Growth of papaya nodal and rooted shoot cultures as affected by ACC, STS, culture vessel size and incubation conditions

Nodal shoot cultures of 'Clone 003', a selected Australian papaya cultivar, were cultured on modified De Fossard medium supplemented with chemicals that either promote ethylene evolution or inhibit action while in culture. Nodal shoot cultures grown in the presence of 1-aminocyclopropane carboxylic acid (ACC, 1.0 mM) resulted in a significant reduction in percent fresh and dry weights, shoot length, leaf area, petiole length and chlorophyll content, but leaf development was significantly increased. In contrast, nodal cultures grown in the presence of silver thiosulphate (STS, 0.5 mM) significantly produced the highest percentage of fresh and dry weights, shoot length, leaf production, leaf area expansion, petiole length and leaf chlorophyll content. Nodal cultures and rooted whole plantlets placed in medium-sized (125 mL) culture vessels had significantly better growth than those cultures placed in small (70 mL) or in large (250 mL) vessels. Cultures grown in medium-sized vessels had higher fresh and dry weights, longer shoots, more leaves and larger leaf area than those cultures placed in smaller or larger vessels. Similarly, values for said growth parameters and for chlorophyll content of the nodal and rooted whole plantlets were higher when they were incubated under high light intensity of 120 mumol m(-2)s(-1) at a prevailing temperature of either 20+/-1 C or 25+/-1 C.

Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression

Shrimp cell lines are yet to be reported and this restricts the prospects of investigating the associated viral pathogens, especially white spot syndrome virus (WSSV). In this context, development of primary cell cultures from lymphoid organs was standardized. Poly-l-lysine-coated culture vessels enhanced growth of lymphoid cells, while the application of vertebrate growth factors did not, except insulin-like growth factor-1 (IGF-1). Susceptibility of the lymphoid cells to WSSV was confirmed by immunofluoresence assay using monoclonal antibody against the 28 kDa envelope protein of WSSV. Expression of viral and immunerelated genes in WSSV-infected lymphoid cultures could be demonstrated by RT-PCR. This emphasizes the utility of lymphoid primary cell culture as a platform for research in virus–cell interaction, virus morphogenesis, up and downregulation of shrimp immune-related genes, and also for the discovery of novel drugs to combat WSSV in shrimp culture

Concise reviews: in vitro-produced pancreas organogenesis models in three dimensions: self-organization from few stem cells or progenitors.

Three-dimensional models of organ biogenesis have recently flourished. They promote a balance between stem/progenitor cell expansion and differentiation without the constraints of flat tissue culture vessels, allowing for autonomous self-organization of cells. Such models allow the formation of miniature organs in a dish and are emerging for the pancreas, starting from embryonic progenitors and adult cells. This review focuses on the currently available systems and how these allow new types of questions to be addressed. We discuss the expected advancements including their potential to study human pancreas development and function as well as to develop diabetes models and therapeutic cells.

Comparative assessment of intimal hyperplasia development after 14 days in two different experimental settings: tissue culture versus ex vivo continuous perfusion of human saphenous vein.

BACKGROUND: Intimal hyperplasia (IH) is a vascular remodeling process which often leads to failure of arterial bypass or hemodialysis access. Experimental and clinical work have provided insight in IH development; however, further studies under precise controlled conditions are required to improve therapeutic strategies to inhibit IH development. Ex vivo perfusion of human vessel segments under standardized hemodynamic conditions may provide an adequate experimental approach for this purpose. Therefore, chronically perfused venous segments were studied and compared to traditional static culture procedures with regard to functional and histomorphologic characteristics as well as gene expression. MATERIALS AND METHODS: Static vein culture allowing high tissue viability was performed as previously described. Ex vivo vein support system (EVVSS) was performed using a vein support system consisting of an incubator with a perfusion chamber and a pump. EVVSS allows vessel perfusion under continuous flow while maintaining controlled hemodynamic conditions. Each human saphenous vein was divided in two parts, one cultured in a Pyrex dish and the other part perfused in EVVSS for 14days. Testing of vasomotion, histomorphometry, expression of CD 31, Factor VIII, MIB 1, alpha-actin, and PAI-l were determined before and after 14days of either experimental conditions. RESULTS: Human venous segments cultured under traditional or perfused conditions exhibited similar IH after 14 days as shown by histomorphometry. Smooth-muscle cell (SMC) was preserved after chronic perfusion. Although integrity of both endothelial and smooth-muscle cells appears to be maintained in both culture conditions as confirmed by CD31, factor VIII, and alpha-actin expression, a few smooth-muscle cells in the media stained positive for factor VIII. Cell-proliferation marker MIB-1 was also detected in the two settings and PAI-1 mRNA expression and activity increased significantly after 14 days of culture and perfusion. CONCLUSION: This study demonstrates the feasibility to chronically perfuse human vessels under sterile conditions with preservation of cellular integrity and vascular contractility. To gain insights into the mechanisms leading to IH, it will now be possible to study vascular remodeling not only under static conditions but also in hemodynamic environment mimicking as closely as possible the flow conditions encountered in reconstructive vascular surgery.

