Experimental Culture System Research Articles

Influence of physiological glucose levels on SIM‐A9 microglial cell immune response

AbstractBackgroundMicroglia play an important role in the early onset of Alzheimer’s disease pathology1. They are responsible for maintaining brain homeostasis, where changes in energy metabolism alter microglial metabolic profile and function2. The majority of cell culture‐based studies have relied on growing microglia under optimal media conditions, not reflecting the brain’s physiological levels of key metabolites. This study aims to define a suitable experimental culture system that reflects the brain’s physiological glucose level, approximately 1mM3, so the effects of altered glucose concentration can be tested.MethodSIM‐A9 mouse immortalised microglial cells were initially grown in DMEM/F‐12 medium (17.5mM glucose) containing 10% FBS and 0.1% Penicillin/Streptomycin (Gibco, UK). After 20 hours cells were re‐seeded in Neurobasal medium containing 1x B‐27 supplement (Gibco, UK) and either 1mM or 5mM glucose and grown for 20 hours. Cell proliferation and viability was assessed over 40 hours using an Incucyte imaging system (Essen BioScience, UK) and Trypan Blue assay. Media glucose concentration, cellular glucose uptake (Promega, UK), microglia activation and metabolic marker expression using qRT‐PCR were measured following stimulation of cells with Lipopolysaccharide (LPS) (2.5ng/ml)ResultMaintaining SIM‐A9 cells in 1mM and 5mM glucose medium showed static proliferation but similar viability over 20 hours compared to growth in DMEM/F12. LPS treatment increased TNFa and IL1b expression in SIM‐A9 cells in all glucose conditions, indicating the cells’ competency to produce a pro‐inflammatory response. No change in pro‐inflammatory activation markers (TNFa, IL1b) was seen after maintenance in 1mM, but in 5mM glucose for 20 hours, compared to LPS treated cells. Media glucose concentration was 0.7mM over 20 hours in 1mM and 1mM in 5mM glucose media. Expression of glucose transporters Glut1 and Glut3 and glycolysis marker PFKFB3 under these glucose conditions was investigated.ConclusionUntil now microglial metabolic studies have not always reflected the physiological concentrations of glucose in the brain. The results show that culturing microglia in 1mM glucose medium does not induce pro‐inflammatory activation, while maintaining minimal cell death and unvaried confluency.

Follicle Rescue From Prepubertal Ovaries After Recent Treatment With Cyclophosphamide-An Experimental Culture System Using Mice to Achieve Mature Oocytes for Fertility Preservation.

Ovarian tissue cryopreservation is the only feasible method for fertility preservation in prepubertal girls that will undergo gonadotoxic chemotherapy. To date, the only clinical use of cryopreserved tissue is by a later tissue retransplantation to the patient. Clinical challenges in fertility preservation of very young patients with cancer include time constraints that do not allow to retrieve the tissue for cryopreservation before starting chemotherapy and the preclusion of future ovarian tissue transplantation due to the risk of reintroduction of malignant cells in patients with systemic diseases. To overcome these two challenges, we investigated using an experimental model the feasibility of retrieving secondary follicles from ovaries of prepubertal mice after cyclophosphamide (CPA) treatment in increasing doses of 50, 75, and 100 mg/kg. The follicles were thereafter cultured and matured in vitro. The main outcomes included the efficiency of the method in terms of obtained matured oocytes and the safety of these potentially fertility preservative procedures in terms of analyses of oocyte competence regarding normality of the spindle and chromosome configurations. Our findings demonstrated that it was feasible to isolate and culture secondary follicles and to obtain mature oocytes from prepubertal mice ovaries recently treated with CPA. The efficiency of this method was highly demonstrated in the 100 mg/kg CPA group, with near 90% follicle survival rate after 12 days’ culture, similarly to control. Around 80% of the follicles met the criteria to put into maturation, and more than 40% of them achieved metaphase II, with normal spindle and chromosome configurations observed. Suboptimal results were obtained in the 50 and 75 mg/kg CPA groups. These paradoxical findings towards CPA dose might probably reflect a more difficult selection of damaged growing follicles from ovaries recently treated with lower doses of CPA and a hampered ability to identify and discard those with reduced viability for the culture.

