Role for protease activity in visceral pain in irritable bowel syndrome

Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-kappaB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor-2 (PAR2). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR2 antagonist and were absent in PAR2-deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR2.

A feasible study of EEG-driven assistive robotic system for stroke rehabilitation

Stroke is a medical emergency and can cause a neurological damage, affecting the motor and sensory systems. Harnessing brain plasticity should make it possible to reconstruct the closed loop between the brain and the body, i.e., association of the generation of the motor command with the somatic sensory feedback might enhance motor recovery. In order to aid reconstruction of this loop with a robotic device it is necessary to assist the paretic side of the body at the right moment to achieve simultaneity between motor command and feedback signal to somatic sensory area in brain. To this end, we propose an integrated EEG-driven assistive robotic system for stroke rehabilitation. Depending on the level of motor recovery, it is important to provide adequate stimulation for upper limb motion. Thus, we propose an assist arm incorporating a Magnetic Levitation Joint that can generate a compliant motion due to its levitation and mechanical redundancy. This paper reports on a feasibility study carried out to verify the validity of the robot sensing and on EEG measurements conducted with healthy volunteers while performing a spontaneous arm flexion/extension movement. A characteristic feature was found in the temporal evolution of EEG signal in the single motion prior to executed motion which can aid in coordinating timing of the robotic arm assistance onset.

Backtracks: a devised performance

Backtracks aimed to investigate critical relationships between audio-visual technologies and live performance, emphasising technologies producing sound, contrasted with non-amplified bodily sound. Drawing on methodologies for studying avant garde theatre, live performance and the performing body, it was informed by work in critical and cultural theory by, for example, Steven Connor and Jonathan Rée, on the body's experience and interpretation of sound. The performance explored how shifting national boundaries, mobile workforces, complex family relationships, cultural pluralities and possibilities for bodily transformation have compelled a re-evaluation of what it means to feel 'at home' in modernity. Using montages of live and mediated images, disrupted narratives and sound, it evoked destablised identities which characterise contemporary lived experience, and enacted the displacement of certainties provided by family and nation, community and locality, body and selfhood. Homer's Odyssey framed the performance: elements could be traced in the mise-en-scène; in the physical presence of Athene, the narrator and Penelope weaving mementoes from the past into her loom; and in voice-overs from Homer's work. The performance drew on personal experiences and improvisations, structured around notions of journey. It presented incomplete narratives, memories, repressed anxieties and dreams through different combinations of sounds, music, mediated images, movement, voice and bodily sound. The theme of travel was intensified by performers carrying suitcases and umbrellas, by soundtracks incorporating travel effects, and by the distorted video images of forms of transport playing across 'screens' which proliferated across the space (sails, umbrellas, the loom, actors' bodies). The performance experimented with giving sound and silence performative dimensions, including presenting sound in visual and imagistic ways, for example by using signs from deaf sign language. Through-composed soundtracks of live and recorded song, music, voice-over, and noise exploited the viscerality of sound and disrupted cognitive interpretation by phenomenological, somatic experience, thereby displacing the impulse for closure/destination/home.

Overexpression of coconut AINTEGUMENTA-like gene, CnANT, promotes in vitro regeneration in transgenic Arabidopsis

Knowledge of the molecular biological changes underlying the process of embryogenesis is important for the improvement of somatic embryogenesis of coconut. Among the transcription factors that control the transition from vegetative to embryogenic growth, members of APETALA2/Ethylene-responsive element binding protein domain family play an important role in promoting embryo development. Significant insights into the role of AP2 genes have been obtained by the ectopic expression of AP2 sub family genes in transgenic Arabidopsis. A homolog of the AINTEGUMENTA-like gene that encodes the two AP2 domains and the linker region was identified in the coconut genome. Phylogenetic analysis showed that this gene, CnANT, encodes a protein that branched with BABY BOOM/PLETHORA clade in the AINTEGUMENTA-like major clade and was similar to the oil palm EgAP2-1 protein. According to real time RT-PCR results, higher expression of CnANT was observed in more mature zygotic embryos. Also, high CnANT expression was recorded in embryogenic callus compared to other stages of somatic embryogenesis. We examined the effect of ectopic CnANT expression on the development and regenerative capacity of transgenic Arabidopsis. Overexpression of CnANT in Arabidopsis induced hormone free regeneration of explants. Furthermore, ectopic expression of CnANT enhanced regeneration in vitro and suggested a role for this gene in cell proliferation during in vitro culture.

