The phylum Platyhelminthes shares a unique population of undifferentiated cells responsible for the proliferation capacity needed for cell renewal, growth, tissue repair and regeneration. These cells have been extensively studied in free-living flatworms, whereas in cestodes the presence of a set of undifferentiated cells, known as germinative cells, has been demonstrated in classical morphology studies, but poorly characterized with molecular biology approaches. Furthermore, several genes have been identified as neoblast markers in free-living flatworms that deserve study in cestode models. Here, different cell types of the model cestode Mesocestoides corti were characterized, identifying differentiated and germinative cells. Muscle cells, tegumental cells, calcareous corpuscle precursor cells and excretory system cells were identified, all of which are non-proliferative, differentiated cell types. Besides those, germinative cells were identified as a population of small cells with proliferative capacity in vivo. Primary cell culture experiments in Dulbecco's Modified Eagle Medium (DMEM), Echinococcus hydatid fluid and hepatocyte conditioned media in non-reductive or reductive conditions confirmed that the germinative cells were the only ones with proliferative capacity. Since several genes have been identified as markers of undifferentiated neoblast cells in free-living flatworms, the expression of pumilio and pL10 genes was analysed by qPCR and in situ hybridization, showing that the expression of these genes was stronger in germinative cells but not restricted to this cell type. This study provides the first tools to analyse and further characterise undifferentiated cells in a model cestode.

A Nutrition Society member-led meeting was held on 9 January 2020 at The University of Surrey, UK. Sixty people registered for the event, and all were invited to participate, either through chairing a session, presenting a ‘3 min lightning talk’ or by presenting a poster. The meeting consisted of an introduction to the topic by Dr Barbara Fielding, with presentations from eight invited speakers. There were also eight lightning talks and a poster session. The meeting aimed to highlight recent research that has used stable isotope tracer techniques to understand human metabolism. Such studies have irrefutably shaped our current understanding of metabolism and yet remain a mystery to many. The meeting aimed to de-mystify their use in nutrition research.

In response to gonadotropins and androgens, testicular cells produce various molecules that control proper proliferation and differentiation of spermatogenic cells through their paracrine and autocrine actions. However, molecules functioning downstream of the hormonal stimulation are poorly understood. Leukaemia inhibitory factor (Lif) is known to maintain the pluripotency of stem cells including embryonic stem cells and primordial germ cells at least in vitro, but its actual roles in vivo remain to be elucidated. To clarify the function of Lif in teleost (medaka) testes, we examined the effects of Lif on spermatogenesis in a newly established cell culture system using a cell line (named Mtp1) derived from medaka testicular somatic cells as feeder cells. We found that addition of baculovirus-produced recombinant medaka Lif to the culture medium or co-culture with Lif-overexpressing Mtp1 cells increased the number of spermatogonia. In situ hybridization and immunohistochemical analyses of the medaka testes showed that mRNAs and proteins of Lif are expressed in spermatogonia and the surrounding Sertoli cells, with higher expression levels in type A (undifferentiated) spermatogonia than in type B (differentiated) spermatogonia. Our findings suggest that Lif regulates spermatogonial cell proliferation in the medaka.

The present study aimed to determine if the long-chain MUFA cetoleic acid (22 : 1n-11) can improve the capacity to synthesise the health-promoting n-3 fatty acids EPA and DHA in human and fish models. Human hepatocytes (HepG2) and salmon primary hepatocytes were first enriched with cetoleic acid, and thereafter their capacities to convert radio-labelled 18 : 3n-3 (α-linolenic acid, ALA) to EPA and DHA were measured. Increased endogenous levels of cetoleic acid led to increased production of radio-labelled EPA + DHA in HepG2 by 40 % and EPA in salmon hepatocytes by 12 %. In order to verify if dietary intake of a fish oil rich in cetoleic acid would have the same beneficial effects on the n-3 fatty acid metabolic pathway in vivo as found in vitro, Atlantic salmon were fed four diets supplemented with either sardine oil low in cetoleic acid or herring oil high in cetoleic acid at two inclusion levels (Low or High). The diets were balanced for EPA + DHA content within the Low and within the High groups. The salmon were fed these diets from 110 to 242 g. The level of EPA + DHA in liver and whole-body retention of docosapentaenoic acid and EPA + DHA relative to what was eaten, increased with increased dietary cetoleic acid levels. Thus, it is concluded that cetoleic acid stimulated the synthesis of EPA and DHA from ALA in human HepG2 and of EPA in salmon hepatocytes in vitro and increased whole-body retention of EPA + DHA in salmon by 15 % points after dietary intake of cetoleic acid.

