Combined Effect of Cryogel Matrix and Temperature-Reversible Soluble-Insoluble Polymer for the Development of in Vitro Human Liver Tissue

Hepatic cell culture on a three-dimensional (3D) matrix or as a hepatosphere appears to be a promising in vitro biomimetic system for liver tissue engineering applications. In this study, we have combined the concept of a 3D scaffold and a spheroid culture to develop an in vitro model to engineer liver tissue for drug screening. We have evaluated the potential of poly(ethylene glycol)-alginate-gelatin (PAG) cryogel matrix for in vitro culture of human liver cell lines. The synthesized cryogel matrix has a flow rate of 7 mL/min and water uptake capacity of 94% that enables easy nutrient transportation in the in vitro cell culture. Youngs modulus of 2.4 kPa and viscoelastic property determine the soft and elastic nature of synthesized cryogel. Biocompatibility of PAG cryogel was evaluated through MTT assay of HepG2 and Huh-7 cells on matrices. The proliferation and functionality of the liver cells were enhanced by culturing hepatic cells as spheroids (hepatospheres) on the PAG cryogel using temperature-reversible soluble-insoluble polymer, poly(N-isopropylacrylamide) (PNIPAAm). Pore size of the cryogel above 100 mu m modulated spheroid size that can prevent hypoxia condition within the spheroid culture. Both the hepatic cells have shown a significant difference (P < 0.05) in terms of cell number and functionality when cultured with PNIPAAm. After 10 days of culture using 0.05% PNIPAAm, the cell number increased by 11- and 7-fold in case of HepG2 and Huh-7 cells, respectively. Similarly, after 10 days of hepatic spheroids culture on PAG cryogel, the albumin production, urea secretion, and CYP450 activity were significantly higher in case of culture with PNIPAAm. The developed tissue mass on the PAG cryogel in the presence of PNIPAAm possess polarity, which was confirmed using F-actin staining and by presence of intercellular bile canalicular lumen. The developed cryogel matrix supports liver cells proliferation and functionality and therefore can be used for in vitro and in vivo drug testing.

Metabolite and transcriptome analysis of Campylobacter jejuni in vitro growth reveals a stationary-phase physiological switch.

Campylobacter jejuni is a prevalent cause of food-borne diarrhoeal illness in humans. Understanding of the physiological and metabolic capabilities of the organism is limited. We report a detailed analysis of the C. jejuni growth cycle in batch culture. Combined transcriptomic, phenotypic and metabolic analysis demonstrates a highly dynamic 'stationary phase', characterized by a peak in motility, numerous gene expression changes and substrate switching, despite transcript changes that indicate a metabolic downshift upon the onset of stationary phase. Video tracking of bacterial motility identifies peak activity during stationary phase. Amino acid analysis of culture supernatants shows a preferential order of amino acid utilization. Proton NMR (1H-NMR) highlights an acetate switch mechanism whereby bacteria change from acetate excretion to acetate uptake, most probably in response to depletion of other substrates. Acetate production requires pta (Cj0688) and ackA (Cj0689), although the acs homologue (Cj1537c) is not required. Insertion mutants in Cj0688 and Cj0689 maintain viability less well during the stationary and decline phases of the growth cycle than wild-type C. jejuni, suggesting that these genes, and the acetate pathway, are important for survival.

A snapshot of digitisation and OCR Developments

One of the goals of Knowledge Exchange (www.knowledge-exchange.info) is to help partners to share knowledge and expertise and facilitate the build of expert networks. In the area of digitisation, including non-textual and 3-D-digitisation, a first step is to provide a snapshot of current activities and challenges in the KE partner countries. This paper is a synthesis of the information gathered in a questionnaire that was sent to 15 infrastructure institutions, e.g. libraries and also funders, within the five partner countries of Knowledge Exchange: Denmark (DK), Finland (FIN), Germany (GER), the Netherlands (NL) and the United Kingdom (UK). The paper is based on the answers provided by 6 respondents from four countries:  DK o Danish Agency of Culture (Henrik Jarl Hansen) o State and University Library (Tonny Skovgård Jensen)  GER o German research foundation, DFG (Franziska Regner)  NL o Royal Library (Hildelies Balk) o Leiden University Library (Saskia van Bergen)  UK o Jisc (Paola Marchionni, Peter Findlay) The absence of Finnish responses may be due to Finland participating in the recent Enumerate Core Survey II that also addressed digitisation. We have included some of the outcomes of this survey to present a richer picture.