Assimilation of shrimp farm sediment by Holothuria scabra: a coupled fatty acid and stable isotope approach

Deposit-feeding sea cucumbers are efficient nutrient recyclers and have the potential to contribute to the limitation of organic matter load in polyculture or integrated aquaculture systems. Assessing how they assimilate organic matter originating from other farmed species is therefore important for the development of such multi-species farming systems. Here, a coupled stable isotope − fatty acid approach was used to characterize the assimilation of organic matter from shrimp (Penaeus stylirostris) farming by Holothuria scabra in an experimental culture system. H. scabra were reared in mesocosms on shrimp farming-originating sediment with and without additional food sources (maize and fish meals). Although fatty acid results did indicate that shrimp-farming sediment was assimilated by holothurids, we found no evidence of maize waste and fish meal contribution to H. scabra organic carbon (no effect on δ13C, no accumulation of meal-specific fatty acids). However, a strong effect of fish meal on H. scabra δ15N was observed, suggesting that this additional food source could represent an alternative source of nitrogen for holothurids. Finally, this study supports the culture of H. scabra as a perspective to reduce sedimentary organic matter excess associated with shrimp farms, and suggest that the addition of selected food sources might contribute to increasing the content in some nitrogen organic compounds in holothurid tissues.

Bioenergetics underlying single-cell migration on aligned nanofiber scaffolds.

Cell migration is centrally involved in a myriad of physiological processes, including morphogenesis, wound healing, tissue repair, and metastatic growth. The bioenergetics that underlie migratory behavior are not fully understood, in part because of variations in cell culture media and utilization of experimental cell culture systems that do not model physiological connective extracellular fibrous networks. In this study, we evaluated the bioenergetics of C2C12 myoblast migration and force production on fibronectin-coated nanofiber scaffolds of controlled diameter and alignment, fabricated using a nonelectrospinning spinneret-based tunable engineered parameters (STEP) platform. The contribution of various metabolic pathways to cellular migration was determined using inhibitors of cellular respiration, ATP synthesis, glycolysis, or glucose uptake. Despite immediate effects on oxygen consumption, mitochondrial inhibition only modestly reduced cell migration velocity, whereas inhibitors of glycolysis and cellular glucose uptake led to striking decreases in migration. The migratory metabolic sensitivity was modifiable based on the substrates present in cell culture media. Cells cultured in galactose (instead of glucose) showed substantial migratory sensitivity to mitochondrial inhibition. We used nanonet force microscopy to determine the bioenergetic factors responsible for single-cell force production and observed that neither mitochondrial nor glycolytic inhibition altered single-cell force production. These data suggest that myoblast migration is heavily reliant on glycolysis in cells grown in conventional media. These studies have wide-ranging implications for the causes, consequences, and putative therapeutic treatments aimed at cellular migration.

Flaviviridae Viruses and Oxidative Stress: Implications for Viral Pathogenesis

Oxidative stress is induced once the balance of generation and neutralization of reactive oxygen species (ROS) is broken in the cell, and it plays crucial roles in a variety of natural and diseased processes. Infections of Flaviviridae viruses trigger oxidative stress, which affects both the cellular metabolism and the life cycle of the viruses. Oxidative stress associated with specific viral proteins, experimental culture systems, and patient infections, as well as its correlations with the viral pathogenesis attracts much research attention. In this review, we primarily focus on hepatitis C virus (HCV), dengue virus (DENV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV) as representatives of Flaviviridae viruses and we summarize the mechanisms involved in the relevance of oxidative stress for virus-associated pathogenesis. We discuss the current understanding of the pathogenic mechanisms of oxidative stress induced by Flaviviridae viruses and highlight the relevance of autophagy and DNA damage in the life cycle of viruses. Understanding the crosstalk between viral infection and oxidative stress-induced molecular events may offer new avenues for antiviral therapeutics.

Abstract 3266: Distinct immune stimulatory functions of anti-human VISTA antibodies are determined by the antibody isotype

