Supplemented Culture Medium Research Articles

Visualizing the interaction of Acanthamoeba castellanii with human retinal epithelial cells by spontaneous Raman and CARS imaging

AbstractImproved understanding of the mechanism of nutrient's uptake can enable targeted manipulation of nutrient sensing pathways in medically important pathogens to a greater degree than is currently possible. In this context, we present the use of spontaneous Raman microspectroscopy and coherent anti‐Stokes Raman spectroscopy to visualize the time‐dependent molecular interactions between the protozoan Acanthamoeba castellanii and host human cells. Human retinal pigment epithelial (ARPE‐19) cells were pre‐labelled with deuterated Phe (L‐Phe[D8]) and the uptake of the host derived L‐Phe(D8) by A. castellanii trophozoites was measured by Raman microspectroscopy for up to 48 hr post infection (hpi). This approach revealed a time‐dependent uptake pattern of this essential amino acid by A. castellanii trophozoites during the first 24 hpi with complete enrichment with L‐Phe(D8) detected in trophozoites at 48 hpi. In contrast, cell free A. castellanii trophozoites showed a modest uptake of only 16–18% L‐Phe(D8) from L‐Phe(D8)–supplemented culture medium after 3, 24, and 48 hr hpi. Coherent anti‐Stokes Raman spectroscopy microscopy was successfully used to monitor the reprogramming of lipids within the trophozoites as they engaged with host cells. The methodology presented here provides new advances in the ability to analyze the kinetic of amino acid acquisition by A. castellanii from host cell and extracellular environment, and to visualize lipid reprogramming within the trophozoite.

Strategy for erythroid differentiation in ex vivo cultures: Lentiviral genetic modification of human hematopoietic stem cells with erythropoietin gene

If cultured in appropriate conditions, such as supplementing culture media with costly cytokines and growth factors, hematopoietic stem/progenitor cells (HSPCs) from different origins have shown to be an adequate source of erythroid cells. This requirement turns erythroid cells production into a complicated process to be scaled-up for future applications. The aim of our work was to genetically modify HSPCs with human erythropoietin (hEPO) sequence by lentiviral transgenesis in order for cells to secrete the hormone into the culture medium. Initially, we evaluated erythroid differentiation in colony forming units (CFU) assays and further analyzed cell expansion and erythroid differentiation throughout time in suspension cultures by flow cytometry and May-Grünwald-Giemsa staining. Additionally, we studied hEPO production and its isoforms profile. The different assessment approaches demonstrated erythroid differentiation, which was attributed to the hEPO secreted by the HSPCs. Our data demonstrate that it is possible to develop culture systems in which recombinant HSPCs are self-suppliers of hEPO. This feature makes our strategy attractive to be applied in biotechnological production processes of erythroid cells that are currently under development.

Abstract 851: Reversion of epithelial to mesenchymal transition under androgen deprived conditions promotes chemoresistance in prostate cancer