Control of bacterial disease in small scale fresh-water aquaculture: Project R0754. Project completion report December 1998 - May 1999

The work presented here represents an 18-month study to examine the relationship between environmental conditions, bacterial load in the water and bacteria levels in tissue macrophages of a range of clinically healthy freshwater fish species, farmed in a range of culture systems in Thailand and Vietnam. Preliminary assessment was made of the clinical significance of the macrophage bacterial load. The aim of this work was to improve production in fresh-water aquaculture through the control of clinical bacterial disease and subclinical infection, and to identify management practices most effective in promoting fish health. [PDF contains 37 pages]

Physical, Metabolic, and Energetic Investigations of Methane-Metabolizing Microbial Communities

Understanding the roles of microorganisms in environmental settings by linking phylogenetic identity to metabolic function is a key challenge in delineating their broad-scale impact and functional diversity throughout the biosphere. This work addresses and extends such questions in the context of marine methane seeps, which represent globally relevant conduits for an important greenhouse gas. Through the application and development of a range of culture-independent tools, novel habitats for methanotrophic microbial communities were identified, established settings were characterized in new ways, and potential past conditions amenable to methane-based metabolism were proposed. Biomass abundance and metabolic activity measures – both catabolic and anabolic – demonstrated that authigenic carbonates associated with seep environments retain methanotrophic activity, not only within high-flow seep settings but also in adjacent locations exhibiting no visual evidence of chemosynthetic communities. Across this newly extended habitat, microbial diversity surveys revealed archaeal assemblages that were shaped primarily by seepage activity level and bacterial assemblages influenced more substantially by physical substrate type. In order to reliably measure methane consumption rates in these and other methanotrophic settings, a novel method was developed that traces deuterium atoms from the methane substrate into aqueous medium and uses empirically established scaling factors linked to radiotracer rate techniques to arrive at absolute methane consumption values. Stable isotope probing metaproteomic investigations exposed an array of functional diversity both within and beyond methane oxidation- and sulfate reduction-linked metabolisms, identifying components of each proposed enzyme in both pathways. A core set of commonly occurring unannotated protein products was identified as promising targets for future biochemical investigation. Physicochemical and energetic principles governing anaerobic methane oxidation were incorporated into a reaction transport model that was applied to putative settings on ancient Mars. Many conditions enabled exergonic model reactions, marking the metabolism and its attendant biomarkers as potentially promising targets for future astrobiological investigations. This set of inter-related investigations targeting methane metabolism extends the known and potential habitat of methanotrophic microbial communities and provides a more detailed understanding of their activity and functional diversity.

The impact of molecular biology on assessment of water quality: advantages and limitations of current techniques

The advent of molecular biology has had a dramatic impact on all aspects of biology, not least applied microbial ecology. Microbiological testing of water has traditionally depended largely on culture techniques. Growing understanding that only a small proportion of microbial species are culturable, and that many microorganisms may attain a viable but non-culturable state, has promoted the development of novel approaches to monitoring pathogens in the environment. This has been paralleled by an increased awareness of the surprising genetic diversity of natural microbial populations. By targeting gene sequences that are specific for particular microorganisms, for example genes that encode diagnostic enzymes, or species-specific domains of conserved genes such as 16S ribosomal RNA coding sequences (rrn genes), the problems of culture can be avoided. Technical developments, notably in the area of in vitro amplification of DNA using the polymerase chain reaction (PCR), now permit routine detection and identification of specific microorganisms, even when present in very low numbers. Although the techniques of molecular biology have provided some very powerful tools for environmental microbiology, it should not be forgotten that these have their own drawbacks and biases in sampling. For example, molecular techniques are dependent on efficient lysis and recovery of nucleic acids from both vegetative forms and spores of microbial species that may differ radically when growing in the laboratory compared with the natural environment. Furthermore, PCR amplification can introduce its own bias depending on the nature of the oligonucleotide primers utilised. However, despite these potential caveats, it seems likely that a molecular biological approach, particularly with its potential for automation, will provide the mainstay of diagnostic technology for the foreseeable future.

Stock evaluation and development of a breeding program for common carp (Cyprinus carpio) in Karnataka, India: progress of a research project

Common carp (Cyprinus carpio) is the single most important species for aquaculture in the state of Karnataka, India, where it is generally grown in polyculture with Indian major carps. Precocious maturation and unwanted reproduction in the species have been identified as constraints to increase production in aquaculture and culture-based fisheries in Karnataka state. Stocks of C. carpio obtained from Hungary (Amur and P3), Indonesia (Rajdanu) and Vietnam (SV) are being assessed alongside two local stocks (L-BRP and L-FRS) in a series of culture performance trials with the objective of setting up a base population for selective breeding. The paper presents progress of research being undertaken at the Fisheries Research Station, University of Agricultural Sciences, Bangalore, India.