Increased plasma α-1 acid glycoprotein (AGP) is correlated with reduced growth rates in neonatal swine. The specific physiological mechanisms contributing to this relationship are unknown. This study was performed to determine if AGP can modify muscle metabolism by examining glucose oxidation and protein synthesis in the C2C12 muscle cell line. Cells were used for experiments 4 days post-fusion as myotubes. Myotubes were exposed to AGP for 24 h, with the last 4 h used to monitor 14C-glucose oxidation or to measure protein synthesis by incorporation of 3H-tyrosine. Treatment of C2C12 myotubes with mouse AGP (100 µg/ml) reduced glucose oxidation (P<0.01, n=3 trials), whereas bovine insulin (1 µM) stimulated glucose oxidation (P<0.05, n=3 trials). Treatment with AGP in combination with insulin reduced 14C-glucose oxidation (P<0.05, n=3 trials), similar to the effect of AGP alone. Glucose transport, as measured by 3H-deoxyglucose uptake, was increased by 38% with 1 µM insulin (P<0.05, n=3 trials), whereas AGP alone increased glucose uptake by 36% (P<0.05, n=3 trials). The combination of insulin and AGP in the medium resulted in an 88% increase in glucose uptake (P<0.01, n=3 trials). Protein synthesis was measured by 3H-tyrosine incorporation into C2C12 myotubes. Insulin stimulated a 18% increase in 3H-tyrosine incorporation (P<0.05, n=6 trials). The incorporation of 3H-tyrosine into myotubes was reduced by 20% with AGP incubation (P<0.01, n=6 trials), like the 20% decrease in 3H-tyrosine incorporation in response to the combination of AGP and insulin (P<0.01, n=6 trials). Protein breakdown, as measured by the release of 3H-tyrosine from C2C12 myotubes, was reduced 27% by insulin (P<0.01, n=6 trials). Treatment with AGP had no effect on protein breakdown (P>0.05, n=6 trials), whereas incubation with both AGP and insulin reduced 3H-tyrosine release by 15% (P<0.01, n=6 trials). First, these data indicate that the acute phase protein AGP can interact with the skeletal muscle to reduce glucose oxidation, but this is not the result of an effect on glucose transport. Second, AGP can specifically reduce protein synthesis. Lastly, AGP can inhibit insulin-stimulated glucose oxidation, protein synthesis and breakdown.

We previously reported that napropamide [2-(α-naphthoxy)-N,N-diethylpropionamide] was highly toxic to achlorophyllous callus of tomato (Lycopersicon esculentum Mill. ‘San Marzano’), whereas foliar application to seedlings was almost without effect. Experiments were performed to ascertain the effects of napropamide on green and achlorophyllous calli of tomato and broadleaf dock (Rumex obtusifolius L.). The growth of achlorophyllous tissues was more severely inhibited. Foliar applications were not as inhibitory in both species. Napropamide was applied to tomato seedlings via roots and cut shoots and root growth was more inhibited than shoot growth. Thus napropamide is toxic to achlorophyllous tissue and is either less toxic to, or detoxified by green tissue.