Abstract Introduction: Metastatic prostate cancer (PC) is one of the leading causes of cancer related male deaths in Australia. Radical prostatectomy followed by androgen deprivation therapy (ADT) upon recurrence of disease is the conventional form of treatment. Upon relapse, administration of androgen targeted therapy (ATT) and subsequent chemotherapy improves quality of life and overall survival of patients. However, it is not curative and the disease ultimately progresses. Epithelial to mesenchymal transition (EMT) is a process that can facilitate tumour growth and progression to metastasis and has also been associated with chemoresistance. We therefore aim to determine how EMT and resultant reversal of this state (MET) influences the chemoresistance profile of PC cells and further determine if and how specific genetic regulators are involved in this process. Methods: Doxycycline hyclate (Dox) inducible Snai1 and Zeb1, LNCaP EMT models were used to determine the effect of the EMT status in response to a clinically relevant panel of drugs using cellular and molecular assays. All assays were performed in both fetal bovine serum (FBS) and charcoal stripped serum (CSS) supplemented culture media. CSS media was used to mimic an ADT-like environment. Using 2D monolayer and 3D spheroid [Happy Cell©] formats. Cells were induced for 5 days of EMT in the presence of Dox and were allowed to revert for 14 days of MET (absence of Dox) before the drug assays were carried out over a period of 72 hours. Live/dead cell staining was used to determine viability percentage in both models. Flow cytometry was used for 2D assays and 3D assays were analysed using either a GE IN Cell Analyzer live cell imaging system or confocal microscopy. Results: Induction of Snai1 and Zeb1 induced an elongated cell shape and increased mRNA and protein expression of mesenchymal markers such as vimentin during EMT. This was associated with a decrease in expression of epithelial markers E-cadherin and EpCam,. An eventual drop in the expression levels of vimentin at around 14 days of dox removal along with an increase in epithelial markers was observed. These changes were also associated with a gradual reversal in the phenotype of these cells. However, this reversal in phenotype and expression of EMT markers was not as striking in cells grown in CSS compared to cells in FBS media (both 2D and 3D). Mitoxantrone concentration-response assays in a 2D format showed that cells that undergo a subsequent MET are more chemoresistant than uninduced or EMT cells when grown in CSS media. This was however, not observed in cells undergoing similar transitions in the FBS media. Conclusions: The chemoresistance profile of prostate cancer cells is altered by their EMT status. This effect is enhanced in the context of ADT. Cells that revert back to a more epithelial phenotype after undergoing an EMT exhibit higher resistance towards chemotherapy drugs. Citation Format: Akanksha Upadhyaya, Nataly Stylianou, Anthony M. Davies, Sarah-Louise Ryan, Brett G. Hollier, Elizabeth D. Williams. Reversion of epithelial to mesenchymal transition under androgen deprived conditions promotes chemoresistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 851. doi:10.1158/1538-7445.AM2017-851

Modelling the effect of water activity reduction by sodium chloride or glycerol on conidial germination and radial growth of filamentous fungi encountered in dairy foods

Water activity (aw) is one of the most influential abiotic factors affecting fungal development in foods. The effects of aw reduction on conidial germination and radial growth are generally studied by supplementing culture medium with the non-ionic solute glycerol despite food aw can also depend on the concentration of ionic solutes such as sodium chloride (NaCl). The present study aimed at modelling and comparing the effects of aw, either modified using NaCl or glycerol, on radial growth and/or conidial germination parameters for five fungal species occurring in the dairy environment. The estimated cardinal values were then used for growth prediction and compared to growth kinetics observed on commercial fresh cheese. Overall, as compared to glycerol, NaCl significantly increased the fungistatic effect resulting from aw reduction by extending latency and/or reducing radial growth rates of Paecilomyces niveus, Penicillium brevicompactum, Penicillium expansum and Penicillium roqueforti but not of Mucor lanceolatus. Besides, NaCl significantly reduced aw range for conidial germination and delayed median germination time of P. expansum but not of P. roqueforti. Despite these observations, cardinal aw values obtained on glycerol-medium yielded similar predictions of radial growth and germination time in commercial fresh cheese as those obtained with NaCl. Thus, it indicates that, for the studied species and aw range used for model validation, the use of NaCl instead of glycerol as a aw depressor had only limited impact for fungal behavior prediction.

Osseous differentiation on freeze casted 10CeTZP-Al2O3 structures

Three-dimensional structures with directionally oriented pore networks were fabricated from a 10mol% ceria-stabilized zirconia and alumina composite (10CeTZP-Al2O3) via freeze casting. Ceramic suspensions of different concentrations (30, 40 and 50wt% solids) were frozen at various rates (2, 5 and 10°C/min) to obtain lamellar structures with aligned tubular pores of different characteristics: porosity (75–84%), pore dimensions (small diameter of the elliptical pores: 10–23μm; large diameter of the elliptical pores: ∼200±70μm), lamella thickness (2.7–4μm) and compression strength (1–12MPa). In vitro assays confirmed the non-cytotoxic nature of the samples. Furthermore, specific osseous differentiation genes were quantified after incubating osteoblasts on different cross sections of the samples during 7days in supplemented culture medium; results demonstrated that the freeze casted structures induce up to nine times more osseous gene expression than tissue culture polystyrene (TCPS), an advanced surface used for optimized in vitro cell growth.

Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro.

The amino acid leucine is thought to be important for skeletal muscle growth by virtue of its ability to acutely activate mTORC1 and enhance muscle protein synthesis, yet little data exist regarding its impact on skeletal muscle size and its ability to produce force. We utilized a tissue engineering approach in order to test whether supplementing culture medium with leucine could enhance mTORC1 signaling, myotube growth, and muscle function. Phosphorylation of the mTORC1 target proteins 4EBP‐1 and rpS6 and myotube hypertrophy appeared to occur in a dose dependent manner, with 5 and 20 mM of leucine inducing similar effects, which were greater than those seen with 1 mM. Maximal contractile force was also elevated with leucine supplementation; however, although this did not appear to be enhanced with increasing leucine doses, this effect was completely ablated by co‐incubation with the mTOR inhibitor rapamycin, showing that the augmented force production in the presence of leucine was mTOR sensitive. Finally, by using electrical stimulation to induce chronic (24 hr) contraction of engineered skeletal muscle constructs, we were able to show that the effects of leucine and muscle contraction are additive, since the two stimuli had cumulative effects on maximal contractile force production. These results extend our current knowledge of the efficacy of leucine as an anabolic nutritional aid showing for the first time that leucine supplementation may augment skeletal muscle functional capacity, and furthermore validates the use of engineered skeletal muscle for highly‐controlled investigations into nutritional regulation of muscle physiology.

Cultivation of Human Oral Mucosal Explants on Contact Lenses

ABSTRACTPurpose/Aim: Autologous cultivated oral mucosal (OM) epithelial transplantation has been successfully used as corneal epithelial replacement in bilateral limbal stem cell deficiency. Recently, lotrafilcon A contact lens (CL) surface was described as a suitable carrier for cultured stem cells in corneal epithelial transplantation. Our aim was to establish explant cultures from human OM on CL carriers that are free of animal-derived materials and feeder cells.Materials and methods: Human cadaveric 2 mm OM explants were sutured onto CL surfaces and cultivated with fetal calf serum (FCS) or human serum (HS) supplemented culture medium without feeder cells. Confluent cultures were harvested and evaluated morphologically with hematoxylin and eosin stain and with immunofluorescence microscopy for the presence of p63, vimentin and cytokeratins (CK) 3, 4, 13 and 14.Results: Confluent cell sheets covering the whole CL surface were produced from OM explants after 2 weeks of culture with HS and after 3 weeks with FCS. A basal layer consisted of small, vimentin, p63 and CK14 positive putative stem/progenitor cells, which were present in the whole cell sheet. Large, CK3, CK4 and CK13 positive, differentiated cells appeared to spread above this confluent layer.Conclusions: We have established an animal-free culture system from human OM explants on CL surface. The cultured OM sheets contain large numbers of putative stem cells including limbal-like CK3 and CK14 positive cells. This method can be adapted to good manufacturing practice (GMP) conditions and has, therefore, great potential for clinical use.

Modification of mitochondrial function, cytoplasmic lipid content and cryosensitivity of bovine embryos by resveratrol.

Resveratrol is a potent activator of NAD-dependent deacetyltransferase sirtuin-1 (SIRT1) and affects lipid metabolism and ATP generation in somatic cells. In the present study, the effects of supplementing culture medium with resveratrol on lipid metabolism, ATP generation, and cryosensitivity of bovine in vitro produced embryos were investigated. Bovine early cleaved-stage embryos were cultured in medium containing 0 or 0.5 µM resveratrol for 1 or 5 days. Resveratrol treatment for both 1 day and 5 days increased the expression levels of SIRT1 and phosphorylated AMP-activated protein kinase (pAMPK) in the embryos. Furthermore, resveratrol treatment was effective to increase ATP generation and reduce lipid content of the embryos. The effects of resveratrol treatment were diminished by the SIRT1 inhibitor “EX527”, and the reduced lipid content was reversed by treatment with etomoxir (a potent inhibitor of beta-oxidation). Blastocysts developed after resveratrol treatment showed low levels reactive oxygen species and increased cryotolerance. These results demonstrate that resveratrol improves in vitro development of bovine embryos, while reducing cytoplasmic lipid content through activation of beta-oxidation, thereby effective for production of bovine blastocysts with enhanced cryotolerance.

