The isolation of primary hepatocytes from human tissue: optimising the use of small non-encapsulated liver resection surplus

Two-step perfusion is considered the gold standard method for isolating hepatocytes from human liver tissue. As perfusion may require a large tissue specimen, which is encapsulated and has accessible vessels for cannulation, only a limited number of tissue samples may be suitable. Therefore, the aim of this work was to develop an alternative method to isolate hepatocytes from non-encapsulated and small samples of human liver tissue. Healthy tissue from 44 human liver resections were graded for steatosis and tissue weights between 7.8 and 600 g were used for hepatocyte isolations. Tissue was diced and underwent a two-step digestion (EDTA and collagenase). Red cell lysis buffer was used to prevent red blood cell contamination and toxicity. Isolated hepatocyte viability was determined by trypan blue exclusion. Western blot and biochemical analyses were undertaken to ascertain cellular phenotype and function. Liver tissue that weighed ≥50 g yielded significantly higher (P < 0.01) cell viability than tissue <50 g. Viable cells secreted urea and displayed the phenotypic hepatocyte markers albumin and cytochrome P450. Presence of steatosis in liver tissue or intra-hepatocellular triglyceride content had no effect on cell viability. This methodology allows for the isolation of viable primary human hepatocytes from small amounts of “healthy” resected liver tissue which are not suitable for perfusion. This work provides the opportunity to increase the utilisation of resection surplus tissue, and may ultimately lead to an increased number of in vitro cellular studies being undertaken using the gold-standard model of human primary hepatocytes.     

Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.     

Novel whole cell adhesion assays of three isolates of the fouling diatom Amphora coffeaeformis reveal diverse responses to surfaces of different wettability

Whole cell, strength of adhesion assays of three different isolates of the fouling diatom Amphora coffeaeformis were compared using a hydrophilic surface viz. acid washed glass (AWG), and a hydrophobic surface viz. a self assembled monolayer (SAM) of undecanethiol (UDT). Assays were performed using a newly designed turbulent flow channel that permits direct observation and recording of cell populations on a test surface. Exposure to continuous shear stress over 3 h revealed that the more motile isolate, WIL2, adhered much more strongly to both test surfaces compared to the other two strains. When the response of the isolates to shear stress after 3 h was compared, there was no significant difference in the percentage of cells removed, irrespective of surface wettability. Cells of the three isolates of A. coffeaeformis varied significantly in their response to different surfaces during initial adhesion, indicating the presence of a wide range of ‘physiological races’ within this species.     

The effect of time from hatch on the yolk conversion efficiency of rainbow trout, Salmo gairdneri

The growth and development of rainbow trout, Salmo gairdneri , from hatch to first feeding depends upon the nutrients stored in the yolk sac. The efficiency ( E ) with which yolk can be converted to larval biomass is a useful measurement in chronic toxicity tests, and it can be simply estimated by comparing the gain in larval weight ( L ) to the loss in yolk weight ( Y ) at hatch (time 0) and at any time t after hatch: E =( L ₁− L ₀)/( Y ₀− Y ₁); E may be calculated on a wet or dry weight basis. Although the rates of weight change were constant for the first 15 days after hatch at 10.5° C, E was not. E increased from near 0 and approached a value of 2.1 on a wet weight basis or 0.70 on a dry weight basis after 5–8 days. The variability of E was also very high shortly after hatch, but declined and stabilized within 5–9 days. For toxicity testing, we recommend that E be measured 10–15 days post-hatch to ensure precision with a minimum bias due to dissection error.     

Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience

Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.     

Exercise therapy for prevention of falls in people with Parkinson's disease: A protocol for a randomised controlled trial and economic evaluation

Background

People with Parkinson's disease are twice as likely to be recurrent fallers compared to other older people. As these falls have devastating consequences, there is an urgent need to identify and test innovative interventions with the potential to reduce falls in people with Parkinson's disease. The main objective of this randomised controlled trial is to determine whether fall rates can be reduced in people with Parkinson's disease using exercise targeting three potentially remediable risk factors for falls (reduced balance, reduced leg muscle strength and freezing of gait). In addition we will establish the cost effectiveness of the exercise program from the health provider's perspective.

