Liquid Suspension Culture Research Articles

Cassava for thefuture: embryogenic liquid cultures suitable for new biotech techniques.

Cassava, a crop of importance for subsistence farming in Africa, Asia, and Latin America, has the potential to benefit from global economic integration as a versatile industrial resource. Enhancing cassava productivity is not just a matter of agricultural competitiveness but a crucial step toward ensuring many communities' food security and livelihoods. Given its high performance in marginal environments, where climate change poses threats, ensuring food security and livelihoods relies on rapidly adapting cassava. This study aimed to develop a protocol that swiftly transitions cassava embryogenic short-period liquid suspension cultures, facilitating the regeneration of genetically stable in vitro plants. The resulting protocol, with its potential to be a foundational component in future technologies employing various genome editing or genetic modification techniques, holds promise for the advancement of cassava biotechnology.

Production and characterization of basic fibroblast growth factor protein in rice suspension cultures

The basic fibroblast growth factor (bFGF) plays a major role in cell growth, survival, and other mitogenic activities. General applications of this protein involve tissue repair, morphogenesis, embryonic development, cellular growth, etc. Due to its importance and high demand, research has been done on producing recombinant bFGF in different expression platforms. We have produced bFGF in transgenic rice (Oryza sativa) cells growing in liquid suspension culture. The gene construct consists of the rice alpha-amylase (RAmy) 3D sugar-inducible promotor and rice glutelin secretion signal peptide for controlled production and release of the bFGF protein by manipulating the sugar concentration for batch or semi-continuous bioreactor runs. A thermal shift assay was performed on the purified rice cell produced bFGF (rbFGF) and the results were compared with E. coli derived bFGF. The purified rbFGF protein from rice cells was tested for propagating human-induced pluripotent stem cells (hiPSCs) up to eight passages. Furthermore, the biological activity measured in both crude lysate and purified rbFGF showed a similar pattern of the extracellular signal-regulated kinase (ERK) activity compared to E. coli derived bFGF (control), indicating that rbFGF is active before purification and the protein activity is not inhibited by other components of the lysate. From the current results, the rice-produced rbFGF protein is biologically active and could serve as a viable product for various applications.

Copper exposure effects on antibiotic degradation in swine manure vary between mesophilic and thermophilic conditions

Antibiotic and heavy metal commonly coexist in manure. This study investigated the effect of Cu exposure on antibiotic dissipation in swine manure under two typical temperature (mesophilic and thermophilic) conditions in composting, focusing on biodegradation behaviors. The results showed that Cu promoted the dissipation of norfloxacin and sulfamethazine (SMZ) in solid swine manure under mesophilic conditions at initial concentrations ranging from 407.8 to 1353.0 mg·kg−1 but insignificantly influenced or even inhibited their dissipation under thermophilic conditions. A liquid manure suspension culture experiment was designed to elucidate the response of SMZ biodegradation to Cu. In this manure suspension, biodegradation was the major mechanism for SMZ removal, but SMZ biodegradation was decreased from 23.2 % to 5.5 % when the Cu concentration increased from 0 to 10 mg L−1. Mesophilic and heat-resistant SMZ-degrading bacterial inoculants were subsequently prepared using 21 SMZ-degrading bacteria that were isolated and identified from manure suspension cultures. Inoculating both mesophilic and heat-resistant SMZ-degrading bacterial inoculants enhanced SMZ degradation in sterilized manure suspensions without Cu addition, however only mesophilic SMZ-degrading inoculum improved SMZ degradation after Cu addition. In the presence of Cu, the heat-resistant SMZ-degrading inoculum failed to enhance SMZ removal in manure suspensions. Our findings can help to answer why Cu has varied effects on antibiotic degradation during manure composting.