Evaluation of milk compositional variables on coagulation properties using partial least squares

The aim of this study was to investigate the effects of numerous milk compositional factors on milk coagulation properties using Partial Least Squares (PLS). Milk from herds of Jersey and Holstein-Friesian cattle was collected across the year and blended (n=55), to maximize variation in composition and coagulation. The milk was analysed for casein, protein, fat, titratable acidity, lactose, Ca2+, urea content, micelles size, fat globule size, somatic cell count and pH. Milk coagulation properties were defined as coagulation time, curd firmness and curd firmness rate measured by a controlled strain rheometer. The models derived from PLS had higher predictive power than previous models demonstrating the value of measuring more milk components. In addition to the well-established relationships with casein and protein levels, CMS and fat globule size were found to have as strong impact on all of the three models. The study also found a positive impact of fat on milk coagulation properties and a strong relationship between lactose and curd firmness, and urea and curd firmness rate, all of which warrant further investigation due to current lack of knowledge of the underlying mechanism. These findings demonstrate the importance of using a wider range of milk compositional variable for the prediction of the milk coagulation properties, and hence as indicators of milk suitability for cheese making.

Clonal expansion of early to mid-life mitochondrial DNA point mutations drives mitochondrial dysfunction during human ageing

Age-related decline in the integrity of mitochondria is an important contributor to the human ageing process. In a number of ageing stem cell populations, this decline in mitochondrial function is due to clonal expansion of individual mitochondrial DNA (mtDNA) point mutations within single cells. However the dynamics of this process and when these mtDNA mutations occur initially are poorly understood. Using human colorectal epithelium as an exemplar tissue with a well-defined stem cell population, we analysed samples from 207 healthy participants aged 17-78 years using a combination of techniques (Random Mutation Capture, Next Generation Sequencing and mitochondrial enzyme histochemistry), and show that: 1) non-pathogenic mtDNA mutations are present from early embryogenesis or may be transmitted through the germline, whereas pathogenic mtDNA mutations are detected in the somatic cells, providing evidence for purifying selection in humans, 2) pathogenic mtDNA mutations are present from early adulthood (<20 years of age), at both low levels and as clonal expansions, 3) low level mtDNA mutation frequency does not change significantly with age, suggesting that mtDNA mutation rate does not increase significantly with age, and 4) clonally expanded mtDNA mutations increase dramatically with age. These data confirm that clonal expansion of mtDNA mutations, some of which are generated very early in life, is the major driving force behind the mitochondrial dysfunction associated with ageing of the human colorectal epithelium.

Effect of blending Jersey and Holstein-Friesian milk on Cheddar cheese processing, composition and quality

The aim of this study was to investigate the effects of numerous milk compositional factors on milk coagulation properties using Partial Least Squares (PLS). Milk from herds of Jersey and Holstein- Friesian cattle was collected across the year and blended (n=55), to maximise variation in composition and coagulation. The milk was analysed for casein, protein, fat, titratable acidity, lactose, Ca2+, urea content, micelles size, fat globule size, somatic cell count and pH. Milk coagulation properties were defined as coagulation time, curd firmness and curd firmness rate measured by a controlled strain rheometer. The models derived from PLS had higher predictive power than previous models demonstrating the value of measuring more milk components. In addition to the well-established relationships with casein and protein levels, CMS and fat globule size were found to have as strong impact on all of the three models. The study also found a positive impact of fat on milk coagulation properties and a strong relationship between lactose and curd firmness, and urea and curd firmness rate, all of which warrant further investigation due to current lack of knowledge of the underlying mechanism. These findings demonstrate the importance of using a wider range of milk compositional variables for the prediction of the milk coagulation properties, and hence as indicators of milk suitability for cheese making.

