurate calibration of a head mounted display (HMD) is essential both for research on the visual system and for realistic interaction with virtual objects. Yet, existing calibration methods are time consuming and depend on human judgements, making them error prone. The methods are also limited to optical see-through HMDs. Building on our existing HMD calibration method [1], we show here how it is possible to calibrate a non-see-through HMD. A camera is placed inside an HMD displaying an image of a regular grid, which is captured by the camera. The HMD is then removed and the camera, which remains fixed in position, is used to capture images of a tracked calibration object in various positions. The locations of image features on the calibration object are then re-expressed in relation to the HMD grid. This allows established camera calibration techniques to be used to recover estimates of the display’s intrinsic parameters (width, height, focal length) and extrinsic parameters (optic centre and orientation of the principal ray). We calibrated a HMD in this manner in both see-through and in non-see-through modes and report the magnitude of the errors between real image features and reprojected features. Our calibration method produces low reprojection errors and involves no error-prone human measurements.

Effects of biologically active tumour-promoting and non-promoting phorbol esters on in vivo growth of melanocytic cells

Sapintoxin A (SAP A), a naturally occurring biologically active but non-promoting phorbol ester, acts as an effective in vitro mitogen for freshly derived human melanocytes. Seven days after addition of 50 nM SAP A there was a four to fivefold increase in melanocyte number over that observed in untreated control cultures comparable to that achieved with a 50 nM concentration of 12-0-tetradecanoylphorbol 13-acetate (TPA). The fluorescent stage 2 promoter sapintoxin D (SAP D) also supported the growth of these cells, with a 50 nM dose producing an increase in cell number comparable to that observed with 200 nM TPA. Similar results were obtained with an established, but non-tumorigenic, line of murine melanocytes. The same compounds exerted a potent anti-proliferative effect against transformed melanocyte lines of murine and human origin associated with morphological alterations and an increase in melanin production consistent with induced cytodifferentiation.

Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5

The 5'-cap-structures of higher eukaryote mRNAs are ribose 2'-O-methylated. Likewise, a number of viruses replicating in the cytoplasm of eukayotes have evolved 2'-O-methyltransferases to modify autonomously their mRNAs. However, a defined biological role of mRNA 2'-O-methylation remains elusive. Here we show that viral mRNA 2'-O-methylation is critically involved in subversion of type-I-interferon (IFN-I) induction. We demonstrate that human and murine coronavirus 2'-O-methyltransferase mutants induce increased IFN-I expression, and are highly IFN-I sensitive. Importantly, IFN-I induction by 2'-O-methyltransferase-deficient viruses is dependent on the cytoplasmic RNA sensor melanoma differentiation-associated gene 5 (MDA5). This link between MDA5-mediated sensing of viral RNA and mRNA 2'-O-methylation suggests that RNA modifications, such as 2'-O-methylation, provide a molecular signature for the discrimination of self and non-self mRNA.

In vitro immunomodulatory activity of Lactobacillus fermentum CECT5716 and Lactobacillus salivarius CECT5713: two probiotic strains isolated from human breast milk

Commensal bacteria, including some species of lactobacilli commonly present in human breast milk, appear to colonize the neonatal gut and contribute to protection against infant infections, suggesting that lactobacilli could potentially modulate immunity. In this study, we evaluated the potential of two Lactobacillus strains isolated from human milk to modulate the activation and cytokine profile of peripheral blood mononuclear cell (PBMC) subsets in vitro. Moreover, these effects were compared to the same probiotic species of non-milk origin. Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716 at 105, 106 and 107 bacteria/mL were co-cultured with PBMC (106/mL) from 8 healthy donors for 24 h. Activation status (CD69 and CD25 expressions) of natural killer (NK) cells (CD56+), total T cells (CD3+), cytotoxic T cells (CD8+) and CD4+ T cells was determined by flow cytometry. Regulatory T cells (Treg) were also quantified by intracellular Foxp3 evaluation. Regarding innate immunity, NK cells were activated by addition of both Lactobacillus strains, and in particular, the CD8+ NK subset was preferentially induced to highly express CD69 (90%, p<0.05). With respect to acquired immunity, approximately 9% of CD8+ T cells became activated after co-cultivation with L. fermentum or L salivarius. Although CD4+ T cells demonstrated a weaker response, there was a preferential activation of Treg cells (CD4+CD25+Foxp3+) after exposure to both milk probiotic bacteria (p<0.05). Both strains significantly induced the production of a number of cytokines and chemokines, including TNFα, IL-1β, IL-8, MIP-1α, MIP-1β, and GM-CSF, but some strain-specific effects were apparent. This work demonstrates that L salivarius CECT5713 and L. fermentum CECT5716 enhanced both natural and acquired immune responses, as evidenced by the activation of NK and T cell subsets and the expansion of Treg cells, as well as the induction of a broad array of cytokines.