Transcriptomic Profiling Analysis of Arabidopsis thaliana Treated with Exogenous Myo-Inositol.

Myo-insositol (MI) is a crucial substance in the growth and developmental processes in plants. It is commonly added to the culture medium to promote adventitious shoot development. In our previous work, MI was found in influencing Agrobacterium-mediated transformation. In this report, a high-throughput RNA sequencing technique (RNA-Seq) was used to investigate differently expressed genes in one-month-old Arabidopsis seedling grown on MI free or MI supplemented culture medium. The results showed that 21,288 and 21,299 genes were detected with and without MI treatment, respectively. The detected genes included 184 new genes that were not annotated in the Arabidopsis thaliana reference genome. Additionally, 183 differentially expressed genes were identified (DEGs, FDR ≤0.05, log2 FC≥1), including 93 up-regulated genes and 90 down-regulated genes. The DEGs were involved in multiple pathways, such as cell wall biosynthesis, biotic and abiotic stress response, chromosome modification, and substrate transportation. Some significantly differently expressed genes provided us with valuable information for exploring the functions of exogenous MI. RNA-Seq results showed that exogenous MI could alter gene expression and signaling transduction in plant cells. These results provided a systematic understanding of the functions of exogenous MI in detail and provided a foundation for future studies.

Substrate Stiffness Combined with Hepatocyte Growth Factor Modulates Endothelial Cell Behavior.

Endothelial cells (ECs) play a crucial role in regulating various physiological and pathological processes. The behavior of ECs is modulated by physical (e.g., substrate stiffness) and biochemical cues (e.g., growth factors). However, the synergistic influence of these cues on EC behavior has rarely been investigated. In this study, we constructed poly(l-lysine)/hyaluronan (PLL/HA) multilayer films with different stiffness and exposed ECs to these substrates with and without hepatocyte growth factor (HGF)-supplemented culture medium. We demonstrated that EC adhesion, migration, and proliferation were positively correlated with substrate stiffness and that these behaviors were further promoted by HGF. Interestingly, ECs on the lower stiffness substrates showed stronger responses to HGF in terms of migration and proliferation, suggesting that HGF can profoundly influence stiffness-dependent EC behavior correlated with EC growth. After the formation of an EC monolayer, EC behaviors correlated with endothelial function were evaluated by characterizing monolayer integrity, nitric oxide production, and gene expression of endothelial nitric oxide synthase. For the first time, we demonstrated that endothelial function displayed a negative correlation with substrate stiffness. Although HGF improved endothelial function, HGF was not able to change the stiffness-dependent manner of endothelial functions. Taken together, this study provides insights into the synergetic influence of physical and biochemical cues on EC behavior and offers great potential in the development of optimized biomaterials for EC-based regenerative medicine.

Bacteriogenic synthesis of selenium nanoparticles by Escherichia coli ATCC 35218 and its structural characterisation.

A biosynthetic method for the production of selenium nanoparticles under ambient temperature and pressure from sodium selenite was developed using Gram-negative bacterial strain Escherichia coli ATCC 35218. Bacteriogenic nanoparticles were methodologically characterized employing UV-vis, XRD, Raman spectroscopy, SEM, TEM, DLS and FTIR techniques. Generation of nanoparticles was visualized from the appearance of red colour in the selenite supplemented culture medium and broad absorption bands in the UV-vis. Biofabricated nanoparticles were spherical, polydisperse, ranged from 100-183 nm and the average particle size was about 155 nm. Based on selected-area electron diffraction, XRD patterns; and Raman spectroscopy the nanospheres were found to be amorphous. IR spectrum revealed the involvement of bacterial proteins in the reduction of selenite and stabilization of nanoparticles. Used bacterial strain demonstrated efficient selenite reduction capability which was evident from 89.2% of selenium removal within 72 h at a concentration of 1 mM. Observation noted in the current study highlight the importance of bacterial reduction in selenium nanoparticle generation which can be scaled up for commercial production. Also, the bacteriogenic, amorphous nanoparticles can also be used as nutritional supplements for humans since selenium nanoparticles of 5-200 nm are bioavailable and known to induce seleno enzymes involved in antioxidant defence.