Abstract The clinical success of immune modulatory antibodies in oncological indications has demonstrated the importance of harnessing the immune system for the treatment of cancer. VISTA (PD-1H) is an Ig superfamily cell surface molecule mainly expressed on myeloid cells, but also to some extent on NK cells and T cells. Substantial published data supports the view that VISTA-binding antibodies or administration of VISTA itself can provide an inhibitory signal to T cells though the mechanism of action remains unclear including whether this activity is mediated directly or indirectly. We present here novel findings regarding the impact of different IgG isotypes on the activity of anti-human VISTA antibodies with immune stimulatory effects on primary human immune cells. The immune stimulatory effects of IgG antibodies were found to differ between variants with an identical antigen-binding domain but with different IgG isotypes (IgG4 and IgG1). First, we observed that both variants increased ongoing immune reactions, while the IgG1-variant was additionally able to activate resting immune cells. In a mixed leukocyte reaction (MLR) with healthy donor PBMCs, the ongoing immune reaction was effectively augmented by both, anti-VISTA-IgG4 and anti-VISTA-IgG1 isotype antibodies, as observed by increased levels of cytokines, e.g. TNF. However, in an experimental culture system of unstimulated PBMC of single healthy donors, only VISTA-IgG1 isotype antibodies induced increased levels of activation markers, e.g. upregulation of HLA-DR on resting myeloid cells. Second, we showed that Fc-receptor binding was a necessary interaction for immune activation as preventing the Fc-gamma-receptor binding to either of the two isotypes abolished the effects. The effects observed were specific as non-VISTA-binding isotype control mAbs were inactive. We then investigated the effects of anti-VISTA-IgG4 isotype antibodies on cells from AML patient samples by pharmacoscopy, a novel microscopy analysis method able to robustly quantify cell-cell proximity as a measure of cellular interactions. Here, VISTA-IgG4 isotype antibodies augmented interactions between multiple immune cell populations as well as between immune cells and AML cancer cells. This augmented interaction was abolished by blockade of Fc-gamma-receptor interactions, confirming our previous results. In summary, the immune stimulatory effects of anti-VISTA antibodies are defined by the antibody isotype and interaction with Fc-gamma-receptors, highlighting the importance of understanding these interactions when designing immune stimulatory antibody therapeutics for IO applications. Citation Format: Sven Mostböck, Helen Wu, Timothy Fenn, Christina Taubert, Gregory Vladimer, Anne Vogt. Distinct immune stimulatory functions of anti-human VISTA antibodies are determined by the antibody isotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3266.

5 Gene expression analysis and DNA methylation patterns of porcine somatic cell nuclear transfer blastocysts with high and low incidence of apoptosis

Somatic cell NT (SCNT) efficiency remains poor, preventing the technology from being regularly used in the agricultural industry. It is believed that faulty epigenetic reprogramming of SCNT embryos leads to the low overall success. A clear apoptotic signature is associated with inappropriate gene expression and epigenomic aberrancies in many experimental cell culture systems, and we hypothesised that an apoptosis biomarker could be used to effectively separate properly reprogrammed porcine SCNT embryos from those that are destined to fail due to incomplete reprogramming. Therefore, our objective was to evaluate global gene expression and DNA methylation patterns in high- and low-apoptosis individual embryos in an effort to characterise the extent of genomic reprogramming that had taken place. Porcine SCNT blastocysts on Day 6 of development were stained with a nontoxic, noninvasive caspase activity reporter, and the top and bottom 20% of detected caspase activity were classified as high and low apoptosis, respectively (3 replicate cloning sessions; n=13 embryos per group). Genomic DNA and total RNA were isolated from each individual blastocyst. The RNA sequencing libraries were prepared using the Ovation SoLo RNA-Seq system (NuGen, San Carlos, CA, USA). Reduced representation bisulfite sequencing libraries were prepared for DNA methylation analysis using a modification of the single-cell reduced representation bisulfite sequencing global DNA methylation analysis approach detailed by Guo et al. (2015 Nat. Protoc. 10, 645-59). The RNA sequencing analysis using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) revealed 175 total differentially expressed genes (fold change ≥1.5; false discovery rate ≤0.05) between the high- and low-apoptosis SCNT embryos. This list of differentially expressed genes was used to perform enrichment analysis to identify overrepresented Gene Ontology (GO) terms or Kyoto Encyclopedia of Genes and Genomes pathways (DAVID Ease version 6.8 (https://david.ncifcrf.gov/) against the Sus scrofa background genome). However, no significantly enriched GO terms or pathways were identified (false discovery rate P>0.05). Analysis of global DNA methylation patterns between high- and low-apoptosis SCNT embryos using MethylKit (Akalin et al. 2012Genome Biol. 13, R87) revealed 335 differentially methylated 100-bp regions with at least 25% difference in methylation (adjusted P ≤ 0.01). Gene transcription start sites associated with these regions were used for enrichment analysis; again, no significant enrichment of GO terms or Kyoto Encyclopedia of Genes and Genomes pathways was identified. Principal component analysis of CpG methylation showed the low-apoptosis embryos clustering more tightly than the high-apoptosis embryos, which were highly scattered. Ongoing comparisons of high- and low-apoptosis cloned embryos with naturally fertilized embryos produced invivo may provide more information about which embryos were properly reprogrammed. Although we are still pursuing a link between reprogramming and gene expression in high- and low-apoptosis embryos, we conclude that these data support a model of stochastic epigenetic reprogramming following SCNT and reinforce the necessity of identifying embryos most likely to be successful due to proper epigenetic reprogramming in order to increase SCNT efficiency.

