Semisolid Culture Research Articles

Mutagenesis of a Thermophilic Alkalibacillus flavidus for Enhanced Production of an Extracellular Acetyl Xylan Esterase in Semi-solid Culture of Linseed Meal

In the present study, 20 different strains of Alkalibacillus flavidus were isolated from soils of industrial, agricultural and mountainous ranges of salt affected areas. After preliminary and secondary screening, isolate Isl-3 was selected as a better producer (5.25 ± 0.52 IU/g) of acetyl xylan esterase (AcXE). The wild-type Isl-3 was mutated to improve its hydrolytic potential by treating it with different EMS concentrations (0.25–1.5 mM). A relatively better enzyme activity (18.95 ± 0.95 IU/g) was observed at 0.5 mM EMS conc. The mutagenic exposure time was varied from 5 to 30 min, and a mutant strain (EMS-t3 was selected as it provided maximum enzyme activity (19.47 ± 0.97 IU/g) in 15 min. The resistance in EMS-t3 was developed by adding low-levels of l-cysteine HCl as a repressing agent. The enzyme activity of wild-type Isl-3 was compared with the putative mutant strain EMS-cys2 in semi-solid culture of linseed meal. The maximum enzyme activity (21.15 ± 1.05 IU/g) was obtained at substrate conc. of 7.5 g/50 ml of MSS by the mutant strain EMS-cys2. An increase in enzyme activity was noticed by both the strains when physical parameters including moisture content (100 ml), time of incubation (48 h), initial pH (9.5) and temperature (45 °C) were optimized. Overall, there was up to 6.8-fold increase in enzyme activity of mutant strain than that of wild-type. The substantial enhancement in enzyme activity by the mutant strain A. flavidus EMS-cys2 (34.65 ± 1.53 IU/g) is highly significant (HS, p ≤ 0.05) and could be a viable alternative (LSD ~ 0.046) for the routine batch culture process.

A new temporary immersion system for commercial micropropagation of banana (Musa AAA cv. Grand Naine)

Temporary immersion systems (TIS) are among the best options for commercial micropropagation. However, although successes have been reported with TIS, it is necessary to establish the most suitable system. The aim of this research was to compare the efficiency of different banana (Musa AAA cv. Grand Naine) micropropagation systems. In addition, a liquid medium with partial immersion and a semi-solid culture medium were evaluated. At 28 d of culture, the number and length of shoots, number of leaves per shoot, chlorophyll content, stomatal index, and the percentage of closed stomata were recorded. Additionally, the survival percentage and ex vitro development of the plantlets were evaluated during acclimatization. Results showed that TIS have a higher multiplication rate with respect to partial immersion and semi-solid medium. The chlorophyll content, stomatal index, and stomata functioning were affected by the different culture systems. The different culture systems had no effect on the survival percentage, although morphological differences were observed during acclimatization. The SETIS™ bioreactor was more efficient and resulted in better development in the plants obtained in vitro and ex vitro, which proved this system to be a useful alternative for banana micropropagation.

2-Alkyl-4-hydroxyquinolines from a Marine-Derived Streptomyces sp. Inhibit Hyphal Growth Induction in Candida albicans.

Four 2-alkyl-4-hydroxyquinoline derivatives (1–4) were isolated from a semisolid rice culture of the marine-derived actinomycete Streptomyces sp. MBTG13. The structures of these compounds were elucidated by a combination of spectroscopic methods, and their data were in good agreement with previous reports. Compounds 1 and 2 exhibited weak to moderate antibacterial activity against pathogenic bacteria. Unexpectedly, we found that compound 1 acted as a potent inhibitor of hyphal growth induction in the dimorphic fungus Candida albicans, with an IC50 value of 11.4 μg/mL. Growth experiments showed that this compound did not inhibit yeast cell growth, but inhibited hyphal growth induction. Semi-quantitative reverse transcription (RT)-PCR analysis of hyphal-inducing signaling pathway components indicated that compound 1 inhibited the expression of mRNAs related to the cAMP-Efg1 pathway. The expression of HWP1 and ALS3 mRNAs (hypha-specific genes positively regulated by Efg1, an important regulator of cell wall dynamics) was significantly inhibited by the addition of compound 1. These results indicate that compound 1 acts on the Efg1-mediated cAMP pathway and regulates hyphal growth in Candida albicans.