Effects of the imidazolinone herbicide AC243 997 [2-(5-isopropyl)-5-methyl-4-oxo-2-imidazolin-2-yl) nicotinic acid] on the growth of corn (Zea mays L. ‘Black Mexican Sweet’) cell suspension cultures were characterized. The herbicide caused half maximal growth inhibition at concentrations in the range of 10 nM to 30 nM. Significant reductions in protein synthesis were observed following treatment of cells with 10 μM herbicide. Analysis of free amino acid pool sizes indicated that levels of the biosyn-thetically related amino acids leucine and valine were substantially decreased in cells exposed to 10 μM herbicide. Supplementation of suspension culture growth media with leucine and valine plus isoleucine (1 mM each) reversed growth inhibitory effects of AC243 997 at levels ranging from 10 nM to 1 mM. The results suggest a specific interaction of this imidazolinone herbicide with leucine, valine, and isoleucine metabolism.

Although in vivo and in vitro approaches appear to be very different, they are related and complementary techniques and both are essential for the investigation of diverse biological topics. The employment of both techniques was considered particularly appropriate to investigate the role of 17β-oestradiol and testosterone in echinoid reproductive biology. The relationship between sex-steroids and echinoid reproduction has not been clearly determined yet, due to the highly variable and sometimes contrasting results obtained from steroid administration experiments. These might be due to the activation of protective metabolic mechanisms that can prevent the exogenous molecules from exerting their biological functions, as observed in our previous research. To clarify these aspects, in the present study we explored sex-steroid involvement in the reproduction of the sea urchin Paracentrotus lividus, employing both in vivo and in vitro approaches: (1) an experiment involving hormone dietary administration was performed and different reproductive parameters were deeply analysed; (2) ovarian cells were cultured in the presence of the same steroids and morphological and biochemical analyses were carried out. According to our results, sex-steroids appear not to be involved in sea urchin gonad development and gamete maturation, as neither in vivo administration nor in vitro exposure influenced gonad and gamete growth. In addition, in vitro hormonal treatment did not affect sea urchin Major Yolk Protein content. Overall, the present work complements our previous research providing information on sex-steroid involvement in echinoid reproduction and illustrates new methodological approaches that will be useful for future research on invertebrate biology and physiology.

The ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP) is an efflux protein involved in the bioavailability and milk secretion of endogenous and exogenous compounds, actively affecting milk composition. A limited number of physiological substrates have been identified. However, no studies have reported the specific effect of this polymorphism on the secretion into milk of compounds implicated in milk quality such as vitamins or endogenous compounds. The bovine ABCG2 Y581S polymorphism is described as a gain-of-function polymorphism that increases milk secretion and decreases plasma levels of its substrates. This work aims to study the impact of Y581S polymorphism on plasma disposition and milk secretion of compounds such as riboflavin (vitamin B2), enterolactone, a microbiota-derived metabolite from the dietary lignan secoisolariciresinol and uric acid. In vitro transport of these compounds was assessed in MDCK-II cells overexpressing the bovine ABCG2 (WT-bABCG2) and its Y581S variant (Y581S-bABCG2). Plasma and milk levels were obtained from Y/Y homozygous and Y/S heterozygous cows. The results show that riboflavin was more efficiently transported in vitro by the Y581S variant, although no differences were noted in vivo. Both uric acid and enterolactone were substrates in vitro of the bovine ABCG2 variants and were actively secreted into milk with a two-fold increase in the milk/plasma ratio for Y/S with respect to Y/Y cows. The in vitro ABCG2-mediated transport of the drug mitoxantrone, as a model substrate, was inhibited by enterolactone in both variants, suggesting the possible in vivo use of this enterolignan to reduce ABCG2-mediated milk drug transfer in cows. The Y581S variant was inhibited to a lesser extent probably due to its higher transport capacity. All these findings point to a significant role of the ABCG2 Y581S polymorphism in the milk disposition of enterolactone and the endogenous molecules riboflavin and uric acid, which could affect both milk quality and functionality.