Genetic diversity in wild stocks of the giant freshwater prawn (Macrobrachium rosenbergii): implications for aquaculture and conservation

The giant freshwater prawn (Macrobrachium rosenbergii) is cultured widely around the world but little is known about the levels and patterns of genetic diversity in either wild or cultured stocks. Studies have suggested that genetic diversity may be relatively low in some cultured stocks due to the history of how they were founded and subsequent exposure to repeated population bottlenecks in hatcheries. In contrast, wild stocks have an extensive distribution that extends from Southern Asia across Southeast (SE) Asia to the Pacific region. Therefore, wild stocks could be an important resource for genetic improvement of culture stocks in the future. Understanding the extent and patterns of genetic diversity in wild giant freshwater prawn stocks will assist decisions about the direction future breeding programs may take. Wild stock genetic diversity was examined using a 472 base-pair segment of the 16S rRNA gene in 18 wild populations collected from across the natural range of the species. Two major clades ("eastern" and "western") were identifi ed either side of Huxley’s line, with a minimum divergence of 6.2 per cent, which implies separation since the Miocene period (5-10 MYA). While divergence estimates within major clades was small (maximum 0.9 per cent), evidence was also found for population structuring at a lower spatial scale. This will be examined more intensively with a faster evolving mtDNA gene in the future.

The History and Literature of America's Oysters

Historically, America's use and enjoyment of the oyster extend far back into prehistoric times. The Native Americans often utilized oysters, more intensively in some areas than in others, and, at least in some areas of the Caribbean and Pacific coast, the invading Spanish sought oysters as eagerly as they did gold-but for the pearls. That was the pearl oyster, Pinctada sp., and signs of its local overexploitation were recorded early in the 16th century. During the 1800's, use of the eastern oyster grew phenomenally and, for a time, it outranked beef as a source of protein in some parts of the nation. Social events grew up around it, as it became an important aspect of culture and myth. Eventually, research on the oyster began to blossom, and scientific literature on the various species likewise bloomed-to the extent that when the late Paul Galtsoff wrote his classic treatise "The American oyster Crassostrea virginica Gmelin" in 1954, he reported compiling an extensive bibliography of over 6,000 subject and author cards on oysters and related subjects which he deposited in the library of the Woods Hole Laboratory of the Bureau of Commercial Fisheries (now NMFS). That large report, volume 64 (480 pages) of the agency's Fishery Bulletin, was a bargain at $2.75, and it has been a standard reference ever since. But the research and the attendant literature have grown greatly since Galtsoff's work was published, and now that has been thoroughly updated.

Water quality management on the enhancement of shrimp (Penaeus monodon Fab.) production in the traditional and improved-traditional ghers of Bangladesh

On-farm research on enhancement of P. monodon production through water quality management was carried out in five ghers of Paikgacha, Khulna. Based on the prevailing condition of the ghers, lime in the form of CaCO(sub 3), urea and TSP were used as the major inputs to minimize the soil-water acidity and to ensure the availability of natural food particles in the water bodies. Exchange of water at required level also practiced for the qualitative improvement of culture water. Ghers of varying sizes showed that water quality management and fertilization have a positive impact on production performance of P. monodon (61.59% increment) that yielded an average production of 385.43 kg/ha/crop against the present traditional rate of 238.50 kg/ha/year.

Socio-economic conditions of the pond owners of Demra, Dhaka

On the basis of fish culture status 96 ponds of the study area were classified into 4 categories like wild stock (27%), extensive culture (24%), improved extensive culture (33%) and semi-intensive culture (16%). Percentage of small, medium and large ponds were 38, 44, and 18 respectively whereas education levels below SSC, below Bachelor and above were 43, 38 and 19 respectively and single owners belonged to 54% of the ponds. Per hectare yields of extensive, improved extensive and semi-intensive categories of culture were 1.3, 2.12 and 4.0 metric tons respectively and their net return were 46, 63 and 92 thousand taka respectively. Considering the problems of fish culture, multiple ownership was found to be the most important one.