Suspension Culture and Somatic Embryogenesis of Korean Pine

Korean pine is an important afforestation tree species in Northeast China, which has a high ecological and economic value. Although regeneration of somatic embryogenesis using immature zygotic embryos of Korean pine as explants has been successful, it cannot be applied to automation and large-scale production. Therefore, we urgently need a method that can increase the output of somatic embryos (SEs) to meet the needs of large-scale production. We used Korean pine 1–1 and 1–100 cell lines as research materials to evaluate the effects of inoculum-density, culture time, orbiting speed, vessel volume, plant growth regulator (PGR) concentration, and carbon source on the proliferation of embryogenic tissue (ET). The somatic embryogenesis ability of ET cultured in different liquid suspension media was also evaluated. We found that during liquid suspension culture of Korean pine ET, the sedimented cell volume (SCV), fresh weight (FW) and dry weight (DW) were affected by inoculum-density, culture time, orbiting speed, 2,4-D concentration, 6-BA concentration and carbon source type. Fourty mg ⋅ mL−1 ET were transferred to a 200 mL Erlenmeyer flask containing 20 mL liquid medium, and cultured at 100 rpm/min for 14 days to obtain the maximum proliferation. In addition, we also found that SCV, FW and DW were higher when PGRs were reduced in the liquid suspension medium. The substitution of maltose for sucrose resulted in slow growth of cultures and limited SE yield (13 SEs g−1 FW). Although culture proliferation was high at 50 rpm, SE yield was inhibited by 48% compared with 100 rpm (50 rpm = 33 SEs g−1 FW; 100 rpm/min = 70 SEs g−1 FW). Cultivation in low-concentration PGR(1.15 μM ⋅ L−1 2,4-D, 0.25 μM ⋅ L−1 6-BA) and sucrose liquid medium at 100 rpm/min (80 SEs g−1 FW) could not only promote culture proliferation but also increase SE yield. The determination of the suspension culture scheme of Korean pine ET provides a reference for further expansion to bioreactor culture in the future and lays a foundation for the automation and scale of somatic embryogenesis of Korean pine.

Culture types and period impact gametophyte morphogenesis and sporophyte formation of eastern bracken

BackgroundLiquid suspension culture efficiently proliferates plant cells and can be applied to ferns because it rapidly increases the fresh weight of gametophytes. This study assessed gametophyte proliferation and sporophyte production of Pteridium aquilinum var. latiusculum using a suspension culture method.ResultsThe growth curve linear phase of gametophyte cells was confirmed between 9 and 18 days of culture, and the subculture cycle was determined to be 2 weeks. A double-strength MS medium (fresh weight, 18.0 g) containing 2% sucrose and NH4+:NO3− (120 mM, 40:80) was found to be the optimal liquid medium. Gametophytes obtained after suspension culture for 18 days did not normally form sporophytes in an ex vitro soil environment. However, this issue was resolved after changing the culture type or extending the culture period to 6 weeks. A short suspension culture period increased the fresh weight of fragmented and homogenized gametophytes but yielded numerous relatively immature gametophytes (globular forms of branching gametophytes, BG). Furthermore, differences in gametophyte morphogenesis and development were indicated by changes in endogenous phytohormone content. BG with immature development exhibited high accumulation of zeatin, jasmonic acid, and salicylic acid, and relatively low levels of abscisic acid and indole-3-acetic acid. The immature development of gametophytes directly affected sporophyte formation.ConclusionsThis study maximized the advantages of liquid suspension culture using eastern bracken gametophytes and provides data to resolve any associated issues, thus facilitating efficient bracken production.

Investigations of solid culture–induced acquisition of desiccation tolerance in liquid suspension culture of Nostoc flagelliforme

Investigations of solid culture–induced acquisition of desiccation tolerance in liquid suspension culture of Nostoc flagelliforme

Food for Thought: The Protein Transformation

Food for Thought: The Protein Transformation

Optimization of culture conditions for in vitro adventitious roots and selected flavonoids production in Boesenbergia rotunda liquid suspension culture

