Comparison of trout hepatocyte culture on different substrates

Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%). This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency. Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David W. Barnes     

Macrophage inhibitory cytokine‐1 is associated with cognitive impairment and predicts cognitive decline – the Sydney Memory and Aging Study

Higher levels of macrophage inhibitory cytokine‐1, also known as growth differentiation factor 15 (MIC‐1/GDF15), are associated with adverse health outcomes and all‐cause mortality. The aim of this study was to examine the relationships between MIC‐1/GDF15 serum levels and global cognition, five cognitive domains, and mild cognitive impairment (MCI), at baseline (Wave 1) and prospectively at 2 years (Wave 2), in nondemented participants aged 70–90 years. Analyses were controlled for age, sex, education, Framingham risk score, history of cerebrovascular accident, acute myocardial infarction, angina, cancer, depression, C‐reactive protein, tumor necrosis factor‐α, interleukins 6 and 12, and apolipoprotein ε4 genotype. Higher MIC‐1/GDF15 levels were significantly associated with lower global cognition at both waves. Cross‐sectional associations were found between MIC‐1/GDF15 and all cognitive domains in Wave 1 (all P < 0.001) and between processing speed, memory, and executive function in Wave 2 (all P < 0.001). Only a trend was found for the prospective analyses, individuals with high MIC‐1/GDF15 at baseline declined in global cognition, executive function, memory, and processing speed. However, when categorizing MIC‐1/GDF15 by tertiles, prospective analyses revealed statistically significant lower memory and executive function in Wave 2 in those in the upper tertile compared with the lower tertile. Receiver operating characteristics (ROC) analysis was used to determine MIC‐1/GDF15 cutoff values associated with cognitive decline and showed that a MIC‐1/GDF15 level exceeding 2764 pg/ml was associated with a 20% chance of decline from normal to MCI or dementia. In summary, MIC‐1/GDF15 levels are associated with cognitive performance and cognitive decline. Further research is required to determine the pathophysiology of this relationship.     

Caffeine-containing energy drink improves sprint performance during an international rugby sevens competition

The aim of this study was to determine the effects of a caffeine-containing energy drink on physical performance during a rugby sevens competition. A second purpose was to investigate the post-competition urinary caffeine concentration derived from the energy drink intake. On two non-consecutive days of a friendly tournament, 16 women from the Spanish National rugby sevens Team (mean age and body mass = 23 ± 2 years and 66 ± 7 kg) ingested 3 mg of caffeine per kg of body mass in the form of an energy drink (Fure^®, ProEnergetics) or the same drink without caffeine (placebo). After 60 min for caffeine absorption, participants performed a 15-s maximal jump test, a 6 × 30 m sprint test, and then played three rugby sevens games against another national team. Individual running pace and instantaneous speed during the games were assessed using global positioning satellite (GPS) devices. Urine samples were obtained pre and post-competition. In comparison to the placebo, the ingestion of the energy drink increased muscle power output during the jump series (23.5 ± 10.1 vs. 25.6 ± 11.8 kW, P = 0.05), running pace during the games (87.5 ± 8.3 vs. 95.4 ± 12.7 m/min, P < 0.05), and pace at sprint velocity (4.6 ± 3.3 vs. 6.1 ± 3.4 m/min, P < 0.05). However, the energy drink did not affect maximal running speed during the repeated sprint test (25.0 ± 1.5 vs. 25.0 ± 1.7 km/h). The ingestion of the energy drink resulted in a higher post-competition urine caffeine concentration than the placebo (3.3 ± 0.7 vs. 0.2 ± 0.1 μg/mL; P < 0.05). In summary, 3 mg/kg of caffeine in the form of a commercially available energy drink considerably enhanced physical performance during a women’s rugby sevens competition.     

White Matter Hyperintensities in Parkinson’s Disease: Do They Explain the Disparity between the Postural Instability Gait Difficulty and Tremor Dominant Subtypes?

Background

Brain white matter hyperintensities (WMHs) commonly observed on brain imaging of older adults are associated with balance and gait impairment and have also been linked to cognitive deficits. Parkinson’s disease (PD) is traditionally sub-classified into the postural instability gait difficulty (PIGD) sub-type, and the tremor dominant (TD) sub-type. Considering the known association between WMHs and axial symptoms like gait disturbances and postural instability, one can hypothesize that WMHs might contribute to the disparate clinical sub-types of patients with PD.

Methods

110 patients with PD underwent a clinical evaluation and a 3T MRI exam. Based on the Unified Parkinson Disease Rating Scale, the patients were classified into motor sub-types, i.e., TD or PIGD, and scores reflecting PIGD and TD symptoms were computed. We compared white matter burden using three previously validated methods: one using a semi-quantitative visual rating scale in specific brain regions and two automated methods.

Results

Overall, MRI data were obtained in 104 patients. The mean WMHs scores and the percent of subjects with lesions in specific brain regions were similar in the two subtypes, p = 0.678. The PIGD and the TD scores did not differ even when comparing patients with a relatively high burden of WMHs to patients with a relatively low burden. Across most of the brain regions, mild to moderate correlations between WMHs and age were found (r = 0.23 to 0.41; p<0.021). Conversely, no significant correlations were found between WMHs and the PIGD score or disease duration. In addition, depressive symptoms and cerebro-vascular risk factors were similar among the two subtypes.

Conclusions

In contrast to what has been reported previously among older adults, the present study could not demonstrate any association between WMHs and the PIGD or TD motor sub-types in patients with PD.     