Adult palatum as a novel source of neural crest-related stem cells

Somatic neural and neural crest stem cells are promising sources for cellular therapy of several neurodegenerative diseases. However, because of practical considerations such as inadequate accessibility of the source material, the application of neural crest stem cells is strictly limited. The secondary palate is a highly regenerative and heavily innervated tissue, which develops embryonically under direct contribution of neural crest cells. Here, we describe for the first time the presence of nestin-positive neural crest-related stem cells within Meissner corpuscles and Merkel cell-neurite complexes located in the hard palate of adult Wistar rats. After isolation, palatal neural crest-related stem cells (pNC-SCs) were cultivated in the presence of epidermal growth factor and fibroblast growth factor under serum-free conditions, resulting in large amounts of neurospheres. We used immunocytochemical techniques and reverse transcriptase-polymerase chain reaction to assess the expression profile of pNC-SCs. In addition to the expression of neural crest stem cell markers such as Nestin, Sox2, and p75, we detected the expression of Klf4, Oct4, and c-Myc. pNC-SCs differentiated efficiently into neuronal and glial cells. Finally, we investigated the potential expression of stemness markers within the human palate. We identified expression of stem cell markers nestin and CD133 and the transcription factors needed for reprogramming of somatic cells into pluripotent cells: Sox2, Oct4, Klf4, and c-Myc. These data show that cells isolated from palatal rugae form neurospheres, are highly plastic, and express neural crest stem cell markers. In addition, pNC-SCs may have the ability to differentiate into functional neurons and glial cells, serving as a starting point for therapeutic studies.

Morphological characterization of periodontium-derived human stem cells

The aim of this study has been to characterize adult human somatic periodontium-derived stem cells (PDSCS) isolated from human periodontium and to follow their differentiation after cell culture. PDSCS were isolated from human periodontal tissue and cultured as spheres in serum-free medium. After 10 days the primary spheres were dissociated and the secondary spheres sub-cultured for another 1-2 weeks. Cells from different time points were analyzed, and immunohistochemical and electron microscopic investigations carried out. Histological analysis showed differentiation of spheres deriving from the PDSCS with central production of extracellular matrix beginning 3 days after sub-culturing. Isolated PDSCS developed pseudopodia which contained actin. Tubulin was found in the central portion of the cells. Pseudopodia between different cells anastomosed, indicating intercellular transport. Immunostaining for osteopontin demonstrated a positive reaction in primary spheres and within extracellular matrix vesicles after sub-culturing. In cell culture under serum-free conditions human PDSCS form spheres which are capable of producing extracellular matrix. Further investigations have do be carried out to investigate the capability of these cells to differentiate into osteogenic progenitor cells.

Global scale transcriptome analysis of Arabidopsis embryogenesis in vitro

Background Somatic embryogenesis (SE) in plants is a process by which embryos are generated directly from somatic cells, rather than from the fused products of male and female gametes. Despite the detailed expression analysis of several somatic-to-embryonic marker genes, a comprehensive understanding of SE at a molecular level is still lacking. The present study was designed to generate high resolution transcriptome datasets for early SE providing the way for future research to understand the underlying molecular mechanisms that regulate this process. We sequenced Arabidopsis thaliana somatic embryos collected from three distinct developmental time-points (5, 10 and 15 d after in vitro culture) using the Illumina HiSeq 2000 platform. Results This study yielded a total of 426,001,826 sequence reads mapped to 26,520 genes in the A. thaliana reference genome. Analysis of embryonic cultures after 5 and 10 d showed differential expression of 1,195 genes; these included 778 genes that were more highly expressed after 5 d as compared to 10 d. Moreover, 1,718 genes were differentially expressed in embryonic cultures between 10 and 15 d. Our data also showed at least eight different expression patterns during early SE; the majority of genes are transcriptionally more active in embryos after 5 d. Comparison of transcriptomes derived from somatic embryos and leaf tissues revealed that at least 4,951 genes are transcriptionally more active in embryos than in the leaf; increased expression of genes involved in DNA cytosine methylation and histone deacetylation were noted in embryogenic tissues. In silico expression analysis based on microarray data found that approximately 5% of these genes are transcriptionally more active in somatic embryos than in actively dividing callus and non-dividing leaf tissues. Moreover, this identified 49 genes expressed at a higher level in somatic embryos than in other tissues. This included several genes with unknown function, as well as others related to oxidative and osmotic stress, and auxin signalling. Conclusions The transcriptome information provided here will form the foundation for future research on genetic and epigenetic control of plant embryogenesis at a molecular level. In follow-up studies, these data could be used to construct a regulatory network for SE; the genes more highly expressed in somatic embryos than in vegetative tissues can be considered as potential candidates to validate these networks.