Impact of various media and organic carbon sources on biofuel production potential from Chlorella spp.

In this study, five Chlorella species (Chlorella vulgaris, Chlorella minutissima, Chlorella pyrenoidosa, Chlorella sp.1 and Chlorella sp.2) were grown in various nutrient medium including BG-11, BBM, Fog’s medium and M4N medium for the evolution of biomass and lipid production potential. Among the tested medium, BG-11 was found most economical and efficient medium for all Chlorella species. To see the impact of organic carbon sources on lipid production potential, all microalgae species were also cultured in selected medium (BG-11) with different organic carbon sources like glucose, glycerol, sodium acetate, and sucrose under mixotrophic condition. The results showed that all Chlorella species performs better under mixotrophic condition, but Chlorella vulgaris achieved maximum lipid productivity (3.5 folds higher) in glycerol supplemented culture medium than control medium among all species.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0434-6) contains supplementary material, which is available to authorized users.

Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II).

BackgroundThe ability to site-specifically conjugate a protein to a payload of interest (e.g., a fluorophore, small molecule pharmacophore, oligonucleotide, or other protein) has found widespread application in basic research and drug development. For example, antibody-drug conjugates represent a class of biotherapeutics that couple the targeting specificity of an antibody with the chemotherapeutic potency of a small molecule drug. While first generation antibody-drug conjugates (ADCs) used random conjugation approaches, next-generation ADCs are employing site-specific conjugation. A facile way to generate site-specific protein conjugates is via the aldehyde tag technology, where a five amino acid consensus sequence (CXPXR) is genetically encoded into the protein of interest at the desired location. During protein expression, the Cys residue within this consensus sequence can be recognized by ectopically-expressed formylglycine generating enzyme (FGE), which converts the Cys to a formylglycine (fGly) residue. The latter bears an aldehyde functional group that serves as a chemical handle for subsequent conjugation.ResultsThe yield of Cys conversion to fGly during protein production can be variable and is highly dependent on culture conditions. We set out to achieve consistently high yields by modulating culture conditions to maximize FGE activity within the cell. We recently showed that FGE is a copper-dependent oxidase that binds copper in a stoichiometric fashion and uses it to activate oxygen, driving enzymatic turnover. Building upon that work, here we show that by supplementing cell culture media with copper we can routinely reach high yields of highly converted protein. We demonstrate that cells incorporate copper from the media into FGE, which results in increased specific activity of the enzyme. The amount of copper required is compatible with large scale cell culture, as demonstrated in fed-batch cell cultures with antibody titers of 5 g · L−1, specific cellular production rates of 75 pg · cell−1 · d−1, and fGly conversion yields of 95–98 %.ConclusionsWe describe a process with a high yield of site-specific formylglycine (fGly) generation during monoclonal antibody production in CHO cells. The conversion of Cys to fGly depends upon the activity of FGE, which can be ensured by supplementing the culture media with 50 uM copper(II) sulfate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0254-0) contains supplementary material, which is available to authorized users.

The long-term survival of Propionibacterium freudenreichii in a context of nutrient shortage.