A human volunteer study to assess the impact of confectionery sweeteners on the gut microbiota composition.

Sweeteners are being sourced to lower the energetic value of confectionery including chocolates. Some, especially non-digestible carbohydrates, may possess other benefits for human health upon their fermentation by the colonic microbiota. The present study assessed non-digestible carbohydrate sweeteners, selected for use in low-energy chocolates, for their ability to beneficially modulate faecal bacterial profiles in human volunteers. Forty volunteers consumed a test chocolate (low-energy or experimental chocolate) containing 22·8 g of maltitol (MTL), MTL and polydextrose (PDX), or MTL and resistant starch for fourteen consecutive days. The dose of the test chocolates was doubled every 2 weeks over a 6-week period. Numbers of faecal bifidobacteria significantly increased with all the three test treatments. Chocolate containing the PDX blend also significantly increased faecal lactobacilli (P = 0·00 001) after the 6 weeks. The PDX blend also showed significant increases in faecal propionate and butyrate (P = 0·002 and 0·006, respectively). All the test chocolates were well tolerated with no significant change in bowel habit or intestinal symptoms even at a daily dose of 45·6 g of non-digestible carbohydrate sweetener. This is of importance not only for giving manufacturers a sugar replacement that can reduce energetic content, but also for providing a well-tolerated means of delivering high levels of non-digestible carbohydrates into the colon, bringing about improvements in the biomarkers of gut health.

Non-genomic effects of PPARgamma ligands: inhibition of GPVI-stimulated platelet activation

BACKGROUND: Peroxisome proliferator-activated receptor-(gamma) (PPAR(gamma)) is expressed in human platelets although in the absence of genomic regulation in these cells, its functions are unclear. OBJECTIVE: In the present study, we aimed to demonstrate the ability of PPAR(gamma) ligands to modulate collagen-stimulated platelet function and suppress activation of the glycoprotein VI (GPVI) signaling pathway. METHODS: Washed platelets were stimulated with PPAR(gamma) ligands in the presence and absence of PPAR(gamma) antagonist GW9662 and collagen-induced aggregation was measured using optical aggregometry. Calcium levels were measured by spectrofluorimetry in Fura-2AM-loaded platelets and tyrosine phosphorylation levels of receptor-proximal components of the GPVI signaling pathway were measured using immunoblot analysis. The role of PPAR(gamma) agonists in thrombus formation was assessed using an in vitro model of thrombus formation under arterial flow conditions. RESULTS: PPAR(gamma) ligands inhibited collagen-stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P-selectin exposure. PPAR(gamma) ligands inhibited thrombus formation under arterial flow conditions. The incorporation of GW9662 reversed the inhibitory actions of PPAR(gamma) agonists, implicating PPAR(gamma) in the effects observed. Furthermore, PPAR(gamma) ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway. PPAR(gamma) was found to associate with Syk and LAT after platelet activation. This association was prevented by PPAR(gamma) agonists, indicating a potential mechanism for PPAR(gamma) function in collagen-stimulated platelet activation. CONCLUSIONS: PPAR(gamma) agonists inhibit the activation of collagen-stimulation of platelet function through modulation of early GPVI signalling.