Controversial role of toll-like receptor 4 in adult stem cells

Adult or somatic stem cells are tissue-resident cells with the ability to proliferate, exhibit self-maintenance as well as to generate new cells with the principal phenotypes of the tissue in response to injury or disease. Due to their easy accessibility and their potential use in regenerative medicine, adult stem cells raise the hope for future personalisable therapies. After infection or during injury, they are exposed to broad range of pathogen or damage-associated molecules leading to changes in their proliferation, migration and differentiation. The sensing of such damage and infection signals is mostly achieved by Toll-Like Receptors (TLRs) with Toll-like receptor 4 being responsible for recognition of bacterial lipopolysaccharides (LPS) and endogenous danger-associated molecular patterns (DAMPs). In this review, we examine the current state of knowledge on the TLR4-mediated signalling in different adult stem cell populations. Specifically, we elaborate on the role of TLR4 and its ligands on proliferation, differentiation and migration of mesenchymal stem cells, hematopoietic stem cells as well as neural stem cells. Finally, we discuss conceptual and technical pitfalls in investigation of TLR4 signalling in stem cells.

Transcriptome analysis for discovering candidate genes involve in embryogenesis in coconut (Cocos nucifera L.) through 454 pyrosequencing

Coconut, Cocos nucifera L. is a major plantation crop, which ensures income for millions of people in the tropical region. Detailed molecular studies on zygotic embryo development would provide valuable clues for the identification of molecular markers to improve somatic embryogenesis. Since there is no ongoing genome project for this species, coconut expressed sequence tags (EST) would be an interesting technique to identify important coconut embryo specific genes as well as other functional genes in different biochemical pathways. The goal of this study was to analyse the ESTs by examining the transcriptome data of the different embryo tissue types together with one somatic tissue. Here, four cDNA libraries from immature embryo, mature embryo, microspore derived embryo and mature leaves were constructed. cDNA was sequenced by the Roche-454 GS-FLX system and assembled into 32621 putative unigenes and 155017 singletons. Of these unigenes, 18651 had significant sequence similarities to non-redundant protein database, from which 16153 were assigned to one or more gene ontology categories. Homologue genes, which are responsible for embryo development such as chitinase, beta-1,3-glucanase, ATP synthase CF0 subunit, thaumatin-like protein and metallothionein-like protein were identified among the embryo EST collection. Of the unigenes, 6694 were mapped into 139 KEGG pathways including carbohydrate metabolism, energy metabolism, lipid metabolism, amino acid metabolism and nucleotide metabolism. This collection of 454-derived EST data generated from different tissue types provides a significant resource for genome wide studies and gene discovery of coconut, a non-model species.

Production of Bovine Hand-Made Cloned Embryos by Zygote-Oocyte Cytoplasmic Hemi-complementation

The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; handmade cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.

Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos

Objective: To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. Design: Comparison between oocyte groups and between early embryo stages. Setting: Laboratories of embryo manipulation and molecular biology from Departamento de Genetica (FMRP) and Departamento de Ciencias Basicas (FZEA) - University of Sao Paulo. Sample(s): Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. Main Outcome Measure(s): Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). Result(s): Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good-and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. Conclusion(s): Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development. (Fertil Steril (R) 2010; 93: 2507-12. (C) 2010 by American Society for Reproductive Medicine.)

Delivery of cloned offspring: experience in Zebu cattle (Bos indicus)

The production of a healthy cloned calf is dependent on a multitude of successful steps, including reprogramming mediated by the oocyte, the development of a functional placenta, adequate maternal-fetal interaction, the establishment of a physiological metabolic setting and the formation of a complete set of well-differentiated cells that will eventually result in well-characterised and fully competent tissues and organs. Although the efficiency of nuclear transfer has improved significantly since the first report of a somatic cell nuclear transfer-derived animal, there are many descriptions of anomalies concerning cloned calves leading to high perinatal morbidity and mortality. The present article discusses some our experience regarding perinatal and neonatal procedures for cloned Zebu cattle (B. indicus) that has led to improved survival rates in Nellore cloned calves following the application of such `labour-intensive technology`.