Macrobenthos off Mahim (Bombay), west coast of India in relation to coastal pollution and aquaculture

Deterioration of water quality clearly indicated a moderate to severe coastal pollution around Mahim whereas a relatively healthy marine environment towards offshore was noticed. Foraminifera, polychaetes, crustaceans and pelecypods were the dominant macro faunal groups encountered in the area. The occasional inhabitants like pennatularians, nemertines, sipunculids, ophiuroids and fish larvae were mostly restricted to offshore regions. Biomass and population density of macro fauna were moderate and showed fluctuating trend. Similarity coefficients of foraminifera (0.89) and polychaetes (0.81) were high for offshore unpolluted stations (3 and 4) as compared to near shore coastal stations (1 and 2). Faunal diversity was relatively more in unpolluted zone. The diversity index (H) was more at station 3 for polychaetes (1.39) and at station 4 for foraminifers (0.54). In general, biomass was high during post monsoon excepting station 1 which was invariably under severe pollution stress throughout the study period. Foraminifera were abundant at sandy bottom while polychaetes preferred muddy (clayey silt) bottom. A noticeable ecological modification associated with faunal abundance and diversity were related to deteriorating marine water quality due to anthropogenic waste disposals. The prevailing water quality around Mahim is unsuitable for any kind of culture practices and also for harvesting economically important marine species.

Use of crude salt in rearing of freshwater giant prawn, Macrobrachium rosenbergii (de Man) larvae

Three different types of culture media: (i) 100% brine (B 100 ), (ii) 75% brine and 25% crude salt (B 75 CS 25 ), and 50% brine and 50% crude salt (B 50 CS 50) were tested to evaluate the possible use of brackish water reconstituted from the crude salt for the production of M. rosenbergii post-larvae. The production rate of 25.26±0.20 PI/l with a corresponding survival rate of 84.20±0.66% was significantly higher (P<0.05) for the larvae reared on B100 than that of 22.10±0.57 Pl/l with a corresponding survival rate of 73.68±1.89% on B50CS50. Larvae cultured on B75CS25 did not show any significant difference (P<0.05) in production as well as in survival of post-larvae than that on B100. The result shows that, for rearing of prawn larvae, use of brine can be replaced up to 25% without any undue reduction in production of post-larvae. However, the production as well as survival rate of post-larvae with 50% replacement (B50CS50) is also appreciable. It is assumed that the mineral constituents of natural seawater might have some triggering effects on prawn larvae in closing their larval cycle.

Effect of pond aeration on growth and survival of Penaeus monodon Fab.

The effect of paddle wheel aeration on shrimp growth and survival were studied at a commercial farm at Chandipur coast of Orissa, India, at different stocking densities of Penaeus monodon. Four different aeration patterns were adopted and evaluated. Influence of individual aeration pattern on average survival rate was not highly significant (p<0.05) at different stocking densities, while different aeration patterns had significant influence (pof P. monodon. It was also estimated that 1.77 hp (aerator) is needed for every 1000 kg shrimp biomass which corresponds to 1 hp/565 kg biomass of shrimp. Higher growth rate was mostly observed during 63-98 days of culture, when six 2 hp aerators were in use. Size variation in growth was higher during initial stage of rearing, while it was reduced to significant level towards the last phase of rearing as number of aerator and hour of operation increased.

Growth performances of three microalgal species in filtered brackishwater with different inorganic media

The growth of three microalgae species, viz., Nannochloropsis oculata, Tetraselmis chui and Chaetoceros muelleri which are commonly used in aquaculture, was investigated using three different inorganic nutrient media: (i) Modified Guillard's f/2 medium (ii) Rix Mix medium and (iii) BFRI medium. Each microalgae species was cultured for 24 days in small- scale with initial inoculation density of 17xl04 cell /ml in the three media with triplicates. N. oculata cultured in modified Guillard's f/2 medium showed superior growth with a mean peak density of 221 ±4.24 x 104 cell/ ml, to Rix Mix medium (141 ± 10.54xl04 cell/ml) and BFRI medium (47±4.94 x 104 cell/ml) on the 16th day of culture at stationary phase. Considering the increase in cell density for 20 days of culture in Rix Mix medium, C. muelleriwas significantly (P<0.05) highest than in other two media. N. oculata cultured in BFRI medium resulted in the poorest growth with a mean peak increase in density of 84±9.19 x 104 cell/ml in 12 days of culture. However, with an increase in cell density, growth of T. chui (182 ± 6.26 x 104 cell/ml) was significantly (P<0.05) higher in BFRI medium than in modified Guillard's f/2 medium. The results of the present study suggest that N. oculata and C. muelleri can be grown very well in both the modified Guillard's f/2 medium and Rix Mix medium. Better growth of T. chui can be obtained while culturing either in BFRI and Rix Mix medium. These three nutrient media used in the present study may be useful for microalgae species culture for establishing green-water culture for suitable target zooplankton, and fish and crustacean larvae in marine and brackishwater hatcheries.