Antioxidants and improved regrowth procedure facilitated cryoconservation of Paphiopedilum insigne Wall. Ex. Lindl. - An Endangered Slipper Orchid

Orchids and their sustainability are very important issues that need global conservation efforts. Paphiopedilum insigne (an endangered orchid), is one of the most excessively exploited species of orchids and is mentioned in the IUCN Red List and Appendix I of CITES. The prospect for conservation and commercialization of this species would be strengthened with the development of improved cryopreservation techniques. This study reports on successful cryopreservation of protocorms of P.insigne after cryopreservation using vitrification (Vit) and encapsulation-vitrification (E-Vit) techniques. The study compared the addition of four antioxidants to the pretreatment and recovery stages, three growth media, and agitated vs. semisolid culture medium for initial recovery. Recovery after cryopreservation for the control was 27% for Vit and 37% for E-Vit. In both cases agitated culture produced improved recovery by about 10%, but with significantly better recovery with E-Vit. The best recovery (51.2 ± 0.9%) was recorded for 0.5 M sucrose precultured encapsulated protocorms treated for 45 min with PVS2 and recovered in ½ MS (L/S) liquid medium for 10 days under agitation, followed by transfer to semi-solid medium. This recovery was further enhanced (62.7 ± 0.5%) with the incorporation of 30 μM glutathione in both liquid preculture and the liquid and semisolid regrowth medium. This new protocol improved the E-Vit cryopreservation recovery from the initial 37%–63%, providing a suitable technique for storage of this threatened orchid.

Improving micropropagation of Mentha × piperita L. using a liquid culture system

In the current study, in vitro shoot proliferation and plant regeneration of Mentha × piperita L. (peppermint) cultivar ‘Black Mitcham’ was compared in semi-solid and liquid culture systems. Shoot tips from field-grown plants were used as explants to study shoot proliferation response on either Murashige and Skoog (MS) or Chee and Pool (C2D) medium containing varying levels of 6-benzylaminopurine (BAP), kinetin, and 6-γ,γ-dimethylallyl aminopurine (2iP). Differences in leaf ultrastructure and antioxidant capacity of greenhouse-grown and micropropagation-derived plants were studied to identify potential changes occurring during in vitro culture. Among the various media treatments tested, the maximum number of shoots was produced on the C2D medium with 4.0 μM BAP (40.7) followed by the MS medium with 4.0 μM BAP (32.2). Among the rooting treatments, shoots on the MS medium with 1.0 μM indole-3-butyric acid (IBA) produced the maximum number of roots (14.4). The number of shoots produced in Liquid Lab Rocker® (LLR) vessels containing liquid C2D medium with BAP (103.4) was significantly higher than that produced on semi-solid medium (40.7). No differences were observed in the leaf ultrastructure and antioxidant capacity of leaf extracts obtained from greenhouse-grown and micropropagation-derived plants. The study indicates that the liquid culture system under the described conditions can enhance peppermint micropropagation, with plant material being potentially valuable for use in herbal supplements and essential oil production.

Investigation of the spore production of Paecilomyces spp. isolated from several agricultural soils with the biocontrol activily against Spodoptera litura

Paecilomyces is a fungus that parasites on various insect species. However, Paecilomyces has not been widely studied and applied in Vietnam. In this study, Paecilomyces spp. were isolated from several agricultural soils and identified based on the morphology and 28S rDNA gene sequencing. Biocontrol activities of Paecilomyces were measured in vitro against Spodoptera litura. The Paecilomyces strains with high biocontrol were studied for the spore acquisition on semi-solid culture. There were five isolated strains belonged to Paecilomyces (strain F01 belonged to P. javanicus, strain F02 belonged to Paecilomyces sp., strain F03 belonged to P. lilacinum and strains F04 and F05 belonged to P. lilacinus) from 33 different samples. In particular, both of F03 and F04 performed high biocontrol activity against S. litura after 10 days of inoculation. Optimization of spores production medium showed that F03 and F04 grew well on a defined semi-solid medium whose the main components were unpolished rice, wheat bran and husk of 55% humidity. The results indicated that the native strains of Paecilomyces were potential for applications to produce bioproducts for pest management strategies.