Visualizing Viral Infection In Vivo by Multi-Photon Intravital Microscopy.

Viral pathogens have adapted to the host organism to exploit the cellular machinery for virus replication and to modulate the host cells for efficient systemic dissemination and immune evasion. Much of our knowledge of the effects that virus infections have on cells originates from in vitro imaging studies using experimental culture systems consisting of cell lines and primary cells. Recently, intravital microscopy using multi-photon excitation of fluorophores has been applied to observe virus dissemination and pathogenesis in real-time under physiological conditions in living organisms. Critical steps during viral infection and pathogenesis could be studied by direct visualization of fluorescent virus particles, virus-infected cells, and the immune response to viral infection. In this review, I summarize the latest research on in vivo studies of viral infections using multi-photon intravital microscopy (MP-IVM). Initially, the underlying principle of multi-photon microscopy is introduced and experimental challenges during microsurgical animal preparation and fluorescent labeling strategies for intravital imaging are discussed. I will further highlight recent studies that combine MP-IVM with optogenetic tools and transcriptional analysis as a powerful approach to extend the significance of in vivo imaging studies of viral pathogens.

Characteristics of Phytoplankton Succession Based on the Functional Group in the Enclosed Culture System

The present study was conducted from August to December 2016 in a cylindrical water tank with a diameter of 1 m, a height of 4 m and a capacity of 3,000 L. The field water and sediment from the Nakdong River were also sampled for the experimental culture (field water+sediment) and control culture (field water), respectively. In this study, we aimed to investigate phytoplankton succession pattern using the phytoplankton functional group in the enclosed culture system. A total of 50 species in 27 genera including Chlorophyceae (30 species), Bacillariophyceae (11 species), Cyanophyceae (7 species), and Cryptophyceae (2 species) were identified in the experimental and control culture systems. A total of 19 phytoplankton functional groups (PFGs) were identified, and these groups include B, C, D, F, G, H1, J, K, Lo, M, MP, N, P, S1, TB, W0, X1, X2 and Y. In particular, W0, J and M groups exhibited the marked succession in the experimental culture system with higher biovolumes compared to those of the control culture system, which may be related to the internal cycling of nutrients by sediment in the experimental culture system. The principal component analyses demonstrated that succession patterns in PFG were associated with the main environmental factors such as nutrients (N, P), water temperature and light intensity in two culture systems. In conclusion, the present study showed the potential applicability of the functional group for understanding the adaptation strategies and ecological traits of the phytoplankton succession in the water bodies of Korea.

In Vitro Modeling of Tumor-Immune System Interaction.

Immunotherapy has emerged during the past two decades as an innovative and successful form of cancer treatment. However, frequently, mechanisms of actions are still unclear, predictive markers are insufficiently characterized, and preclinical assays for innovative treatments are poorly reliable. In this context, the analysis of tumor/immune system interaction plays key roles, but may be unreliably mirrored by in vivo experimental models and standard bidimensional culture systems. Tridimensional cultures of tumor cells have been developed to bridge the gap between in vitro and in vivo systems. Interestingly, defined aspects of the interaction of cells from adaptive and innate immune systems and tumor cells may also be mirrored by 3D cultures. Here we review in vitro models of cancer/immune cell interaction and we propose that updated technologies might help develop innovative treatments, identify biologicals of potential clinical relevance, and select patients eligible for immunotherapy treatments.