Boesenbergia rotunda (L.) Mansf. is one of the unique monocotyledonous perennial plant species belonging to the ginger (Zingiberaceae) family. Locally known as ‘Temu Kunci’ in Malaysia and Indonesia, this medicinal plant has been widely used in Asian dishes, particularly as a condiment or as traditional natural medicines. The important medicinal properties of B. rotunda majorly derived from flavonoids which are highly sought as pharmaceuticals. In this study, culture conditions for the growth of adventitious roots in liquid suspension cultures were optimized. The highest adventitious root production was achieved when cultured with initial inoculum density of 1.5 g and pH value at 5.8 after five weeks of culture. HPLC analysis discovered that production of valuable flavonoid compounds (pinostrobin, cardamonin and panduratin A) was significantly higher when the adventitious roots were cultured with initial inoculum density of 1.5 g whereas the initial pH medium did not significantly affect flavonoid production.

Tailored Cytokine Optimization for ex vivo Culture Platforms Targeting the Expansion of Human Hematopoietic Stem/Progenitor Cells.

Umbilical cord blood (UCB) has been established as an alternative source for hematopoietic stem/progenitor cells (HSPC) for cell and gene therapies. Limited cell yields of UCB units have been tackled with the development of cytokine-based ex vivo expansion platforms. To improve the effectiveness of these platforms, namely targeting clinical approval, in this study, we optimized the cytokine cocktails in two clinically relevant expansion platforms for HSPC, a liquid suspension culture system (CS_HSPC) and a co-culture system with bone marrow derived mesenchymal stromal cells (BM MSC) (CS_HSPC/MSC). Using a methodology based on experimental design, three different cytokines [stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin (TPO)] were studied in both systems during a 7-day culture under serum-free conditions. Proliferation and colony-forming unit assays, as well as immunophenotypic analysis were performed. Five experimental outputs [fold increase (FI) of total nucleated cells (FI TNC), FI of CD34+ cells, FI of erythroid burst-forming unit (BFU-E), FI of colony-forming unit granulocyte-monocyte (CFU-GM), and FI of multilineage colony-forming unit (CFU-Mix)] were followed as target outputs of the optimization model. The novel optimized cocktails determined herein comprised concentrations of 64, 61, and 80 ng/mL (CS_HSPC) and 90, 82, and 77 ng/mL (CS_HSPC/MSC) for SCF, Flt-3L, and TPO, respectively. After cytokine optimization, CS_HSPC and CS_HSPC/MSC were directly compared as platforms. CS_HSPC/MSC outperformed the feeder-free system in 6 of 8 tested experimental measures, displaying superior capability toward increasing the number of hematopoietic cells while maintaining the expression of HSPC markers (i.e., CD34+ and CD34+CD90+) and multilineage differentiation potential. A tailored approach toward optimization has made it possible to individually maximize cytokine contribution in both studied platforms. Consequently, cocktail optimization has successfully led to an increase in the expansion platform performance, while allowing a rational side-by-side comparison among different platforms and enhancing our knowledge on the impact of cytokine supplementation on the HSPC expansion process.

In vitro propagation of bulblets and LC\u2013MS/MS analysis of isosteroidal alkaloids in tissue culture derived materials of Chinese medicinal herb Fritillaria cirrhosa D. Don

BackgroundFritillaria cirrhosa, an important Chinese medicinal herb, is a Class-III protected and highly exploited species by pharmaceutical industry. Dwindling wild populations of species are unable to meet market demand. Therefore, this study was carried out to develop an in vitro propagation method for bulblet production. Also, the study aimed to carry out LC–MS/MS analysis of tissue culture-derived bulblets and callus for the presence of isosteroidal alkaloids (peimissine, verticine, and verticinone), and compare its quantities with commercially available crude drug samples.ResultsIn vitro seed germination (91%) of F. cirrhosa was achieved on Murashige and Skoog’s basal medium (MSBM) supplemented with 6-benzylaminopurine (1 mg L−1) and α-naphthalene-acetic-acid (0.4 mg L−1). On transfer of germinated seeds from Petri-dishes to glass bottles containing hormone-free MSBM, 37.5% of seedlings developed bulblets after 3 months of incubation. Regeneration and multiplication of bulblets were achieved by culture of transverse sections of bulblets on 1/2 X MSBM. By repeated subcultures at an interval of 2 months, 3072 bulblets weighing 1270 g could be produced at the end of 5th subculture. LC–MS/MS analysis showed a significant presence of peimissine in in vitro bulblets while callus incubated in the dark showed presence of peimissine and verticine.ConclusionThe study reports an efficient in vitro propagation method of bulblets production of F. cirrhosa and presence of some isosteroidal alkaloids in tissue culture-derived bulblets and callus. The study could be of immense help in production of F. cirrhosa bulblets and callus under laboratory conditions round the year. Also, these results can be used further to investigate production of isosteroidal alkaloids in bioreactors at commercial scale using liquid and cell suspension cultures. Thus, we not only can reduce our dependence on collections from natural habitats, but also can help in in situ conservation of this important species.