Microtubule analysis is of significant value for a better understanding of normal and pathological cellular processes. Although immunofluorescence microscopic techniques have proven useful in the study of microtubules, comparative results commonly rely on a descriptive and subjective visual analysis. We developed an objective and quantitative method based on image processing and analysis of fluorescently labeled microtubular patterns in cultured cells. We used a multi-parameter approach by analyzing four quantifiable characteristics to compose our quantitative feature set. Then we interpreted specific changes in the parameters and revealed the contribution of each feature set using principal component analysis. In addition, we verified that different treatment groups could be clearly discriminated using principal components of the multi-parameter model. High predictive accuracy of four commonly used multi-classification methods confirmed our method. These results demonstrated the effectiveness and efficiency of our method in the analysis of microtubules in fluorescence images. Application of the analytical methods presented here provides information concerning the organization and modification of microtubules, and could aid in the further understanding of structural and functional aspects of microtubules under normal and pathological conditions.

The selection of human immature oocytes destined for in vitro maturation (IVM) is performed according to their cumulus–oocyte complex (COC) morphology. In animal models, oocyte pre-selection with brilliant cresyl blue (BCB) staining improves fertilization and blastocyst rates and even increases the number of calves born. As the granulosa cells and cumulus cells (GCs and CCs) have a close relationship with the oocyte and are available in in vitro fertilization (IVF) programs, applying BCB staining to these cells may help to elucidate whether BCB shows toxicity to human oocytes and to determine the safest protocol for this dye. GCs and CCs were isolated from 24 patients who underwent controlled ovarian stimulation. After 48 h, cells were exposed to: Dulbecco's Modified Eagle Medium (DMEM) with or without phenol red, DPBS and mDPBS for 60 min; 13, 20 and 26 μM BCB for 60 min; and 60, 90 or 120 min to 13 μM BCB. Cellular viability was tested using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. The 20 and 26 μM BCB exposures resulted in lower cell viability, similar to when cells were exposed to BCB for 90 or 120 min. GCs and CCs viabilities were equal among control group and 13 μM BCB group after 60 min. BCB staining was not toxic to GCs and CCs when the regime of 13 μM BCB for 60 min was used. Due to the close molecular/biochemical relationship between these cells and the gamete, we propose that it is unlikely that the use of BCB could interfere with the viability/health of human oocytes.

Growth and propagation of fish-infecting microsporidians within cell culture has been more difficult to achieve than for insect- and human-infecting microsporidians. Fish microsporidia tend to elicit xenoma development rather than diffuse growth in vivo, and this process likely increases host specificity. We present evidence that the fish microsporidian, Loma salmonae, has the capacity to develop xenomas within a rainbow trout gill epithelial cell line (RTG-1). Spore numbers increased over a 4 weeks period within cell culture flasks. Xenoma-like structures were observed using phase contrast microscopy, and then confirmed using transmission electron microscopy. Optimization of the L. salmonae-RTG-1 cell model has important implications in elucidating the process of xenoma development induced by microsporidian parasites.

From the view of tissue engineering, the deficiency in porosity has impeded further application of bacterial cellulose (BC) as a super biomaterial. In this study, we used a combination method consisting of acetic acid treatment and freeze-drying operation to improve the porous profile of BC, as well as a simple and fast method to measure the thickness, density, and porosity of BC. Results have shown a significant improvement in the porosity of the inner structure of BC treated with acetic acid and freeze-drying. Microscopic observation by scanning electron microscopy exhibited explicit evidences that more orderly porous layer-by-layer structures and more pores were formed along the cross section of modified BC as compared with the control. The enhancement of mechanical properties and crystallinity of modified BC was also demonstrated due to the improvement of material porosity in the particular extent from 50.3 to 76.43%. Cell culture of human fibroblast cells exhibited good cell viability on modified BC, suggesting that a better porous profile of BC on the surface and cross section helps facilitate cells to attach, as well as potentially promotes cells to grow in. These significant results may open the possibility of producing BC nanomaterials for tissue engineering with desirable properties.