Assessment of the relative contribution of dietary nitrogen from fish meal and biofloc meal to the growth of Pacific white shrimp (Litopenaeus vannamei )

The relative contribution of the dietary nitrogen supplied by fish meal and a biofloc meal to the growth of Pacific white shrimp was evaluated using stable isotope analysis. Biofloculated material was obtained from an experimental tilapia culture system. Five formulated diets were supplied. Two of them consisted in isotopic controls having only fish meal or biofloc meal as protein source. Three mixed diets were formulated with varying proportions of these ingredients on a dietary nitrogen basis (75:25, 50:50 and 25:75). At the end of the trial, survival rates were similar (92–100%) but significant differences in mean final weight were observed and a negative correlation between the inclusion of biofloc meal and weight gain was evidenced. Mean final weight in shrimp fed on diet containing only fish meal was 2.8 g, while mean final weight of animals fed on diet containing 50% biofloc was 1.9 g. Isotopic mixing models indicated that all diets contributed higher proportions of dietary nitrogen from fish meal than from biofloc meal. Dietary nitrogen available in diets containing 25%, 50% and 75% of biofloc meal was incorporated in muscle tissue as 5%, 41% and 64% respectively. Diet supplying 25% of nitrogen from biofloc was the only mixed diet eliciting growth comparable to diet containing only fish meal. Lower growth and nitrogen deposition in shrimp fed on diets containing high proportions of biofloc meal were possibly associated to the use of only two protein sources and a restriction of essential amino acids.

Non-antagonistic influence of Krumeich's intrastromal corneal ring in an experimental tissue culture system

Intrastromal insertion of Krumeich's corneal ring between graft and residual host corneal tissue appears to impair preripheral, superficial and superfluous vascularization of donor corneal tissue. The purpose of this study was to investigate the cytotoxic effects of Krumeich's ring using tissue cultures composed of primary human dermal microvascular endothelial cells from adult donors (HMVEC). Soluble growth medium extracts of the individual components of Krumeich's ring alloy were prepared and HMVEC were exposed to these extracts in triplicate for 1 day followed by investigation with 3‑(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Furthermore, HMVEC were cultured for 5 days on either Krumeich's ring or polypropylene (PP) discs coated with individual components of the Krumeich's ring alloy followed by double vital staining with fluorescein diacetate (FDA) and propidium iodide (PI). The MTT assays revealed that higher doses of the extracts appeared to reduce the viability of HMVEC, while highly diluted extracts of molybdenum (Mo) powder appeared to increase the metabolic activity of HMVEC. The FDA-PI staining showed only a few live HMVEC on either cobalt (Co) or Mo-coated PP discs, compared to the respective titanium (Ti) and chromium (Cr) counterparts. Viable HMVEC appeared to attach to Krumeich's ring after a 5‑day incubation period. The results confirm that Krumeich's ring does not exert measurable cytotoxic effects in our chosen assay system. High dilutions of medium-soluble Mo powder extracts appear to increase the metabolic activity of HMVEC.

Sink and swim: a status review of thecosome pteropod culture techniques

The widespread distribution of pteropods, their role in ocean food webs and their sensitivity to ocean acidification and warming has renewed scientific interest in this group of zooplankton. Unfortunately, their fragile shell, sensitivity to handling, unknowns surrounding buoyancy regulation and poorly described feeding mechanisms make thecosome pteropods notoriously difficult to maintain in the laboratory. The resultant high mortality rates and unnatural behaviours may confound experimental findings. The high mortality rate also discourages the use of periods of acclimation to experimental conditions and precludes vital long-term studies. Here we summarize the current status of culture methodology to provide a comprehensive basis for future experimental work and culture system development.

Studies on culture and osteogenic induction of human mesenchymal stem cells under CO2-independent conditions.

Human mesenchymal stem cells (hMSCs) are one of the important factors that regulate bone anabolism. Osteoporosis resulting from microgravity during spaceflight may possibly be due to a decrease in osteogenesis mediated by hMSCs. This speculation should be verified through culture and osteogenic induction of hMSCs in a microgravity environment during spaceflight. Control of CO2 is a key component in current experimental protocols for growth, survival, and proliferation of in vitro cultured cells. However, carrying CO2 tanks on a spaceflight and devoting space/mass allowances for classical CO2 control protocols make experimentation on culture and osteogenesis difficult during most missions. Therefore, an experimental culture and osteogenic medium was developed through modifying the components of buffer salts in conventional culture medium. This experimental medium was used to culture and induce hMSCs under CO2-independent conditions. The results showed that culture and induction of hMSCs with conventional culture medium and conventional osteogenic medium under CO2-independent conditions resulted in an increase of pH in medium. The proliferation of hMSCs was also inhibited. hMSCs cultured with experimental culture medium under CO2-independent conditions showed a proliferation potential that was the same as those cultured with conventional culture medium under CO2-dependent conditions. The experimental osteogenic medium could promote hMSCs to differentiate into osteoblast-like cells under CO2-independent conditions. Cells induced by this induction system showed high alkaline phosphatase activity. The expression levels of osteogenic genes in cells induced with experimental osteogenic medium under CO2-independent conditions were not significantly different from those cells induced with conventional osteogenic medium under CO2-dependent conditions. These results suggest that the experimental culture and induction system could be used to culture hMSCs and induce the osteogenesis of hMSCs in the atmospheric conditions common to spaceflights without additional CO2.