Comparative assessement of intimal hyperplasia development after 14 days in two different experimental settings : tissue culture versus ex vivo continuous perfusion of human saphenous vein

Résumé de l'article : L'hyperplasie intimale est un processus de remodelage vasculaire ubiquitaire après une lésion, pouvant menacer la perméabilité de tout type de reconstruction vasculaire. Les mécanismes physiopathologiques impliqués dans le développement de l'hyperplasie intimale ne sont que partiellement élucidés. Il est par conséquent nécessaire d'effectuer des recherches complémentaires afin d'en améliorer la compréhension et ainsi permettre l'élaboration de nouvelles stratégies thérapeutiques médicamenteuses. La culture de veines en milieu statique permet le développement de l'hyperplasie intimale. Ce modèle maintient la viabilité tissulaire, comme décrit précédemment dans d'autres études, mais empêche l'analyse des paramètres hémodynamiques. La mise au point d'un modèle de perfusion in vitro permettant la perfusion de segments vasculaires représente une approche expérimentale intégrant les différents facteurs hémodynamiques. Le système de perfusion (Ex Vivo Vein Support System) que nous avons élaboré conserve l'intégrité pariétale ainsi que les propriétés vasomotrices des veines pour une durée de 14 jours. Cette étude démontre que les deux modèles permettent le développement de l'hyperplasie intimale. Toutefois, les propriétés vasomotrices ainsi que l'influence des paramètres hémodynamiques ne peuvent être analysées que par l'utilisation du système de perfusion. Ce dernier a permis de perfuser des vaisseaux humains sans contamination bactérienne tout en maintenant l'intégrité cellulaire. Ce modèle de perfusion se rapproche plus des conditions hémodynamiques rencontrées in vivo que le modèle statique. Abstract : Background. Intimal hyperplasia (IH) is a vascular remodeling process which often leads to failure of arterial bypass or hemodialysis access. Experimental and clinical work have provided insight in IH development; however, further studies under precise con-trolled conditions are required to improve therapeutic strategies to inhibit IH development. Ex vivo perfusion of human vessel segments under standardized hemodynamic conditions may provide an adequate experimental approach for this purpose. Therefore, chronically perfused venous segments were studied and compared to traditional static culture procedures with regard to functional and histomorphologic characteristics as well as gene expression. Materials and methods. Static vein culture allowing high tissue viability was performed as previously described. Ex vivo vein support system (EVVSS) was performed using a vein support system consisting of an incubator with a perfusion chamber and a pump. EVVSS allows vessel perfusion under continuous flow while maintaining controlled hemodynamic conditions. Each human saphenous vein was divided in two parts, one cultured in a Pyrex dish and the other part perfused in EVVSS for 14 days. Testing of vasomotion, histomorphometry, expression of CD 31, Factor VIII, MIB 1, α-actin, and PAI-1 were determined before and after 14 days of either experimental conditions. Results, Human venous segments cultured under traditional or perfused conditions exhibited similar IH after 14 days as shown by histomorphometry. Smooth-muscle cell ( SMC) was preserved after chronic perfusion. Although integrity of both endothelial and smooth-muscle cells appears to be maintained in both culture conditions as confirmed by CD31, factor VIII and α-actin expression, a few smooth-muscle cells in the media stained positive for factor VIII. Cell-proliferation marker MIB-1 was also detected in the two settings and PAI-1 mRNA expression and activity increased significantly after 14 days of culture and perfusion. Conclusion. This study demonstrates the feasibility to chronically perfuse human vessels under sterile conditions with preservation of cellular integrity and vascular contractility. To gain insights into the mechanisms leading to IH, it will now be possible to study vascular remodeling not only under static conditions but also in hemodynamic environment mimicking as closely as possible the flow conditions encountered in reconstructive vascular surgery.