Propionibacterium freudenreichii is an actinobacterium widely used in dairy industry during the ripening process of Swiss-type cheeses and which presents probiotic properties. P. freudenreichii is reportedly a hardy bacterium, able to survive during the cheese-making process and when subjected to digestive stresses. During this study the long-term survival (LTS) of P. freudenreichii was investigated for 11 days by means of phenotypic characterization in a culture medium without the addition of any nutrients. For 11 days, in a non-nutrient supplemented culture medium, eight strains were monitored by measuring their optical density, counting colony-forming units (CFU) and using LIVE/DEAD staining and microscopy observation. Under these conditions, all strains displayed high survival rates in the culture medium, their culturability reaching more than 9 log10 CFU ml(-1) after 2 days. After 11 days, this value ranged from 7·8 to 8·2 log10 CFU ml(-1) depending on the strain, and at least 50% of the P. freudenreichii population displayed an intact envelope. As lysis of part of a bacterial population may be a microbial strategy to recover nutrients, in CIRM-BIA 138 (the strain with the highest population at day 11), cell lysis was assessed by quantifying intact bacterial cells using qPCR targeting the housekeeping gene tuf. No lysis was observed. Taken together, our results suggest that P. freudenreichii strains use a viable but nonculturable state to adapt to the LTS phase. Assessing the viability of P. freudenreichii and understanding their mechanisms for survival should be of great interest regarding their potential probiotic applications.

Comparative Analysis of KnockOut™ Serum with Fetal Bovine Serum for the In Vitro Long-Term Culture of Human Limbal Epithelial Cells.

The limbal epithelial cells can be maintained on 3T3 feeder layer with fetal bovine serum supplemented culture medium, and these cells have been used to successfully treat limbal stem cell deficiency. However, fetal bovine serum contains unknown components and displays quantitative and qualitative lot-to-lot variations. To improve the culture condition, the defined KnockOut serum replacement was investigated to replace fetal bovine serum for culturing human limbal epithelial cell. Human primary limbal epithelial cells were cultured in KnockOut serum and fetal bovine serum supplemented medium, respectively. The cell growth rate, gene expression, and maintenance of limbal epithelial stem cells were studied and compared between these two groups. Human primary limbal epithelial cells were isolated and successfully serially cultivated in this novel KnockOut serum supplemented medium; the cell proliferation and stem cell maintenance were similar to those of cells grown in fetal bovine serum supplemented medium. These data suggests that this KnockOut serum supplemented medium is an efficient replacement to traditional fetal bovine serum supplemented medium for limbal epithelial cell culture, and this medium has great potential for long term maintenance of limbal epithelial cells, limbal epithelial stem cells transplantation, and tissue regeneration.

The role of Coenzyme Q10on the Total Antioxidant Capacity of Mouse Vitrified Pre-Antral Follicles

Occurrenceofoxidative stress (OS) following in vitro culture is inescapable. Therefore, the aim of the present study was to determine the efficacy of Coenzyme Q 10 (CoQ 10 ) supplementation medium on developments and Total Antioxidant Capacity (TAC) of mouse vitrified pre-antral follicles. Fresh and vitrified-warmed isolated preantral follicles from ovaries of 14-16 day s -old mice were cultured in supplemented α- MEM medium with or without CoQ 10 .On the twelfth day of culture period,ovulation was induced by adding 1.5 IU/ml human Chorionic Gonadotropin (hCG). Rates of survival, antrum formation and developmental stages of released oocytes (GV, MI, MII) were evaluated. Separately,total antioxidant capacity (TAC) levels were measured at initial time, 24, 48, 72 and 96 hours of culture periodby ferric reducing/antioxidant power (FRAP) assay. The results showed that the mean diameter and rates of survival, antrum formation, ovulation and MII oocytes of fresh and vitrified-warmed isolated preantral follicles with pretreatment of CoQ 10 were significantly higher than those of respective groups without pretreatment ofCoQ 10 . TAC levels significantly increased up to 96hours in fresh and vitrified-warmed preantral follicles with pre-treatment of CoQ 10 compared with thosewhich were cultured without CoQ 10 . Supplemented culture medium with CoQ 10 promotes TAC levels and development of both fresh and vitrified-warmed isolated preantral follicles.

Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation.

Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a “ribozyme”) and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed “retrohoming”. Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms.

Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

IntroductionEfficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs.ResultsThe findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential.ConclusionThe presented isolation method and subsequent cell expansion in platelet lysate supplemented culture medium permits suitable large-scale propagation of PB-ECFC. For optimal use of PB-ECFCs in clinical settings, our data suggest that 15–20 CPDL is the most adequate maturation stage.