The potential of Yucca schidigera extract to reduce the ammonia pollution from shrimp farming

This study investigated the feasibility of using Yucca schidigera extract (YUPE) to reduce ammonia pollution resulting from shrimp farming. YUPE was added at ratios of 18, 36, and 72mgL−1 to effluent from an experimental shrimp culture system containing total ammonia nitrogen (TAN) of 0.592, 0.672, and 0.718mgL−1. TAN reduction increased with an increase in YUPE, and at 18mgL−1 reduced TAN by 71–72% and 86–87% at 6 and 24h, respectively. Shrimp feed containing YUPE at 300, 600, and 1200mgkg−1 was submerged, allowing the ammonia to leach out. The addition of YUPE in feed reduced TAN leaching by 50–83% compared with the control. The application of YUPE in water or feed could considerably reduce the accumulation of ammonia in waste water discharge during shrimp production, thereby providing a feasible solution to ammonia pollution in coastal environments.

Female parthenogenetic apomixis and androsporogenesis in Douglas-fir embryonal initials in an artificial sporangium

Control of female parthenogenetic apomixis and androsporogenesis of Douglas-fir embryonal initials was studied using an experimental culture system in which changes in growth condition can mediate changes in cell identity and outcomes. This culture system constitutes an artificial sporangium in which myriad culture conditions can be simulated and should be applicable for research on a variety of gymnosperms. In this study, embryonal initials from developing seeds from two Douglas-fir trees were rescued and became reprogrammed for female parthenogenetic apomixis (fPA) and parthenogenetic androsporogenesis (mPA). Female PA was initiated by endomitosis forming a binucleate cell with a diploid egg-equivalent and an apoptotic ventral canal nucleus in an archegonial tube. Egg-equivalent nuclei formed cells (parthenotes) that were discharged into an aqueous culture medium. Parthenotes developed axial tiers atypical of early embryogenesis in seeds. Earlier in the year, androsporangial tubes were parthenogenetically differentiated and released monads, dyads, triads, and tetrads into the culture medium. Spores showed chromosomal aberrations. PA demonstrated a temporal separation in gender expression (dichogamy). Embryonal initials brought forward and by-passed the long juvenile phases normally needed for cells to develop into trees and express reproductive maturity. Expressions of fPA and mPA indicated that the specialized culture flasks served as an artificial sporangium (AS). Awareness is raised for the value of an AS for research in gymnosperm life cycles and as a teaching and research laboratory.

Reproductive performance of the crayfish Procambarus ( Austrocambarus) acanthophorus Villalobos 1948 under controlled conditions

Aspects of the reproductive biology of the crayfish Procambarus acanthophorus were documented under controlled conditions to determine this species’ culture potential. A completely random experimental design with three replicates was used to evaluate three different male/female sex ratios (1 M:1F, 1 M:3F and 1 M:5F) with 12 organisms (TL > 30 mm) per replicate. The experimental culture system was a closed recirculating system with constant water flow (2 L .min − 1 ), and once daily ad libitum feeding with a 35% protein commercial diet. Recorded reproductive parameters were peak reproduction season, fertility index, egg viability and female length at first sexual maturity. Peak reproduction activity occurred during November–December, the lowest activity in February–March, and no differences were observed between treatments. Fertility was estimated as a function of egg count per female ( n = 25) and hatch rate. The highest ( p < 0.05) number of ovigerous females was produced at the 1 M:3F ratio, and the lowest at the 1 M:5F. Average egg count per female was 240.9 (± 93.1) and average egg viability was 29.1% (± 31.7). The relationship between total length and egg count was positive and significant ( r 2 = 0.654), although the relationship between length and egg viability was not ( r 2 = 0.1132). Average female length at first sexual maturity was 34 mm. The study results indicate P. acanthophorus can be effectively reproduced in captivity under controlled conditions and is therefore a candidate for use in commercial aquaculture activities.