Safety Culture and Maritime Personnel’s Safety Attitudes: Interview Report

”METKU –projektissa” (Merenkulun turvallisuuskulttuurin kehittäminen) tutkitaan kansainvälisen turvallisuusjohtamiskoodin (ISM-koodin) vaikutuksia merenkulun turvallisuuteen ja etsitään kehittämiskohteita merenkulun turvallisuusjohtamisen parantamiseksi. Tämä haastatteluraportti on laadittu METKU –projektin yhteistyössä työpakettien 1 ja 2 kesken. Tähän raporttiin haastateltiin yhteensä 94 merenkulun ammattilaista. Suurimman osan haastateltavista muodostivat aktiiviset merenkulkijat: miehistön jäsenet, päällystö ja alusten päälliköt. Haastattelukohteena oli seitsemän suomalaista varustamoa. Haastatteluissa kerättiin merenkulkijoiden kokemuksia ja mielipiteitä ISM-koodin vaikutuksesta heidän käytännön työhönsä. Suomalaiset merenkulkijat uskovat, että tänä päivänä varustamoiden johtajat ovat hyvin sitoutuneita turvallisuuteen. Myös miehistön asenteet turvallisuuteen ovat ISM-koodin käytön myötä parantuneet. Haasteltavien yhteinen huoli kohdistui jatkuvan parantamisen toimivuuteen. Kaikki haastatellut ryhmät olivat samaa mieltä siitä, että poikkeamien raportointi ei ISMkoodin vaatimuksesta huolimatta toimi kunnolla. ISM-koodin käyttöön otosta on ollut merenkululle selkeää hyötyä. Haastateltavat esittivät hyötyinä parantuneen yhteistyön ja tiedonkulun alusten ja varustamon välillä sekä sen, että merenkulun toiminnan laatu on parantunut. Monet haastateltavat korostivat, että ISM-koodin selkeät turvallisuusvastuut yhtiölle on ollut merkittävä hyöty. Itse ISM-koodiin merenkulkijoilla ei ollut juurikaan huomauttamista. Sen sijaan turvallisuusjohtamisen käytännön toteutuksessa nähtiin parantamisen varaa. ISMkoodin aiheuttamina ongelmina mainittiin mm. lisääntynyt byrokratia ja liian monimutkaiset ja yksityiskohtaiset turvallisuuskäsikirjat. Monet haastateltavat toivovat, että ISM-koodin käytännön soveltamiseen laadittaisiin ohjeita.

Human sepsis-associated Escherichia coli (SEPEC) is able to adhere to and invade kidney epithelial cells in culture

The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.

Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.

A two-stage continuous culture system to study the effect of supplemental alpha-lactalbumin and glycomacropeptide on mixed cultures of human gut bacteria challenged with enteropathogenic Escherichia coli and Salmonella serotype Typhimurium

Aims: Certain milk factors may promote the growth of a gastrointestinal microflora predominated by bifidobacteria and may aid in overcoming enteric infections. This may explain why breast-fed infants experience fewer intestinal infections than their formula-fed counterparts. The effect of formula supplementation with two such factors was investigated in this study. Methods and Results: Infant faecal specimens were used to ferment formulae supplemented with glycomacropeptide (GMP) and alpha-lactalbumin (alpha-la) in a two-stage compound continuous culture model. At steady state, all fermenter vessels were inoculated with 5 ml of 0.1 M phosphate-buffered saline (pH 7.2) containing 10(8) CFU ml(-1) of either enteropathogenic Escherichia coli 2348/69 (O127:H6) or Salmonella serotype Typhimurium (DSMZ 5569). Bacteriology was determined by independent fluorescence in situ hybridization. Vessels that contained breast milk (BM), as well as alpha-la and GMP supplemented formula had stable total counts of bifidobacteria while lactobacilli increased significantly only in vessels with breast milk. Bacteroides, clostridia and E. coli decreased significantly in all three groups prior to pathogen addition. Escherichia coli counts decreased in vessels containing BM and alpha-la while Salmonella decreased significantly in all vessels containing BM, alpha-la and GMP. Acetate was the predominant acid. Significance and Impact of the Study: Supplementation of infant formulae with appropriate milk proteins may be useful in mimicking the beneficial bacteriological effects of breast milk.