Preservation of cyanobacteria Glaucospira laxissima at low temperature

This short paper addresses viability and renewed growth of cyanobacteria Glaucospira laxissima after 1-year preservation under -80°C in a deep freezer. To storage in liquid suspension cultures with min 40% viability were used 10%-glucose and DMSO solution.

In situ and non-destructive detection of the lipid concentration of Scenedesmus obliquus using hyperspectral imaging technique

Microalgae can be used as a raw material for biodiesel production. Monitoring the lipid accumulation process of microalgae is crucial for optimizing the microalgae culture environment and the breeding of microalgae species with a high yield of lipid. Current methods for the detection of the lipid accumulation of microalgae are tedious and require trained professionals. Thus, a fast visual and intuitive approach for the detection of the lipid concentration of microalgae with a living status is desirable for microalgae culture and bioprocess control. In this study, a fast and nondestructive method based on hyperspectral imaging (HSI) was proposed to obtain the concentration and distribution of microalgae lipids from liquid suspension culture. Several data preprocessing, regression and model development methods were tested. Savitzky-Golay data smoothing followed by a competitive adaptive reweighted sampling in a multiple linear regression model performed the best. Finally, a chemical map was provided to visualize the lipid distribution of microalgae in a suspension. The distribution map of microalgae lipids showed a dynamic process of lipid accumulation, which could be useful for the investigation of the mechanism of lipid accumulation in microalgae. Hyperspectral imaging is a promising approach to inspect the lipid concentration of microalgae in an intuitive way, which can be widely used for the screening of strains and monitoring the lipid production in microalgae industries.