Methods/Design

230 community-dwelling participants with idiopathic Parkinson's disease will be recruited. Eligible participants will also have a history of falls or be identified as being at risk of falls on assessment. Participants will be randomly allocated to a usual-care control group or an intervention group which will undertake weight-bearing balance and strengthening exercises and use cueing strategies to address freezing of gait. The intervention group will choose between the home-based or support group-based mode of the program. Participants in both groups will receive standardized falls prevention advice. The primary outcome measure will be fall rates. Participants will record falls and medical interventions in a diary for the duration of the 6-month intervention period. Secondary measures include the Parkinson's Disease Falls Risk Score, maximal leg muscle strength, standing balance, the Short Physical Performance Battery, freezing of gait, health and well being, habitual physical activity and positive and negative affect schedule.

Discussion

No adequately powered studies have investigated exercise interventions aimed at reducing falls in people with Parkinson's disease. This trial will determine the effectiveness of the exercise intervention in reducing falls and its cost effectiveness. This pragmatic program, if found to be effective, has the potential to be implemented within existing community services.

Trial registration

The protocol for this study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12608000303347).     

Glacier ecosystem response to episodic nitrogen enrichment in Svalbard, European High Arctic

We describe the climatology, hydrology and biogeochemistry of an extreme nitrogen deposition event that occurred in the highly glacierised environment of the European High Arctic during June 1999. Meteorological analysis, three-dimensional air mass trajectories and a 3D transport model show that blocking high pressures over Scandinavia and the rapid advection of western European pollution toward Svalbard were sufficient to cause the most concentrated (1.15 ppm NO₃–N and 1.20 ppm NH₄–N), high magnitude (total 26 mm and up to 2.4 mm h^?1 at 30 m above sea level) nitrogen deposition event on record in this sensitive, high Arctic environment (78.91° N, 11.93° E). Since the event occurred when much of the catchment remained frozen or under snow cover, microbial utilisation of nitrogen within snowpacks and perennially unfrozen subglacial sediments, rather than soils, were mostly responsible for reducing N export. The rainfall event occurred long before the annual subglacial outburst flood and so prolonged (ca. 10 day) water storage at the glacier bed further enhanced the microbial assimilation. When the subglacial outburst eventually occurred, high runoff and concentrations of NO₃ ^? (but not NH₄ ⁺) returned in the downstream rivers. Assimilation accounted for between 53 and 72% of the total inorganic nitrogen deposited during the event, but the annual NO₃ ^? and NH₄ ⁺ runoff yields were still enhanced by up to 5 and 40 times respectively. Episodic atmospheric inputs of reactive nitrogen can therefore directly influence the biogeochemical functioning of High Arctic catchments, even when microbial activity takes place beneath a glacier at a time when terrestrial soil ecosystems remain frozen and unresponsive.     

Chemical weathering and solute export by meltwater in a maritime Antarctic glacier basin

Solute yields, laboratory dissolution data and both chemical and isotopic markers of rock weathering reactions are used to characterise the biogeochemistry of glacial meltwaters draining a maritime Antarctic glacier. We find that delayed flowpaths through ice-marginal talus and moraine sediments are critical for the acquisition of solute from rock minerals because delayed flowpaths through subglacial sediments are absent beneath this small, cold-based glacier. Here the mechanisms of weathering are similar to those reported in subglacial environments, and include sub-oxic conditions in the early summer and increasingly oxic conditions thereafter. Up to 85% of the NO₃ ^? and 65% of the SO₄ ^2? are most likely produced by bacterially mediated reactions in these ice marginal sediments. However, reactive pyrite phases are sparse in the host rocks, limiting the export of Fe, SO₄ ^2? and cations that may be removed by weathering once pyrite oxidation has taken place. This means that dissolution of Ca²⁺ and Na⁺ from carbonate and silicate minerals dominate, producing moderate cationic denudation yields from Tuva Glacier (163 Σ*meq⁺ m^?2 a^?1) compared to a global range of values (94–4,200 Σ*meq⁺ km^?2 a^?1). Overall, crustally derived cations represent 42% of the total cationic flux, the rest being accounted for by snowpack sources.     

Phylogenetic Diversity of Marine Cyanophage Isolates and Natural Virus Communities as Revealed by Sequences of Viral Capsid Assembly Protein Gene g20

In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different Synechococcus groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity.