Variation in composition of media and reagents used in the preparation of cell cultures from human and other animal tissues: Dulbecco’s,Earle’s,and Hanks’ balanced salt solutions

Summary When comparison was made of directions given for three salt solutions commonly used in cell culture preparations and identified as Dulbecco’s, Earle’s, and Hanks’, variations in composition were found. Some significantly alter the suitability of the materials for the intended use. Other differences may have less effect. This brief review reveals a tendency among researchers to follow procedures obtained from colleagues for the preparation of laboratory reagents and media, to cite the original publication rather than their colleagues’ work as the source of the information, and to fail to compare the two for differences. Some failures in cell culture propagation may be attributed to similar instances with other published but incorrectly cited work. Tables are provided that facilitate comparison of the correct original formulations with variants from selected published sources. This research was supported by Storrs Agricultural Experiment Station, University of Connecticut, Scientific Contribution Number 1032.     

Study of the persistence and viability of Metarhizium acridum in Mexico’s agricultural area

Locust is one of the most predominant agricultural pests in Mexico, followed by non-migratory grasshoppers. Their devastating effects cover numerous Mexican states, generating important economic losses. Metarhizium acridum (Hypocreales: Clavicipitaceae) has been employed in Mexico as a microbial agent for biological control for some time. However, questions related to the long-term persistence and viability of the fungus after it has been sprayed over crop fields remain to be elucidated. To resolve these questions, we performed a study in a crop field where an abundant population of the grasshopper Sphenarium purpurascens (Orthoptera: Pyrgomorphidae) is present. The fungus M. acridum was sprayed during August and October 2007. Its presence in the air, soil and vegetative cover was subsequently monitored using molecular methods and culture techniques. The results showed that the fungus was viable on the ground during the follow-up period (66 weeks). Its population fluctuated markedly, however, reaching four times its initial concentration and later decreasing to nearly half its initial concentration by the end of the study. The initial concentrations of the fungus in the air and in the vegetative cover decayed steadily (with some small, isolated recoveries) until its complete disappearance after 8 months.     

Genetic diversity and assessment of 23 microsatellite markers for parentage testing of Santa Inês hair sheep in Brazil

Santa Inês is the most common hair sheep breed in Brazil and probably has the highest genetic diversity among sheep breeds in this country. Successful breeding programs for Brazilian sheep breeds are not common for various reasons, including a lack of control of parentage in the flocks. We developed an allele frequency database for 23 STR loci for the Santa Inês breed based on 285 animals sampled from five populations distributed across the central-western and north-eastern regions of Brazil. The marker set included seven microsatellites used in the 2011 International Society for Animal Genetics sheep genotyping comparison tests and all eight microsatellites currently approved by the Brazilian Agricultural Ministry laboratory accreditation guidelines for sheep identification. The microsatellites had an average of 10 alleles and a mean expected heterozygosity of 0.745. Combined paternity exclusion probabilities when no parent or one parent was known were >99.99%. A small proportion (5.8%) of the existing genetic variation was found to be among the Santa Inês populations, possibly derived from genetic drift and selection. We found that the marker panel proposed by the Agricultural Ministry, although generally useful, should be enhanced by including more markers for improved exclusionary power in parentage testing. This database provides a useful tool for parentage testing of this major Brazilian breed, contributing to improved management and breeding of existing herds.     

Should human chondrocytes fly? The impact of electromagnetic irradiation on chondrocyte viability and implications for their use in tissue engineering

A significant logistic factor as to the successful clinical application of the autologous tissue engineering concept is efficient transportation: the donor cells need to be delivered to tissue processing facilities which in most cases requires air transportation. This study was designed to evaluate how human chondrocytes react to X-ray exposure. Primary cell cultures were established, cultured, incubated and exposed to different doses and time periods of radiation. Subsequently, quantitative cell proliferation assays were done and qualitative evaluation of cellular protein production were performed. Our results show that after irradiation of chondrocytes with different doses, no significant differences in terms of cellular viability occurred compared with the control group. These results were obtained when chondrocytes were exposed to luggage transillumination doses as well as exposure to clinically used radiation doses. Any damage affecting cell growth or quality was not observed in our study. However, information about damage of cellular DNA remains incomplete.     