The main cell types of the adult bee fat body are trophocytes and oenocytes; however, in pupae of some newly emerged bees, trophocytes are modified into cells called urocytes, which possibly function as a substitute for Malpighian tubules during metamorphosis when larval tubules are not functional and/or storage of urate salts is required. This study evaluated the morphology of urocytes in the stingless bee Melipona quadrifasciata and the possibility of maintaining these cells in primary culture. The urocytes M. quadrifasciata are white spherical cells with an irregular surface as observed by stereomicroscopy. They may be found individually or in groups associated with tracheae. Urocytes have a single, small, and spherical nucleus and cytoplasm rich in neutral polysaccharides, lipid droplets, protein, and granules containing calcium and urate salts. Our findings suggest that urocytes play a role in storage of neutral polysaccharides and calcium in M. quadrifasciata pupae and that these cells can be cultured for 72 h.

A culture's confluency is a fundamental measure in the field of biology, and routine quantification of confluence in cell culture protocols, biological assays and tissue engineering work is important. However, current techniques for obtaining confluency are either subjective, destructive, not simple enough, or time-consuming. We developed an image processing method for automated confluency measurement from a single microscope image without any chemical staining. To demonstrate utility we monitored the confluency of three cell types: NIH3T3 fibroblasts, C2C12 myoblasts, and 3T3L1 pre-adipocytes for 5 days, twice a day. The captured micrographs had different and uneven illumination, the cell types varied in cell-to-background contrast, and the confluency ranged between 10% and 100%. Despite these variable conditions, our method was shown to be practical, economic, and easy to implement, providing quantitative confluency measurements over time in each culture case. The method is hence suitable for routine automatic determination of confluency to standardize handling of cells, achieve reproducibility across trials, and improve accuracy in experimental outcome measures.

Feeding a low protein (LP) diet in rat pregnancy is associated with impaired cardiovascular health and function, possibly as a result of tissue remodelling. To assess whether cardiomyocytes retain differences induced by protein restriction, cells from neonatal rats exposed to control or LP diets in utero were cultured for a period of 10 days. At the end of this period, no differences in cell size, proliferation differentiation or metabolic function were noted. When treated with dexamethasone (0.1–10 μM) for 2 days, it was noted that insulin-stimulated glucose uptake was enhanced, but only in cells from LP rats. Increased glucocorticoid sensitivity of cardiomyocytes from LP rats could not be explained by differential expression of the glucocorticoid receptor or the glucose transporters, GLUT1 and GLUT4. The findings of the study suggest that sensitivity to endocrine signals may be permanently programmed by undernutrition through mechanisms that are preserved in vitro.

Somatic cells derived from zebrafish haploid larval (both androgenetic and gynogenetic) cultures were used as donors for nuclear transplant into non-enucleated oocytes. Nuclei were transplanted either before or simultaneously with oocyte activation in the central region and in the incipient animal pole, respectively. Against expected results, 20% of transplanted embryos during oocyte activation using cells of gynogenetic origin reached the 100% epiboly stage, even two survived for up to 5 days, whereas no development was observed when cells from androgenetic origin were used. Results derived from this work open a novel possibility of studying somatic cell reprogramming and imprinting phenomena in zebrafish.

When cells are allowed to age in vitro, or are taken from aged normal donors or subjects with features of accelerated aging (progeria or Werner syndrome), they may be considered 'old'. Such old cells have reduced growth rates in culture compared to early or mid-passage cells from young normal donors. During exponential growth the rate constants for protein synthesis were not significantly different between young and old cells (0.023±0.002 h1 vs. 0.021±0.002 h1, respectively), yet growth rates (i.e. protein accretion) were only 0.013±0.003 h1 in old cells compared to 0.022±0.002 h1 in young cells. Thus, the reduced rate of protein accumulation during growth of old cells compared to young cells was associated with increased protein degradation (0.01+0.002 h1 vs. 0.001±0.002 h1; P<0.05) rather than reduced rates of protein synthesis. When cells entered quiescence from density dependent inhibition of growth, protein synthetic rates decreased in both young and old cells to comparable levels (0.013±0.002 h1) with the result that rates of growth (0.003±0.0003 h1) and degradation (0.01±0.003h1) were not significantly different between the two groups. Thus, a difference in protein turnover between young and old cells was only seen during exponential growth, where degradation was increased in old cells. The causal relationship between increased protein degradation and decreased growth rates in old cells is not known.