The quantitation of lipoprotein lipase mRNA in biopsies of human adipose tissue, using the polymerase chain reaction, and the effect of increased consumption of n-3 polyunsaturated fatty acids

Objective: To examine the effects of the consumption of fish oils on the gene expression of lipoprotein lipase (LPL, EC 3.1.1.34) in human adipose tissue. In order to measure LPL mRNA in adipose tissue samples obtained by needle biopsy from human volunteers a competitive, reverse transcriptase PCR (RT-PCR) protocol was developed. Design: A randomised controlled, single blind cross over dietary study which compared the effects of a low level n-3 polyunsaturated fatty acids (PUFA) using normal foods enriched with eicosapentaenoic (EPA) and docosahexaenoic (DHA) (test diet), with non-enriched but otherwise identical foods (control). The diets were consumed for a period of 22 d with a wash out period of 5 months between the diets. Setting: Free-living individuals associated with the University of Surrey. Subjects: Six male subjects with a mean (±sd) age of 51.2±3.6 y were recruited. Major Outcome Measures: Pre-and postprandial blood samples were taken for the measurement of triacylglycerol (TAG), postheparin LPL activity and adipose tissue samples for the measurement of LPL mRNA levels. Results: Mean LPL expression values were 4.12´105 molecules of LPL mRNA per ng total RNA on the control diet and 4.60´105 molecules of LPL mRNA per ng total RNA on the n-3 PUFA enriched (test) diet. There was no significant difference between the levels of LPL expression following each diet, consistent with the lack of change in TAG levels in response to increased dietary n-3 PUFA intake. However, the change in LPL expression (Test-Control diet) correlated significantly with the change in fasting TAG levels (P=0.03, R=-0.87 and R2=0.75) and with the total area under the TAG-time response curve (P=0.003, R=-0.96 and R2=0.92) in individuals. Conclusions: These findings, although based on a small number of subjects, suggest that LPL expression may be a determinant of plasma TAG levels. The development of this methodology should allow further elucidation of the effects of dietary manipulation and disease processes on lipid clearance and regulation in human subjects.

Modelling human dynamics in-situ for rehabilitation and therapy robots

This paper outlines some rehabilitation applications of manipulators and identifies that new approaches demand that the robot make an intimate contact with the user. Design of new generations of manipulators with programmable compliance along with higher level controllers that can set the compliance appropriately for the task, are both feasible propositions. We must thus gain a greater insight into the way in which a person interacts with a machine, particularly given that the interaction may be non-passive. We are primarily interested in the change in wrist and arm dynamics as the person co-contracts his/her muscles. It is observed that this leads to a change in stiffness that can push an actuated interface into a limit cycle. We use both experimental results gathered from a PHANToM haptic interface and a mathematical model to observe this effect. Results are relevant to the fields of rehabilitation and therapy robots, haptic interfaces, and telerobotics

Adipose tissue metabolism in pregnancy: the lipolytic effect of human placental lactogen

In vitro adipose tissue lipolysis was investigated in pregnant and non-pregnant women. Basal and hormone-stimulated rates of lipolysis were significantly higher in the pregnant group. Human placental lactogen (HPL) was shown to be lipolytic on adipose tissue from both pregnant and non-pregnant women; the response being more marked in the pregnant group. We postulate that HPL is responsible for this increased lipolytic sensitivity in pregnancy, the result of which is the elevation in levels of plasma free fatty acids in the third trimester.

Crosstalk with insulin and dependence on PI3K/Akt/mTOR rather than MAPK pathways in upregulation of basal growth following long-term oestrogen deprivation in three human breast cancer cell lines