Association of Shh and Ptc with keratin localization in the initiation of the formation of circumvallate papilla and von Ebner’s gland

The development of gustatory papillae in mammalian embryos requires the coordination of a series of morphological events, such as proliferation, differentiation and innervation. In mice, the circumvallate papilla (CVP) is a specialized structure that develops in a characteristic spatial and temporal pattern in the posterior region of the tongue dorsal surface. The distinct expression patterns of Shh and Ptc, which play important roles in the development of other epithelial appendages, have been localized in the trench wall that gives rise to von Ebner’s gland (VEG). To define the cellular mechanisms responsible for morphogenesis and differentiation during early development of CVP and VEG, the localization patterns of keratins (cytokeratins) K7, K8, K18, K19, K14 and connexin-43, which are dependent on Shh expression in other developmental systems, have been examined in detail. The distinct localization of keratins K7, K8, K18, K19, K14 and connexin-43 in the epithelium giving rise to the CVP and VEG suggests that cytodifferentiation is established prior to morphological changes. Interestingly, the localization of proliferating cell nuclear antigen, a marker for cell proliferation, is similar to that of Shh. An understanding of the regulatory roles of cell-cell interactions and signalling molecules in orchestrating a mutual network will bring us nearer to defining the molecular and cellular mechanisms underlying morphogenesis in mammalian taste bud development.M.-J. Lee and J.-Y. Kim contributed equally to this work.This work was supported by grant no. R01-2003-000-11649 from the Korea Science and Engineering Foundation.     

Vitamin D and melatonin protect the cell’s viability and ameliorate the CCl4 induced cytotoxicity in HepG2 and Hep3B hepatoma cell lines

Carbon tetrachloride (CCl₄) is widely used to induce liver toxicity in in vitro/in vivo models. Lipid peroxidation (LPO) begins with toxicity and affects cell viability. Recently, the beneficial effects of melatonin and Vitamin D on cell proliferation in human normal and cancer cells were found. This study was planned to evaluate antioxidant and cytoprotective activity of melatonin and Vitamin D in CCl₄ induced cytotoxicity in HepG2 and Hep3B hepatoma cell lines. Based on the cytotoxicity assay, melatonin and Vitamin D were evaluated for cytotoprotective potential against CCl₄ induced toxicity in HepG2 and Hep3B liver cell lines by monitoring cell viability, LPO and glutathione (GSH) level. Different dosages of CCl₄ (0.1, 0.2, 0.3 and 0.4 % v/v) were applied to HepG2 and Hep3B cells in order to determine the most toxic dosage of it in a time dependent manner. The same experiments were repeated with exogenously applied melatonin (MEL) and Vitamin D to groups treated with/without CCL₄. Cell viability was determined with MTT measurements at the 2nd, 24th and 48th h. GSH content and Malondialdehyde levels were measured from the cell lysates. As a result, both melatonin and Vitamin D administration during CCl₄ exposure protected liver cells from CCl₄ induced cell damage. Increase in LPO and decrease in GSH were found in the CCl₄ groups of both cells. Contrary to these results administration of MEL and Vitamin D on cells exhibited results similar to the control groups. Therefore, melatonin and Vitamin D might be a promising therapeutic agent in several toxic hepatic diseases.     