Plant growth promotion due to rhizobacterial volatiles – An effect of CO 2?

Serratia odorifera, an antagonistic rhizobacterium, emits a diverse and complex bouquet of volatiles. Three different in vitro experimental culture systems indicated that these volatiles promote the growth of Arabidopsis thaliana. CO 2 trapping and significant rise of CO 2 levels (390–3000 ppm CO 2 within 24 h) due to bacterial growth in sealed Petri dishes verified the enhanced effects of rhizobacterial CO 2 on A. thaliana’s growth. In contrast, open cocultivations abrogated growth promotion, and inhibitory effects come to the fore at ambient CO 2 concentrations.

Arsenic as an Endocrine Disruptor: Effects of Arsenic on Estrogen Receptor–Mediated Gene Expression In Vivo and in Cell Culture

Arsenic (As) contamination of drinking water is considered a serious worldwide environmental health threat that is associated with increased disease risks including skin, lung, bladder, and other cancers; type 2 diabetes; vascular and cardiovascular diseases; reproductive and developmental effects; and neurological and cognitive effects. Increased health risks may occur at as low as 10-50 ppb, while biological effects have been observed in experimental animal and cell culture systems at much lower levels. We previously reported that As is a potent endocrine disruptor, altering gene regulation by the closely related glucocorticoid, mineralocorticoid, progesterone, and androgen steroid receptors (SRs) at concentrations as low as 0.01 microM ( approximately 0.7 ppb). Very low doses enhanced hormone-mediated gene transcription, whereas slightly higher but still noncytotoxic doses were suppressive. We report here that As also disrupts the more distally related estrogen receptor (ER) both in vivo and in cell culture. At noncytotoxic doses (1-50 micromol/kg arsenite) As strongly suppressed ER-dependent gene transcription of the 17beta-estradiol (E2)-inducible vitellogenin II gene in chick embryo liver in vivo. In cell culture, noncytotoxic levels (0.25-3 microM, approximately 20-225 ppb) of As significantly inhibited E2-mediated gene activation of an ER-regulated reporter gene and the native ER-regulated GREB1 gene in human breast cancer MCF-7 cells. While the effects of As on ER-dependent gene regulation were generally similar to As effects on the other SRs, there were specific differences, particularly the lack of significant enhancement at the lowest doses, that may provide insights into possible mechanisms.

Dietary carotenoids, connexins and cancer: what is the connection?

Carotenoids and retinoids are chemically related; indeed a major source of vitamin A in humans occurs through enzymic cleavage of beta-carotene. However, most dietary carotenoids cannot be converted into retinoids. Retinoids have demonstrated cancer-preventive activities in humans and experimental models; however, their toxicity has precluded wide-scale clinical use. In contrast, carotenoids are essentially non-toxic and their cancer-preventive activities, although strongly supported by epidemiological studies, have only been satisfactorily demonstrated in experimental systems. We have shown that in an experimental cell culture system consisting of carcinogen-treated 10T1/2 cells, both retinoids and all dietary carotenoids examined can reversibly inhibit neoplastic transformation in the post-initiation phase of carcinogenesis. This activity strongly correlates with their ability to increase gap junctional intercellular communication by up-regulating the expression of the gene CX43 (connexin43). Connexins comprise the structural unit of gap junctions, organelles which allow direct transfer of signals, nutrients and waste products between contacting cells. CX43 is the most widely expressed member of the gap junction family of genes, and we have demonstrated that its expression is strongly down-regulated in human cancers and in several premalignant conditions. When several human tumour cell lines were genetically engineered to conditionally express CX43 under the influence of a tetracycline promoter, their neoplastic phenotype was strongly attenuated. Specifically, induced cells were inhibited from growing in an anchorage-independent manner and, additionally, growth as xenografts in immunocompromised animals was also strongly attenuated. Growth inhibition in suspension was associated both with increased G(1) cell-cycle arrest and with increased apoptosis. We propose a model whereby junctional communication allows the transfer of growth inhibitory signals from normal to neoplastic cells and that retinoids and carotenoids, by increasing signal transfer, act to prevent cancer.