Background: MCF-7, T-47-D, ZR-75-1 human breast cancer cell lines are dependent on oestrogen for growth but can adapt to grow during long-term oestrogen deprivation. This serves as a model for identification of therapeutic targets in endocrine-resistant breast cancer. Methods: An overlooked complication of this model is that it involves more than non-addition of oestrogen, and inadequate attention has been given to separating molecular events associated with each of the culture manipulations. Results: Insulin and oestradiol were shown to protect MCF-7 cells against upregulation of basal growth, demonstrating a crosstalk in the growth adaptation process. Increased phosphorylation of p44/42MAPK and c-Raf reflected removal of insulin from the medium and proliferation of all three cell lines was inhibited to a lesser extent by PD98059 and U0126 following long-term oestrogen/insulin withdrawal, demonstrating a reduced dependence on the MAPK pathway. By contrast, long-term oestrogen/insulin deprivation did not alter levels of phosphorylated Akt and did not alter the dose-response of growth inhibition with LY294002 in any of the three cell lines. The IGF1R inhibitor picropodophyllin inhibited growth of all MCF-7 cells but only in the long-term oestrogen/insulin-deprived cells was this paralleled by reduction in phosphorylated p70S6K, a downstream target of mTOR. Long-term oestrogen/insulin-deprived MCF-7 cells had higher levels of phosphorylated p70S6K and developed increased sensitivity to growth inhibition by rapamycin. Conclusions: The greater sensitivity to growth inhibition by rapamycin in all three cell lines following long-term oestrogen/insulin deprivation suggests rapamycin-based therapies might be more effective in breast cancers with acquired oestrogen resistance. Keywords Akt, breast cancer cells, endocrine resistance, insulin, MAPK, MCF-7 cells, mTOR, oestrogen, oestrogen-deprived, PI3K, picropodophyllin, rapamycin, T-47-D cells, ZR-75-1 cells