Antipathogenic activity of probiotics against Salmonella Typhimurium and Clostridium difficile in anaerobic batch culture systems: is it due to synergies in probiotic mixtures or the specificity of single strains?

Probiotics are currently being investigated for prevention of infections caused by enteric pathogens. The aim of this in vitro study was to evaluate the influence of three single probiotics: Lactobacillus casei NCIMB 30185 (PXN 37), Lactobacillus acidophilus NCIMB 30184 (PXN 35), Bifidobacterium breve NCIMB 30180 (PXN 25) and a probiotic mixture containing the above strains plus twelve other strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera on the survival of Salmonella Typhimurium and Clostridium difficile using pH-controlled anaerobic batch cultures containing mixed fecal bacteria. Changes in relevant bacterial groups and effects of probiotic addition on survival of the two pathogens were assessed over 24 h. Quantitative analysis of bacterial populations revealed that there was a significant increase in lactobacilli and/or bifidobacteria numbers, depending on probiotic addition, compared with the control (no added probiotic). There was also a significant reduction in S. Typhimurium and C. difficile numbers in the presence of certain probiotics compared with controls. Of the probiotic treatments, two single strains namely L. casei NCIMB 30185 (PXN 37), and B. breve NCIMB 30180 (PXN 25) were the most potent in reducing the numbers of S. Typhimurium and C. difficile. In addition, the supplementation with probiotics into the systems influenced some fermentations parameters. Acetate was found in the largest concentrations in all vessels and lactate and formate were generally detected in higher amounts in vessels with probiotic addition compared to controls.

In Vitro Fermentation of NUTRIOSE® FB06, a wheat dextrin soluble fibre, in a continuous culture human colonic model system

Wheat dextrin soluble fibre may have metabolic and health benefits, potentially acting via mechanisms governed by the selective modulation of the human gut microbiota. Our aim was to examine the impact of wheat dextrin on the composition and metabolic activity of the gut microbiota. We used a validated in vitro three-stage continuous culture human colonic model (gut model) system comprised of vessels simulating anatomical regions of the human colon. To mimic human ingestion, 7 g of wheat dextrin (NUTRIOSE® FB06) was administered to three gut models, twice daily at 10.00 and 15.00, for a total of 18 days. Samples were collected and analysed for microbial composition and organic acid concentrations by 16S rRNA-based fluorescence in situ hybridisation and gas chromatography approaches, respectively. Wheat dextrin mediated a significant increase in total bacteria in vessels simulating the transverse and distal colon, and a significant increase in key butyrate-producing bacteria Clostridium cluster XIVa and Roseburia genus in all vessels of the gut model. The production of principal short-chain fatty acids, acetate, propionate and butyrate, which have been purported to have protective, trophic and metabolic host benefits, were increased. Specifically, wheat dextrin fermentation had a significant butyrogenic effect in all vessels of the gut model and significantly increased production of acetate (vessels 2 and 3) and propionate (vessel 3), simulating the transverse and distal regions of the human colon, respectively. In conclusion, wheat dextrin NUTRIOSE® FB06 is selectively fermented in vitro by Clostridium cluster XIVa and Roseburia genus and beneficially alters the metabolic profile of the human gut microbiota.

Use of batch culture and a two-stage continuous culture system to study the effect of supplemental a-lactalbumin and glycomacropeptide on mixed populations of human gut bacteria

Certain milk factors can promote the growth of a host-friendly gastrointestinal microflora. This may explain why breast-fed infants experience fewer intestinal infections than their formula-fed counterparts. The effect of formula supplementation with two such factors was investigated in this study. Infant faecal specimens were used to ferment formulas supplemented with glycomacropeptide and α-lactalbumin in a two-stage compound continuous culture model. Bacteriology was determined by fluorescence in situ hybridisation. Vessels that contained breast milk as well as α-lactalbumin and glycomacropeptide had stable counts of bifidobacteria while lactobacilli increased significantly only in vessels with breast milk. Bacteroides, clostridia and Escherichia coli decreased significantly in all runs. Acetate was the principal acid found along with high amounts of propionate and lactate. Supplementation of infant formulas with appropriate milk proteins may be useful in simulating the beneficial bacteriological effects of breast milk.