Seasonality of Antarctic minke whale (Balaenoptera bonaerensis) calls off the western Antarctic Peninsula

The Antarctic minke whale (Balaenoptera bonaerensis) is a difficult species to study because of its low visual detectability and preference for living within the sea ice habitat, accessible only by ice‐strengthened vessels. Recent identification of the Antarctic minke whale as the source of the seasonally ubiquitous bio‐duck call has allowed the use of this sound, as well as downsweeps, to investigate seasonality trends and diel patterns in Antarctic minke whale call production, and their relationship to sea ice cover. Passive acoustic data were collected using an autonomous Acoustic Recording Package (ARP) off the western Antarctic Peninsula. Bio‐duck calls were classified into four distinct call variants, with one variant having two subtypes. Bio‐duck calls were detected between April and November, with increasing call duration during the austral winter, indicating a strong seasonality in call production. Downsweeps, which were also attributed to Antarctic minke whales, were present throughout most months during the recording period, with a peak in July, and an absence in March and April. Both bio‐duck and downsweeps were significantly correlated with sea ice cover. No diel patterns were observed in bio‐duck calls or in downsweep call production at this site.     

Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures

Mycoplasma contamination events in biomanufacturing facilities can result in loss of production and costly cleanups. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and may penetrate the 0.2 µm filters often used in the primary clarification of harvested cell culture fluid. Culture cell-based and indicator cell-based assays that are used to detect mycoplasma are highly sensitive but can take up to 28 days to complete and cannot be used for real-time decision making during the biomanufacturing process. To support real-time measurements of mycoplasma contamination, there is a push to explore nucleic acid testing. However, cell-based methods measure growth or colony forming units and nucleic acid testing measures genome copy number; this has led to ambiguity regarding how to compare the sensitivity of the methods. In addition, the high risk of conducting experiments wherein one deliberately spikes mycoplasma into bioreactors has dissuaded commercial groups from performing studies to explore the multiple variables associated with the upstream effects of a mycoplasma contamination in a manufacturing setting. Here we studied the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G1 (IgG1) antibody. We examined M. arginini growth and detection by culture methods, as well as the effects of M. arginini on mammalian cell health, metabolism, and productivity. We compared process parameters and controls normally measured in bioreactors including dissolved oxygen, gas mix, and base addition to maintain pH, to examine parameter changes as potential indicators of contamination. Our work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Importantly, how the M. arginini contamination impacts the CHO cells is influenced by the concentration of CHO cells and rate of perfusion at the time of M. arginini spike. Careful evaluation of dissolved oxygen, pH control parameters, ammonia, and arginine over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before a read-out from a traditional method.     

Tracing the trilobite tree from the root to the tips: A model marriage of fossils and phylogeny

Trilobites are a highly diverse group of extinct arthropods that persisted for nearly 300 million years. During that time, there was a profusion of morphological form, and they occupied a plethora of marine habitats. Their diversity, relative abundance, and complex morphology make them excellent candidates for phylogenetic analysis, and partly as a consequence they have been the subject of many cladistic studies. Although phylogenetic knowledge is certainly incomplete, our understanding of evolutionary patterns within the group has dramatically increased over the last 30 years. Moreover, trilobites have formed an important component of various studies of macroevolutionary processes. Here, we summarize the phylogenetic breadth of knowledge on the Trilobita, and present various hypotheses about phylogenetic patterns within the group, from the highest to the lowest taxonomic levels. Key topics we consider include the question of trilobite monophyly, the phylogenetic position of trilobites vis à vis extant arthropod groups, and inter- and intra-ordinal relationships.     

DCX, a new mediator of the JNK pathway

Mutations in the X-linked gene DCX result in lissencephaly in males, and abnormal neuronal positioning in females, suggesting a role for this gene product during neuronal migration. In spite of several known protein interactions, the involvement of DCX in a signaling pathway is still elusive. Here we demonstrate that DCX is a substrate of JNK and interacts with both c-Jun N-terminal kinase (JNK) and JNK interacting protein (JIP). The localization of this signaling module in the developing brain suggests its functionality in migrating neurons. The localization of DCX at neurite tips is determined by its interaction with JIP and by the interaction of the latter with kinesin. DCX is phosphorylated by JNK in growth cones. DCX mutated in sites phosphorylated by JNK affected neurite outgrowth, and the velocity and relative pause time of migrating neurons. We hypothesize that during neuronal migration, there is a need to regulate molecular motors that are working in the cell in opposite directions: kinesin (a plus-end directed molecular motor) versus dynein (a minus-end directed molecular motor).     

Co-aggregation of penicillin g acylase and polyionic polymers: an easy methodology to prepare enzyme biocatalysts stable in organic media

A novel type of biocatalyst that combines the good properties of cross-linked enzyme aggregates (CLEAs) and hydrophilic microenvironments has been developed. Dextran sulfate- and polyethyleneimine-coated CLEAs of penicillin acylase (CLEA-GDP) were prepared by adding the polymers of different sizes before the precipitation stage of the enzyme. This study presents the development and optimization of a protocol to produce such a biocatalyst using penicillin acylase as a model. Experiments show that CLEA-GDPs have a highly increased stability in organic media. The average half-life of the preparations was much higher than standard CLEA without a microenvironment (CLEA-G), (e.g., more than 25-fold) in the presence of dioxane. However, their thermal stability was not increased, which leads to the conclusion that the stability of CLEA-GDPs in organic media is due to the hydrophilic microenvironment that surrounds the protein enzyme more than to a conformational stiffening effect. This is further supported by solvation experiments that show a preferential hydration of CLEA when polymers are used to coat the enzyme. CLEA-GDPs are clearly better than other biocatalysts in terms of solvent stability.