Technologies for biological containment of GM and Non-GM crops

International Perspective The development of GM technology continues to expand into increasing numbers of crops and conferred traits. Inevitably, the focus remains on the major field crops of soybean, maize, cotton, oilseed rape and potato with introduced genes conferring herbicide tolerance and/or pest resistance. Although there are comparatively few GM crops that have been commercialised to date, GM versions of 172 plant species have been grown in field trials in 31 countries. European Crops with Containment Issues Of the 20 main crops in the EU there are four for which GM varieties are commercially available (cotton, maize for animal feed and forage, and oilseed rape). Fourteen have GM varieties in field trials (bread wheat, barley, durum wheat, sunflower, oats, potatoes, sugar beet, grapes, alfalfa, olives, field peas, clover, apples, rice) and two have GM varieties still in development (rye, triticale). Many of these crops have hybridisation potential with wild and weedy relatives in the European flora (bread wheat, barley, oilseed rape, durum wheat, oats, sugar beet and grapes), with escapes (sunflower); and all have potential to cross-pollinate fields non-GM crops. Several fodder crops, forestry trees, grasses and ornamentals have varieties in field trials and these too may hybridise with wild relatives in the European flora (alfalfa, clover, lupin, silver birch, sweet chestnut, Norway spruce, Scots pine, poplar, elm, Agrostis canina, A. stolonifera, Festuca arundinacea, Lolium perenne, L. multiflorum, statice and rose). All these crops will require containment strategies to be in place if it is deemed necessary to prevent transgene movement to wild relatives and non-GM crops. Current Containment Strategies A wide variety of GM containment strategies are currently under development, with a particular focus on crops expressing pharmaceutical products. Physical containment in greenhouses and growth rooms is suitable for some crops (tomatoes, lettuce) and for research purposes. Aquatic bioreactors of some non-crop species (algae, moss, and duckweed) expressing pharmaceutical products have been adopted by some biotechnology companies. There are obvious limitations of the scale of physical containment strategies, addressed in part by the development of large underground facilities in the US and Canada. The additional resources required to grow plants underground incurs high costs that in the long term may negate any advantage of GM for commercial productioNatural genetic containment has been adopted by some companies through the selection of either non-food/feed crops (algae, moss, duckweed) as bio-pharming platforms or organisms with no wild relatives present in the local flora (safflower in the Americas). The expression of pharmaceutical products in leafy crops (tobacco, alfalfa, lettuce, spinach) enables growth and harvesting prior to and in the absence of flowering. Transgenically controlled containment strategies range in their approach and degree of development. Plastid transformation is relatively well developed but is not suited to all traits or crops and does not offer complete containment. Male sterility is well developed across a range of plants but has limitations in its application for fruit/seed bearing crops. It has been adopted in some commercial lines of oilseed rape despite not preventing escape via seed. Conditional lethality can be used to prevent flowering or seed development following the application of a chemical inducer, but requires 100% induction of the trait and sufficient application of the inducer to all plants. Equally, inducible expression of the GM trait requires equally stringent application conditions. Such a method will contain the trait but will allow the escape of a non-functioning transgene. Seed lethality (‘terminator’ technology) is the only strategy at present that prevents transgene movement via seed, but due to public opinion against the concept it has never been trialled in the field and is no longer under commercial development. Methods to control flowering and fruit development such as apomixis and cleistogamy will prevent crop-to-wild and wild-to-crop pollination, but in nature both of these strategies are complex and leaky. None of the genes controlling these traits have as yet been identified or characterised and therefore have not been transgenically introduced into crop species. Neither of these strategies will prevent transgene escape via seed and any feral apomicts that form are arguably more likely to become invasives. Transgene mitigation reduces the fitness of initial hybrids and so prevents stable introgression of transgenes into wild populations. However, it does not prevent initial formation of hybrids or spread to non-GM crops. Such strategies could be detrimental to wild populations and have not yet been demonstrated in the field. Similarly, auxotrophy prevents persistence of escapes and hybrids containing the transgene in an uncontrolled environment, but does not prevent transgene movement from the crop. Recoverable block of function, intein trans-splicing and transgene excision all use recombinases to modify the transgene in planta either to induce expression or to prevent it. All require optimal conditions and 100% accuracy to function and none have been tested under field conditions as yet. All will contain the GM trait but all will allow some non-native DNA to escape to wild populations or to non-GM crops. There are particular issues with GM trees and grasses as both are largely undomesticated, wind pollinated and perennial, thus providing many opportunities for hybridisation. Some species of both trees and grass are also capable of vegetative propagation without sexual reproduction. There are additional concerns regarding the weedy nature of many grass species and the long-term stability of GM traits across the life span of trees. Transgene stability and conferred sterility are difficult to trial in trees as most field trials are only conducted during the juvenile phase of tree growth. Bio-pharming of pharmaceutical and industrial compounds in plants Bio-pharming of pharmaceutical and industrial compounds in plants offers an attractive alternative to mammalian-based pharmaceutical and vaccine production. Several plantbased products are already on the market (Prodigene’s avidin, β-glucuronidase, trypsin generated in GM maize; Ventria’s lactoferrin generated in GM rice). Numerous products are in clinical trials (collagen, antibodies against tooth decay and non-Hodgkin’s lymphoma from tobacco; human gastric lipase, therapeutic enzymes, dietary supplements from maize; Hepatitis B and Norwalk virus vaccines from potato; rabies vaccines from spinach; dietary supplements from Arabidopsis). The initial production platforms for plant-based pharmaceuticals were selected from conventional crops, largely because an established knowledge base already existed. Tobacco and other leafy crops such as alfalfa, lettuce and spinach are widely used as leaves can be harvested and no flowering is required. Many of these crops can be grown in contained greenhouses. Potato is also widely used and can also be grown in contained conditions. The introduction of morphological markers may aid in the recognition and traceability of crops expressing pharmaceutical products. Plant cells or plant parts may be transformed and maintained in culture to produce recombinant products in a contained environment. Plant cells in suspension or in vitro, roots, root cells and guttation fluid from leaves may be engineered to secrete proteins that may be harvested in a continuous, non-destructive manner. Most strategies in this category remain developmental and have not been commercially adopted at present. Transient expression produces GM compounds from non-GM plants via the utilisation of bacterial or viral vectors. These vectors introduce the trait into specific tissues of whole plants or plant parts, but do not insert them into the heritable genome. There are some limitations of scale and the field release of such crops will require the regulation of the vector. However, several companies have several transiently expressed products in clinical and pre-clinical trials from crops raised in physical containment.