In vitro evaluation of the fermentation properties and potential probiotic activity of Lactobacillus plantarum C4 in batch culture systems

Lactobacillus plantarum C4 has been tested in in vitro pH-controlled anaerobic faecal batch cultures as compared to Lactobacillus rhamnosus GG to determine changes caused to the composition of faecal bacteria. Effects upon major groups of the microbiota and levels of short-chain fatty acids (SCFA) were assessed over 24 h. Concomitantly, hydrophobic character and ability of both bacterial cells to adhere in vitro to Caco-2 cells were investigated. Quantitative analysis of bacterial populations revealed that there was a significant increase in Lactobacillus/Enterococcus numbers in vessels with probiotic supplemented with fructooligosaccharides (FOS), compared to the negative control. L. plantarum C4 showed to have more hydrophilic behaviour and fulfilled better adhesive properties, compared to L. rhamnosus GG. Thus, L. plantarum C4 can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of SCFA.

Xylella fastidiosa: An in vivo system to study possible survival strategies within citrus xylem vessels based on global gene expression analysis

Xylella fastidiosa inhabits the plant xylem, a nutrient-poor environment, so that mechanisms to sense and respond to adverse environmental conditions are extremely important for bacterial survival in the plant host. Although the complete genome sequences of different Xylella strains have been determined, little is known about stress responses and gene regulation in these organisms. In this work, a DNA microarray was constructed containing 2,600 ORFs identified in the genome sequencing project of Xylella fastidiosa 9a5c strain, and used to check global gene expression differences in the bacteria when it is infecting a symptomatic and a tolerant citrus tree. Different patterns of expression were found in each variety, suggesting that bacteria are responding differentially according to each plant xylem environment. The global gene expression profile was determined and several genes related to bacterial survival in stressed conditions were found to be differentially expressed between varieties, suggesting the involvement of different strategies for adaptation to the environment. The expression pattern of some genes related to the heat shock response, toxin and detoxification processes, adaptation to atypical conditions, repair systems as well as some regulatory genes are discussed in this paper. DNA microarray proved to be a powerful technique for global transcriptome analyses. This is one of the first studies of Xylella fastidiosa gene expression in vivo which helped to increase insight into stress responses and possible bacterial survival mechanisms in the nutrient-poor environment of xylem vessels.

Cell-free cryopreserved arterial allografts from multiorgan donors: a new strategy to fabricate artificial blood vessels suited for peripheral vascular surgery

Critical lower limb ischemia is a severe disease. A common approach is infrainguinal bypass. Synthetic vascular prosthesis, are good conduits in high-flow low-resistance conditions but have difficulty in their performance as small diameter vessel grafts. A new approach is the use of native decellularized vascular tissues. Cell-free vessels are expected to have improved biocompatibility when compared to synthetic and are optimal natural 3D matrix templates for driving stem cell growth and tissue assembly in vivo. Decellularization of tissues represent a promising field for regenerative medicine, with the aim to develop a methodology to obtain small-diameter allografts to be used as a natural scaffold suited for in vivo cell growth and pseudo-tissue assembly, eliminating failure caused from immune response activation. Material and methods. Umbilical cord-derived mesenchymal cells isolated from human umbilical cord tissue were expanded in advanced DMEM. Immunofluorescence and molecular characterization revealed a stem cell profile. A non-enzymatic protocol, that associate hypotonic shock and low-concentration ionic detergent, was used to decellularize vessel segments. Cells were seeded cell-free scaffolds using a compound of fibrin and thrombin and incubated in DMEM, after 4 days of static culture they were placed for 2 weeks in a flow-bioreactor, mimicking the cardiovascular pulsatile flow. After dynamic culture, samples were processed for histological, biochemical and ultrastructural analysis. Discussion. Histology showed that the dynamic culture cells initiate to penetrate the extracellular matrix scaffold and to produce components of the ECM, as collagen fibres. Sirius Red staining showed layers of immature collagen type III and ultrastructural analysis revealed 30 nm thick collagen fibres, presumably corresponding to the immature collagen. These data confirm the ability of cord-derived cells to adhere and penetrate a natural decellularized tissue and to start to assembly into new tissue. This achievement makes natural 3D matrix templates prospectively valuable candidates for clinical bypass procedures