Generation and Molecular Characterization of Human Ring Sideroblasts

(Background) Sideroblastic anemias are heterogeneous congenital and acquired refractory anemias characterized by bone marrow ring sideroblasts, reflecting excess mitochondrial iron deposition. While the disease is commonly associated with myelodysplastic syndrome, the congenital forms of sideroblastic anemias comprise a diverse class of syndromic and non-syndromic disorders, which are caused by the germline mutation of genes involved in iron-heme metabolism in erythroid cells. Although the only consistent feature of sideroblastic anemia is the bone marrow ring sideroblasts, evidence on the detailed molecular characteristics of ring sideroblasts is scarce owing to a lack of the biological models. We have recently established ring sideroblasts by inducing ALAS2 gene mutation based on human-induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells (ASH 2017) and have further extended the molecular characterization of human ring sideroblasts to gain new biological insights.(Method) We targeted the GATA-1-binding region of intron 1 of the human ALAS2 gene in HiDEP cells and established two independent clones [X-linked sideroblastic anemia (XLSA) clones]. A co-culture with OP9 stromal cells (ATCC) was conducted with IMDM medium supplemented with FBS, erythropoietin, dexamethasone, MTG, insulin-transferrin-selenium, and ascorbic acid. To obtain human primary erythroblasts, CD34-positive cells isolated from cord blood were induced in a liquid suspension culture (Fujiwara et al. JBC 2014). Bone marrow glycophorin A (GPA)-positive erythroblasts of patients with XLSA and normal individuals were separated using the MACS system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) after obtaining written informed consent. For transcription profiling, Human Oligo chip 25K (Toray) was used.(Results) We previously demonstrated that co-culture with OP9 cells in the medium supplemented with 100 uM sodium ferrous citrate (SFC) promoted erythroid differentiation of XLSA clones, which enabled the establishment of ring sideroblasts (ASH 2017). To confirm the importance of SFC in terminal erythroid differentiation, we further demonstrated that the addition of SFC, and not transferrin-loaded iron, induced the frequency of GPA+ cells and TfR1-GPA+ mature erythroid population, based on primary erythroblasts derived from human CD34-positive cells. Subsequently, to reveal the molecular mechanism by which abnormal iron mitochondrial iron accumulation occurs by co-culture with SFC, we evaluated the expressions of various metal transporters, demonstrating that the addition of SFC significantly increased the expressions of mitoferrin 1 (MFRN1; a ferrous iron transporter in mitochondria), divalent metal transporter 1 (DMT1), and Zrt- and Irt-like protein 8 (ZIP8; a transmembrane zinc transporter, recently known as a ferrous iron transporter) in the XLSA clone than the wild-type cells, which would have contributed to the formation of ring sideroblasts.Moreover, we performed expression analyses to elucidate the biochemical characteristics of ring sideroblasts. After co-culture with OP9 in the presence of SFC, ring sideroblasts exhibited more than two-fold upregulation and downregulation of 287 and 143 genes, respectively, than the wild-type cells. Interestingly, when compared with the expression profiling results before co-culture (ASH 2017), we noticed prominent upregulation of gene involved in anti-apoptotic process (p = 0.000772), including HSPA1A, superoxide dismutase (SOD) 1, and SOD2. In addition, we conducted a microarray analysis based on GPA-positive erythroblasts from an XLSA patient and a normal individual. The analysis revealed significant upregulation of genes involved in the apoptosis process, as represented by apoptosis enhancing nuclease, DEAD-box helicase 47, and growth arrest and DNA-damage-inducible 45 alpha, and anti-apoptotic genes, such as HSPA1A and SOD2. Concomitantly, when the XLSA clone was co-cultured with OP9 in the presence of SFC, the apoptotic cell frequency as well as DNA fragmentation were significantly reduced compared with the XLSA clone co-cultured without SFC, indicating that ring sideroblasts avoid cell death by inducing anti-apoptotic properties.(Conclusion) Further characterization of the XLSA model would help clarify its molecular etiology as well as establish novel therapeutic strategies. DisclosuresFukuhara:Celgene: Research Funding; Chugai: Research Funding; Daiichi-Sankyo: Research Funding; Boehringer Ingelheim: Research Funding; Eisai: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Janssen: Honoraria, Research Funding; Japan Blood Products Organization: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Mitsubishi Tanabe: Research Funding; Mundipharma: Honoraria, Research Funding; MSD: Research Funding; Nippon-shinyaku: Research Funding; Novartis pharma: Research Funding; Ono: Honoraria, Research Funding; Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; Sanofi: Research Funding; Symbio: Research Funding; Solasia: Research Funding; Sumitomo Dainippon: Research Funding; Taiho: Research Funding; Teijin Pharma: Research Funding; Zenyaku Kogyo: Honoraria, Research Funding; Takeda: Honoraria; Baxalta: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Bayer Yakuhin: Research Funding; Alexionpharma: Research Funding; AbbVie: Research Funding; Astellas: Research Funding; Nihon Ultmarc: Research Funding.

Label-free, simultaneous quantification of starch, protein and triacylglycerol in single microalgal cells