Microbial analysis of raw cows’ milk used for cheese-making: influence of storage treatments on microbial composition and other technological traits

Raw milk used to produce Grana cheese was subjected to several treatment regimes, including varying temperatures and storage times. Milk from morning and evening milking were transferred to a dairy factory separately (double delivery) or together (single delivery), after storage at the farm for 12 h; in the former case, milk was stored at 12 or 8°C, whereas, in the latter, it was kept at ambient temperature or 18°C. Values of pH of the vat milk were lower for milk samples kept at room temperature, while other physico-chemical parameters and rheological characteristics tested did not show significant differences linked to the different storage temperatures of milk used for “Grana Trentino” cheese production. Total microorganisms and several microbial groups (psychrotrophic bacteria, coliforms, mesophilic and termophilic lactic acid bacteria, including enterococci, pseudomonads and clostridia) were detected and quantified by classical (plate count and most probable number) techniques, after each technological treatment for a total of 212 milk and cream samples. The application of a culture-independent microbiological strategy, consisting of denaturing gradient gel electrophoresis, allowed the recognition of several bacterial genera and species.     

Comparison of ‘ignition’ and ‘extraction’ methods for the determination of organic phosphate in rocks and soils

Summary Ignition markedly increased the solubility in dilute acid of iron- and aluminium-bound inorganic phosphate in samples of weathered New Zealand greywacke rock. This observation supports the view that ignition methods may sometimes overestimate the total organic phosphate of soils.     

Dependence of red blood cell dynamics in microvessel bifurcations on the endothelial surface layer’s resistance to flow and compression

Red blood cells (RBCs) make up 40–45% of blood and play an important role in oxygen transport. That transport depends on the RBC distribution throughout the body, which is highly heterogeneous. That distribution, in turn, depends on how RBCs are distributed or partitioned at diverging vessel bifurcations where blood flows from one vessel into two. Several studies have used mathematical modeling to consider RBC partitioning at such bifurcations in order to produce useful insights. These studies, however, assume that the vessel wall is a flat impenetrable homogeneous surface. While this is a good first approximation, especially for larger vessels, the vessel wall is typically coated by a flexible, porous endothelial glycocalyx or endothelial surface layer (ESL) that is on the order of 0.5–1 µm thick. To better understand the possible effects of this layer on RBC partitioning, a diverging capillary bifurcation is analyzed using a flexible, two-dimensional model. In addition, the model is also used to investigate RBC deformation and RBC penetration of the ESL region when ESL properties are varied. The RBC is represented using interconnected viscoelastic elements. Stokes flow equations (viscous flow) model the surrounding fluid. The flow in the ESL is modeled using the Brinkman approximation for porous media with a corresponding hydraulic resistivity. The ESL’s resistance to compression is modeled using an osmotic pressure difference. One cell passes through the bifurcation at a time, so there are no cell–cell interactions. A range of physiologically relevant hydraulic resistivities and osmotic pressure differences are explored. Decreasing hydraulic resistivity and/or decreasing osmotic pressure differences (ESL resistance to compression) produced four behaviors: (1) RBC partitioning nonuniformity increased slightly; (2) RBC deformation decreased; (3) RBC velocity decreased relative to blood flow velocity; and (4) RBCs penetrated more deeply into the ESL. Decreasing the ESL’s resistance to flow and/or compression to pathological levels could lead to more frequent cell adhesion and clotting as well as impaired vascular regulation due to weaker ATP and nitric oxide release. Potential mechanisms that can contribute to these behaviors are also discussed.

     

Comparative Evaluation of Borelli’s Lactritmel Agar and Lowenstein-Jensen Agar for Conidiation in the Trichophyton mentagrophytes and Trichophyton rubrum Complexes

The characteristics of macroconidia are the key to the identification of dermatophytic fungi. In this study, we compared Lowenstein-Jensen agar (LJA) with Borelli’s lactritmel agar (BLA) for the ability to induce macroconidia production in a collection of Trichophyton mentagrophytes (12) and Trichophyton rubrum (12) complexes and in Arthroderma isolates (4). We evaluated 28 strains from across the world, which were mainly obtained from the culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, The Netherlands. All cultures were incubated at 26 °C on the bench, and conidia formation was investigated every 5 days, over a period of 30 days after inoculation. At 15 days, strains grown on BLA demonstrated better macroconidia production than those grown on LJA, 23 (82.1 %) versus 16 (57.1 %) isolates, respectively. Thus, the use of BLA, an inexpensive medium that can be prepared in the laboratory, should facilitate conidiation and the presumptive identification of dermatophytic fungi in mycology and clinical microbiology laboratories.     