Characterization and review of MTHFD1 deficiency: four new patients, cellular delineation and response to folic and folinic acid treatment

In the folate cycle MTHFD1, encoded by MTHFD1, is a trifunctional enzyme containing 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase activity. To date, only one patient with MTHFD1 deficiency, presenting with hyperhomocysteinemia, megaloblastic anaemia, hemolytic uremic syndrome (HUS) and severe combined immunodeficiency, has been identified (Watkins et al J Med Genet 48:590-2, 2011). We now describe four additional patients from two different families. The second patient presented with hyperhomocysteinemia, megaloblastic anaemia, HUS, microangiopathy and retinopathy; all except the retinopathy resolved after treatment with hydroxocobalamin, betaine and folinic acid. The third patient developed megaloblastic anaemia, infection, autoimmune disease and moderate liver fibrosis but not hyperhomocysteinemia, and was successfully treated with a regime that included and was eventually reduced to folic acid. The other two, elder siblings of the third patient, died at 9weeks of age with megaloblastic anaemia, infection and severe acidosis and had MTFHD1 deficiency diagnosed retrospectively. We identified a missense mutation (c.806C > T, p.Thr296Ile) and a splice site mutation (c.1674G > A) leading to exon skipping in the second patient, while the other three harboured a missense mutation (c.146C > T, p.Ser49Phe) and a premature stop mutation (c.673G > T, p.Glu225*), all of which were novel. Patient fibroblast studies revealed severely reduced methionine formation from [(14)C]-formate, which did not increase in cobalamin supplemented culture medium but was responsive to folic and folinic acid. These additional cases increase the clinical spectrum of this intriguing defect, provide in vitro evidence of disturbed methionine synthesis and substantiate the effectiveness of folic or folinic acid treatment.

The effect of ascorbic acid and fluid flow stimulation on the mechanical properties of a tissue engineered pelvic floor repair material.

Synthetic non-degradable meshes used in pelvic floor surgery can cause serious complications such as tissue erosion. A repair material composed of an autologous oral fibroblast seeded degradable polylactic acid scaffold may be a viable alternative. The aims of this study were to investigate the effects of media supplementation with additives (ascorbic acid-2-phosphate, glycolic acid and 17-β-oestradiol) on the mechanical properties of these scaffolds. Oral fibroblasts were isolated from buccal mucosa. The effects of the three additives were initially compared in two-dimensional culture to select the most promising collagen stimulating additive. Sterile electrospun scaffolds were seeded with 500,000 oral fibroblasts and fixed in 6-well plates and subjected to ascorbic acid-2-phosphate (the best performing additive) and/or mechanical stimulation. Mechanical stimulation by fluid shear stress was induced by rocking scaffolds on a platform shaker for 1 h/day for 10 of 14 days of culture. In two-dimensional culture, ascorbic acid-2-phosphate (concentrations from 0.02 mM to 0.04 M) and glycolic acid (10 µM) led to significantly greater total collagen production, but ascorbic acid-2-phosphate at 0.03 mM produced the greatest stimulation (of the order of >100%). In three-dimensional culture, mechanical stimulation alone gave non-significant increases in stiffness and strength. Ascorbic acid-2-phosphate (0.03 mM) significantly increased collagen production in the order 280% in both static and mechanically stimulated scaffolds (p < 0.0001). There was no additional effect of mechanical stimulation. Dense collagen I fibres were observed with ascorbic acid-2-phosphate supplementation. Uniaxial tensiometry showed that strength (p < 0.01) and stiffness (p <0.05) both improved significantly. A combination of ascorbic acid-2-phosphate and mechanical stimulation led to further non-signficant increases in strength and stiffness. In conclusion, a pelvic floor repair material with improved mechanical properties can be developed by supplementing culture media with ascorbic acid-2-phosphate to increase collagen I production. Future studies will assess the change in mechanical properties after implantation in an animal model.