BackgroundCurrent approaches for quantification of major energy-storage forms in microalgae, including starch, protein and lipids, generally require cell cultivation to collect biomass followed by tedious and time-consuming analytical procedures. Thus, label-free, non-destructive and simultaneous quantification of such macromolecules at single-cell resolution is highly desirable in microalgal feedstock development and bioprocess control.ResultsHere, we established a method based on single-cell Raman spectra (SCRS) that simultaneously quantifies the contents of starch, protein, triacylglycerol (TAG) and lipid unsaturation degree in individual Chlamydomonas reinhardtii cells. Measurement accuracy for the contents based on full SCRS spectrum each reached 96.86–99.24%, all significantly higher than single peak-based models. However, accuracy and reliability of measurement are dependent on the number of cells sampled, thus a formal mathematical framework was proposed and validated to rationally define “minimal sampling depth” for a given state of cellular population. Furthermore, a barcode consisting of 13 marker Raman peaks was proposed to characterize the temporal dynamics of these energy-storage products, which revealed that the average contents of starch and TAG increased, while their heterogeneity indices decreased, with those of protein being exactly the opposite. Finally, our method is widely applicable, as measurements among cells from liquid suspension culture, wet paste and frozen dried powder all exhibited excellent consistency.ConclusionsWhen sampled at proper depth, SCRS can serve as a quantitative and generally applicable tool for characterization and screening of strains and bioprocesses based on the profile of energy-storage macromolecules and their among-cell heterogeneity.

Translation complex profile sequencing to study the in vivo dynamics of mRNA-ribosome interactions during translation initiation, elongation and termination.

Messenger RNA (mRNA) translation is a tightly controlled process that is integral to gene expression. It features intricate and dynamic interactions of the small and large subunits of the ribosome with mRNAs, aided by multiple auxiliary factors during distinct initiation, elongation and termination phases. The recently developed ribosome profiling method can generate transcriptome-wide surveys of translation and its regulation. Ribosome profiling records the footprints of fully assembled ribosomes along mRNAs and thus primarily interrogates the elongation phase of translation. Importantly, it does not monitor multiple substeps of initiation and termination that involve complexes between the small ribosomal subunit (SSU) and mRNA. Here we describe a related method, termed 'translation complex profile sequencing' (TCP-seq), that is uniquely capable of recording positions of any type of ribosome-mRNA complex transcriptome-wide. It uses fast covalent fixation of translation complexes in live cells, followed by RNase footprinting of translation intermediates and their separation into complexes involving either the full ribosome or the SSU. The footprints derived from each type of complex are then deep-sequenced separately, generating native distribution profiles during the elongation, as well as the initiation and termination stages of translation. We provide the full TCP-seq protocol for Saccharomyces cerevisiae liquid suspension culture, including a data analysis pipeline. The protocol takes ∼3 weeks to complete by a researcher who is well acquainted with standard molecular biology techniques and who has some experience in ultracentrifugation and the preparation of RNA sequencing (RNA-seq) libraries. Basic Bash and UNIX/Linux command skills are required to use the bioinformatics tools provided.

Parthenolide and DMAPT Induce Cell Death in Chronic Myeloid Leukemia Cells Via Generation of Reactive Oxygen Species

