All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

Background & Aims

Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen.

Methods

Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity.

Results

Cell preparation yielded the following cell counts per gram of liver tissue: 2.0±0.4×10⁷ hepatocytes, 1.8±0.5×10⁶ Kupffer cells, 4.3±1.9×10⁵ liver sinusoidal endothelial cells, and 3.2±0.5×10⁵ stellate cells. Hepatocytes were identified by albumin (95.5±1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5±1.2%) and exhibited phagocytic activity, as determined with 1μm latex beads. Endothelial cells were CD146⁺ (97.8±1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1±1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence.

Conclusions

Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.     

Impact of Ramadan intermittent fasting on cognitive function in trained cyclists: a pilot study

This study assessed selected measures of cognitive function in trained cyclists who observed daylight fasting during Ramadan. Eleven cyclists volunteered to participate (age: 21.6±4.8 years, VO₂max: 57.7±5.6 ml kg⁻¹·min⁻¹) and were followed for 2 months. Cognitive function (Cambridge Neuropsychological Test Automated Battery (CANTAB), Reaction Time index (RTI) and Rapid Visual Information Processing (RVP) tests) and sleep architecture (ambulatory EEG) were assessed: before Ramadan (BR), in the 1st week (RA1) and 4th week of Ramadan (RA4), and 2 weeks post-Ramadan (PR). Both cognitive tests were performed twice per day: before and after Ramadan at 8-10 a.m. and 4-6 p.m., and during Ramadan at 4-6 p.m. and 0-2 a.m., respectively. Training load (TL) by the rating of perceived exertion (RPE) method and wellness (Hooper index) were measured daily. If the TL increased over the study period, this variable was stable during Ramadan. The perceived fatigue and delayed onset muscle soreness (DOMS) increased at RA4. Sleep patterns and architecture showed clear disturbances, with significant increases in the number of awakenings and light sleep durations during Ramadan (RA1 and RA4), together with decreased durations of deep and REM sleep stages at PR. RTI (simple and multiple reaction index) reaction and movement times did not vary over the study period. The RVP test showed reduced false alarms during Ramadan, suggesting reduced impulsivity. Overall accuracy significantly increased at RA1, RA4 and PR compared to baseline. At RA4, the accuracy was higher at 0-2 a.m. compared to 4-6 p.m. Despite the observed disturbances in sleep architecture, Ramadan fasting did not negatively impact the cognitive performance of trained cyclists from the Middle East.     

Discovery and biological evaluation of benzo[a]carbazole-based small molecule agonists of the thrombopoietin (Tpo) receptor

A novel series of benzo[a]carbazole-based small molecule agonists of the thrombopoietin (Tpo) receptor is reported. Starting from a 3.4 microM high throughput screen hit, members of this series have been identified which are full agonists with functional potency <50 nM and oral bioavailability in mice.     

Early-Life Socioeconomic Status and Adult Physiological Functioning: A Life Course Examination of Biosocial Mechanisms

A growing literature has demonstrated a link between early-life socioeconomic conditions and adult health at a singular point in life. No research exists, however, that specifies the life course patterns of socioeconomic status (SES) in relation to the underlying biological processes that determine health. Using an innovative life course research design consisting of four nationally representative longitudinal datasets that collectively cover the human life span from early adolescence to old age (Add Health, MIDUS, NSHAP, and HRS), we address this scientific gap and assess how SES pathways from childhood into adulthood are associated with biophysiological outcomes in different adult life stages. For each dataset, we constructed standardized composite measures of early-life SES and adult SES and harmonized biophysiological measurements of immune and metabolic functioning. We found that the relative importance of early-life SES and adult SES varied across young, mid, and late adulthood, such that early-life SES sets a life course trajectory of socioeconomic well-being and operates through adult SES to influence health as adults age. We also documented evidence of the detrimental health effects of downward mobility and persistent socioeconomic disadvantage. These findings are the first to specify the life course patterns of SES that matter for underlying biophysiological functioning in different stages of adulthood. The study thus contributes new knowledge critical for improving population health by identifying the particular points in the life course at which interventions might be most effective in preventing disease and premature mortality.     

Localization of an Ataxia-Telangiectasia Gene to an −500-kb Interval on Chromosome 11q23.1: Linkage Analysis of 176 Families by an International Consortium

We describe a 20-point linkage analysis map of chromosome 11q22-23 that is based on genotyping 249 families (59 CEPH and 190 A-T). Monte Carlo linkage analyses of 176 ataxia-telangiectasia (A-T) families localizes the major A-T locus to the region between S1819(A4) and S1818(A2). When seven nonlinking families were excluded from subsequent analyses, a 2-lod support interval of ~500 kb was identified between S1819(A4) and S1294. No recombinants were observed between A-T and markers S384, B7, S535, or S1294. Only 17 of the international consortium families have been assigned to complementation groups. The available evidence favors either a cluster of A-T genes on chromosome 11 or intragenic defects in a single gene.     

Single molecule spectroscopy on the light-harvesting complex II of higher plants.

Spectroscopic and polarization properties of single light-harvesting complexes of higher plants (LHC-II) were studied at both room temperature and T < 5 K. Monomeric complexes emit roughly linearly polarized fluorescence light thus indicating the existence of only one emitting state. Most probably this observation is explained by efficient triplet quenching restricted to one chlorophyll a (Chl a) molecule or by rather irreversible energy transfer within the pool of Chl a molecules. LHC-II complexes in the trimeric (native) arrangement bleach in a number of steps, suggesting localization of excitations within the monomeric subunits. Interpretation of the fluorescence polarization properties of trimers requires the assumption of transition dipole moments tilted out of the symmetry plane of the complex. Low-temperature fluorescence emission of trimers is characterized by several narrow spectral lines. Even at lowest excitation intensities, we observed considerable spectral diffusion most probably due to low temperature protein dynamics. These results also indicate weak interaction between Chls belonging to different monomeric subunits within the trimer thus leading to a localization of excitations within the monomer. The experimental results demonstrate the feasibility of polarization sensitive studies on single LHC-II complexes and suggest an application for determination of the Chl transition-dipole moment orientations, a key issue in understanding the structure-function relationships.     

Biologically meaningful expression profiling across species using heterologous hybridization to a cDNA microarray

Background  

Unravelling the path from genotype to phenotype, as it is influenced by an organism's environment, is one of the central goals in biology. Gene expression profiling by means of microarrays has become very prominent in this endeavour, although resources exist only for relatively few model systems. As genomics has matured into a comparative research program, expression profiling now also provides a powerful tool for non-traditional model systems to elucidate the molecular basis of complex traits.