A comparative analysis of the molecular genetic processes in the pathogenesis of psoriasis and Crohn’s disease

Molecular Biology - A comparative bioinformatics analysis of the molecular genetic processes in the pathogenesis of multifactorial diseases—psoriasis and Crohn’s disease—was...     

The use of cluster and discriminant analysis in the investigations of the role of trace metals in the pathogenesis of Parkinson’s disease

X-ray fluorescence microscopy was applied for two-dimensional elemental analysis of substantia nigra (SN) tissue. The samples representing Parkinson’s disease (PD) and control cases were examined at HASYLAB beamline L and at ESRF beamline ID22. Two-dimensional mapping of P, S, Cl, K, Ca, Fe, Cu, Zn, Se and Br was done with the spatial resolution of 15 and 5 μm. The masses per unit area of elements in neuromelanin reach nerve cells of SN were determined.The elemental data were processed using two multivariate techniques, namely cluster and discriminant analysis. The statistical methods were used for data reduction, both unsupervised and supervised classification as well as for the creation of a model that would simplify case identification based on the elemental analysis of SN tissue. The results of cluster analysis confirmed the statistical significance of the differences in elemental composition of PD and control SN nerve cells. Based on the results of discriminant analysis, the elements (P, Cl, Fe, Cu and Zn) that played the greatest role in the process of differentiation between neurons from examined groups were determined.     

Dynamic changes and molecular analysis of cell death in the spinal cord of SJL mice infected with the BeAn strain of Theiler’s murine encephalomyelitis virus

Theiler’s murine encephalomyelitis (TME) is caused by the TME virus (TMEV) and represents an important animal model for multiple sclerosis (MS). Oligodendroglial apoptosis and reduced apoptotic elimination of encephalitogenic leukocytes seem to participate in autoimmune demyelination in MS. The present study quantified apoptotic cells in BeAn–TMEV-induced spinal cord white matter lesions at 14, 42, 98, and 196 days post infection (dpi) using immunostaining. Apoptotic cells were identified by transmission electron microscopy and double-immunofluorescence. The mRNA expression of apoptosis-related genes was investigated using microarray analysis. Oligodendroglial apoptosis was already detected in the predemyelinating phase at 14 dpi. Apoptotic cell numbers peaked at 42 dpi and decreased until 196 dpi partly due to reduced T cell apoptosis. In addition to genes involved in the classical pathways of apoptosis induction, microarray analysis detected the expression of genes related to alternative mechanisms of cell death such as pyroptosis, necroptosis, and endoplasmic reticulum stress. Consequently, oligodendroglial apoptosis is involved in the initiation of the TME demyelination process, whereas the development of apoptosis resistance of T cells potentially favors the maintenance of inflammation and myelin loss.     

Assessment of the Efficacy of Solutes from Extremophiles on Protein Aggregation in Cell Models of Huntington’s and Parkinson’s Diseases

Protein misfolding and deposition in the brain are implicated in the etiology of numerous neurodegenerative disorders. Here, organic solutes characteristic of microorganisms adapted to hot environments, were tested on experimental cell models of Huntington’s and Parkinson’s diseases. Diglycerol phosphate, di-myo-inositol phosphate, mannosylglycerate, and mannosylglyceramide were not toxic to the cells, at 10 mM concentration, but caused a decrease in cell density, which suggested an effect on proliferation. In contrast, mannosyl-lactate, an artificial analogue of mannosylglycerate, had a negative impact on cell viability. Concerning protein aggregation, inclusions of mutant huntingtin were reduced in the presence of diglycerol phosphate and di-myo-inositol phosphate, increased with mannosylglycerate, while mannosyl-lactate and mannosylglyceramide had no significant effect. α-Synuclein aggregation was not affected by the solutes tested, except for di-myo-inositol phosphate that led to a slight increased percentage of cells displaying visible aggregates. These solutes might be useful in the development of therapies for protein misfolding diseases.     