Chronic Myeloid Leukemia (CML) is a malignant hematologic disease characterized by the presence of a chromosomal alteration known as the Philadelphia Chromosome (Ph). Ph originates in Leukemia Stem Cells (LSC) and is caused by the reciprocal translocation between chromosome 9 and 22, giving rise to the Bcr-Abl oncogene, which produces a constitutively active tyrosine kinase -the BCR-ABL oncoprotein. This CML feature has made it possible the development of Tyrosine Kinase Inhibitors (TKI's) Imatinib, Dasatinib, and Nilotinib, which are able to inhibit BCR-ABL and take patients into molecular remission. However TKI's are not able to eliminate the population of LSC, which cause patients to relapse if treatment is discontinued. This has made it necessary search for alternative ways to eliminate LSC in CML patients.Parthenolide (PTL), a sesquiterpene lactone extracted from the feverfew plant (Thanacethum partenium), which has been used in folk medicine to treat inflammatory symptoms, has shown activity against LSC in Acute Myeloid Leukemia (AML). Recently Guzman and collaborators have developed an analog of PTL, named DMAPT, which is orally bioavailable. Both molecules have shown to be active inhibitors of NFkB and induce high levels of Reactive Oxygen Species (ROS) in LSC of AML. In the present study we asked whether CML cells (LSC, Progenitors and CML cell lines) are sensitive to PTL and DMAPT.Cell lines K562, Kcl-22 and HL60 (as an AML control) were cultured in liquid suspension cultures with increasing concentrations of PTL or DMAPT at a cell density of 300,000 cells per ml. At 24 hours of exposure, PTL and DMAPT showed significant induction of apoptosis at 7.5 µM and 10 µM, respectively. To determine whether NFkB activity is inhibited by PTL and DMAPT, K562, Kcl-22, HL60 cell lines, as well as CD34+ cells from a CML patient in Chronic phase where exposed to 7.5 µM PTL or 10 µM DMAPT. After 6 hours, protein lysis was induced and Western blots were performed with protein extracts from 250,000 cells per lane. Membranes where probed for Phosphorilated-p65, total p65 and b-actin as a loading control. We observed a decrease in the levels of phosphorylated p65 in cells treated with PTL and DMAPT, compared with untreated cells, while levels of total p65 remained constant.We had previosly used DCFDA stain and HMOX expression, to observe ROS induction in CML cells exposed to PTL. To asses if ROS induction is necessary for CML cell death we pretreated cells with N-acetyl cysteine (NAC), precursor of glutathione, for 1 hour before treating cells with 7.5 µM PTL or 10 µM DMAPT, and evaluated ROS levels by staining with Cell rox deep red, Mitosox-PE, and also assessing cell viability with YoPro-1 and 7AAD stain. After 6 hours of PTL treatment, we found that NAC pretreatment diminished ROS levels in cytoplasm and mitochondrial dysfunction when cells were treated with PTL. Pretreatment with NAC was able to rescue the majority of cells from apoptosis induced by PTL. Similar results were observed in cultures treated with DMAPT.Taken together, our results indicate that PTL and DMAPT were able to induce cell death via apoptosis in CML cells. This process seems to involve inhibition of p65 phosphorylation and an increase in ROS levels, which we found to be necessary for cell death to occur. DisclosuresNo relevant conflicts of interest to declare.

Somatic Embryogenesis in Lisianthus (Eustoma russellianum Griseb.).

Somatic embryogenesis is, for the main floricultural crops, a promising system for commercial scale-up, providing cloned material to be traded as seedlings. Somatic embryos, having the contemporary presence of root apical meristem and shoot apical meristem, can be readily acclimatized. For Lisianthus it is possible to induce embryogenic callus from leaf fragments of selected genotypes and to obtain embryos either in agarized substrate or in liquid suspension culture. The production of somatic embryos in liquid medium is high and can be modulated in order to synchronize the cycle and the size of the neoformed structures. The possibility to use the liquid substrate with high propagation rates reduces labor costs and could support the costs of eventual automation. In this paper we report a stepwise protocol for somatic embryogenesis in the species Eustoma russellianum.

An oil-soluble extract of Rubus idaeus cells enhances hydration and water homeostasis in skin cells.

Raspberry plants, belonging to the species of Rubus idaeus, are known for their excellent therapeutic properties as they are particularly rich in compounds with strong antioxidant activity, which promote health and well-being of human cells. Besides their high content of phenolic compounds, Rubus plants are rich in oil-soluble compounds, which are also primary components of the hydrolipidic film barrier of the skin. As plant cell cultures represented a valuable system to produce interesting compounds and ingredients for cosmetic applications, we developed liquid suspension cultures from Rubus idaeus leaves and used them to obtain an active ingredient aimed at improving hydration and moisturization capacity in the skin. Rubus idaeus cells, grown in the laboratory under sterile and controlled conditions as liquid suspension cultures, were processed to obtain an oil-soluble (liposoluble) extract, containing phenolic compounds and a wide range of fatty acids. The extract was tested on cultured keratinocytes and fibroblasts and then on the skin in vivo, to assess its cosmetic activities. When tested on skin cell cultures, the extract induced the genes responsible for skin hydration, such as aquaporin 3, filaggrin, involucrin and hyaluronic acid synthase, and stimulated the expression and the activity of the enzyme glucocerebrosidase, involved in ceramide production. Moreover, the liposoluble extract increased the synthesis of the extracellular matrix components in cultured fibroblasts and showed a remarkable skin-hydrating capacity when tested on human skin in vivo. Thanks to these activities, the Rubus idaeus liposoluble extract has several potential applications in skin care cosmetics: it can be used as hydrating and moisturizing ingredient in face and body lotions, and as anti-ageing product in face creams specifically designed to fight wrinkle formation.