Comparative analysis of the structure,suberin and wax composition and key gene expression in the epidermis of ‘Dangshansuli’ pear and its russet mutant

Mature fruit of ‘Dangshansuli’ pear has yellow-green skin, while its mutant ‘Xiusu’ has russet fruit skin, which is a genetic variation. To explore the mechanism underlying the russet formation, the fruit spot and epidermal structure were observed, the color, texture, and wax and suberin components were evaluated, and the gene expression levels were confirmed. In the present study, the color, texture and fruit spot of the epidermis differed significantly between ‘Dangshansuli’ and ‘Xiusu’ at 25 days after full bloom (DAFB). The cuticular wax components were alkanes, olefins, alkanoic acids, alcohols and terpenes, and the suberin was composed of fatty acid, α,ω-diacids, ω-hydroxy fatty acids, mainly ferulic acid and primary alcohols in the epidermis of ‘Dangshansuli’ and ‘Xiusu’, which exhibited significant differences at most stages of the development of pear fruits. Moreover, the expression levels of genes involved in wax and suberin were consistent with morphological and biochemical analyses. The results indicated that the suberization of epidermal cells occurred when pear fruit was young and that wax and suberin might contribute to the russet formation on the epidermis of ‘Xiusu’, leading to the significant differences in color, texture, fruit spot, and exocarp structure between ‘Dangshansuli’ and ‘Xiusu’ pears.     

Substitution of dietary oleic acid for myristic acid increases the tissue storage of α-linolenic acid and the concentration of docosahexaenoic acid in the brain, red blood cells and plasma in the rat

Various strategies have been developed to increase the cellular level of (n-3) polyunsaturated fatty acids in animals and humans. In the present study, we investigated the effect of dietary myristic acid, which represents 9% to 12% of fatty acids in milk fat, on the storage of α-linolenic acid and its conversion to highly unsaturated (n-3) fatty acid derivatives. Five isocaloric diets were designed, containing equal amounts of α-linolenic acid (1.3% of dietary fatty acids, i.e. 0.3% of dietary energy) and linoleic acid (7.0% of fatty acids, i.e. 1.5% of energy). Myristic acid was supplied from traces to high levels (0%, 5%, 10%, 20% and 30% of fatty acids, i.e. 0% to 6.6% of energy). To keep the intake of total fat and other saturated fatty acids constant, substitution was made with decreasing levels of oleic acid (76.1% to 35.5% of fatty acids, i.e. 16.7% to 7.8% of energy) that is considered to be neutral in lipid metabolism. After 8 weeks, results on physiological parameters showed that total cholesterol and low-density lipoprotein-cholesterol did not differ in the diets containing 0%, 5% and 10% myristic acid, but were significantly higher in the diet containing 30% myristic acid. In all the tissues, a significant increasing effect of the substitution of oleic acid for myristic acid was shown on the level of both α-linolenic and linoleic acids. Compared with the rats fed the diet containing no myristic acid, docosahexaenoic acid significantly increased in the brain and red blood cells of the rats fed the diet with 30% myristic acid and in the plasma of the rats fed the diet with 20% myristic acid. Arachidonic acid also increased in the brain of the rats fed the diet with 30% myristic acid. By measuring Δ6-desaturase activity, we found a significant increase in the liver of the rats fed the diet containing 10% of myristic acid but no effect at higher levels of myristic acid. These results suggest that an increase in dietary myristic acid may contribute in increasing significantly the tissue storage of α-linolenic acid and the overall bioavailability of (n-3) polyunsaturated fatty acids in the brain, red blood cells and plasma, and that mechanisms other than the single Δ6-desaturase activity are involved in this effect.