Production of Uniformly Labeled 13C‐Lutein and 13C‐α‐Tocopherol in vitro Using Carrot Cell Suspension Culture

The xanthophyll lutein (Lut) is present in high concentrations in primate retinae, protecting against photooxidation and enhancing visual performance. Interestingly, in the elderly, diets high in Lut slow cognitive decline and circulating levels of both Lut and the lipid‐soluble antioxidant α‐tocopherol (αT) are inversely associated with cognitive impairment. However, Lut deposition, metabolism, and function in the brain are poorly understood, and cost‐effective isotopically labeled Lut tracers are not commercially available. The goal of this work, therefore, is to establish a system for efficient bioproduction of uniformly labeled 13C‐Lut and 13C‐αT. Seven carrot (Daucus carota) cultivars were germinated, separated into tissue subtypes, dedifferentiated into solid callus, and initiated in liquid suspension culture. Conditions for production of Lut and αT in suspension culture were optimized and uniformly labeled 13C‐glucose was provided as the sole media carbon source for four serial growth cycles. Lut and αT yields were 2.6 and 3.3 μg/g cells, respectively, as measured by reverse‐phase HPLC. Cell extracts were fractionated by preparatory reverse‐phase HPLC and analyzed for 13C enrichment by mass spectrometry. 58% of Lut and 50% of αT in carrot extract fractions were uniformly labeled with 13C. These tracers can be utilized in studies of Lut and αT in the brain, enhancing our understanding of their function in cognition.

Chloracidobacterium thermophilum gen. nov., sp. nov.: an anoxygenic microaerophilic chlorophotoheterotrophic acidobacterium.

A novel anoxygenic photoheterotrophic member of the phylum Acidobacteria , Chloracidobacterium thermophilum strain B sp. nov., was isolated from a cyanobacterial enrichment culture derived from microbial mats associated with Octopus Spring, Yellowstone National Park, WY. C. thermophilum sp. nov. was a Gram-stain-negative rod (diameter, approximately 0.8-1.0 µm; variable length, approximately 2.5 µm), which formed greenish-brown liquid suspension cultures. It was a moderately thermophilic microaerophile and grew in a defined medium at 51 °C (T(opt); range 44 to 58 °C) and in the pH range 5.5 to 9.5 (pH(opt) = ~7.0). The DNA G+C content was 61.3 mol%, and phylogenetic analysis, based on the 16S rRNA sequence, showed that C. thermophilum sp. nov. belongs to subdivision 4 ( Acidobacteriaceae ) of the Acidobacteria . C. thermophilum sp. nov. was unable to synthesize branched-chain amino acids, L-lysine, and vitamin B12, which were required for growth. Although the organism lacked genes/enzymes for autotrophic carbon fixation, bicarbonate was required. Growth was stimulated by other amino acids and 2-oxoglutarate. Cells produced chlorosomes containing a diverse mixture of bacteriochlorophyll (BChl) c derivatives, and additionally, synthesized BChl a P, Chl a PD, and Zn-BChl a'P, which occurred in type-1 homodimeric reaction centres. The carotenoids included echinenone, canthaxanthin, lycopene, γ-carotene and β-carotene. C. thermophilum sp. nov. produced iso-diabolic acid as its major fatty acid and synthesized three hopanoids (diploptene, bacteriohopanetetrol and bacteriohopanetetrol cyclitol ether). Based upon its phenotypic and genotypic properties, the name Chloracidobacterium thermophilum gen. nov., sp. nov. is proposed for this isolate; the type strain is C. thermophilum strain B(T) (ATCC BAA-2647 = JCM 30199).