Resveratrol improves ex vivo expansion of CB-CD34+ cells via downregulating intracellular reactive oxygen species level.

Intracellular reactive oxygen species (ROS) play important roles in the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). In this study, the effects of resveratrol (RES), on the ex vivo expansion of HSPCs were investigated by analyzing CD34+ cells expansion and biological functions, with the objective to optimize ex vivo culture conditions for CD34 + cells. Among the five tested doses (0, 0.1, 1, 10, 20, and 50 μM), 10 μM RES was demonstrated to be the most favorable for ex vivo CD34 + cells expansion. In the primary cultures, 10 μM RES favored higher expansion folds of CD34 + cells, CD34 + CD38 - cells, and colony-forming units (CFUs) ( P < 0.05). It was found that the percentages of primitive HSPCs (CD34 + CD38 - CD45R - CD49f + CD90 + cells) in 10 μM RES cultures were higher than those without RES. Further, in the secondary cultures, expanded CD34 + cells derived from primary cultures with 10 μM RES exhibited significantly higher total cells and CD34 + cells expansion ( P < 0.05). In the semisolid cultures, the frequency of CFU-GM and total CFUs of 10 μM RES group were both higher than those of without RES group, demonstrating that CD34 + cells expanded with 10 μM RES possessed better biological function. Furthermore, the addition of 10 μM RES downregulated the intracellular ROS level via strengthening the scavenging capability of ROS, and meanwhile reducing the percentages of apoptotic cells in cultures. Collectively, RES could stimulate the ex vivo expansion of CD34 + cells, preserved more primitive HSPCs and maintain better biological function by alleviating intracellular ROS level and cell apoptosis in cultures.

High-frequency protocorm-like bodies and shoot regeneration through a combination of thin cell layer and RITA® temporary immersion bioreactor in Cattleya forbesii Lindl.

An efficient in vitro mass propagation through protocorm-like bodies (PLBs) was established in Cattleya forbesii Lindl., a commercially important orchid. Whole PLBs (W-PLB) and transverse thin cell layers of PLB (tTCL-PLB) explants were cultured in RITA® bioreactors based on temporary immersion system. Explants were transferred in semi-solid or RITA® bioreactor for protocorm production or shoot regeneration. The effect of different immersion frequencies, medium volumes and inoculum densities were studied and optimized. RITA® bioreactor cultures were found to be superior compared with semi-solid cultures regarding PLB production and shoot regeneration. tTCL-PLB explant types cultured in the RITA® bioreactor with immersion for 1 min/4 h, 250 mL of medium and 20 explants showed the highest number of PLBs per RITA® (2237 PLBs) and per explant (111.9 PLBs). The highest number of PLBs per explant was 21 times higher than those from semi-solid culture. The highest number of shoots per RITA® (3998 shoots) and per explant (199.9 shoots) were observed on tTCL-PLB cultured in RITA® bioreactor (1 min/4 h; 150 mL of medium and 20 explants). The highest number of shoots per explant was 95 times higher than those grown on semi-solid culture. Mass propagation of PLBs and shoots of C. forbesii Lindl. using combined thin cell layer and RITA® temporary immersion has been adapted in commercial practice.

PLB Regeneration of Paphiopedilum rothschildianum using Callus and Liquid Culture System

This research was conducted to rapidly propagate Paphiopedilum rothschildianum using semi-solid and liquid culture systems. Calli were induced from seed, leaf segments (LS), seed-derived protocorms (SDP) and secondary protocorms (SP) cultured on half-strength semi-solid MS media supplemented with 0-22.6 µM 2,4-dichlorophenoacetic acid (2,4-D) and 4.54 µM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ). Regeneration of PLB from callus was optimized on semisolid medium and were evaluated in various concentrations and types of PGRs. PLB regeneration was further optimized using callus originating from a different induction medium, and cultured on different concentrations (0, 15 and 58 mM) of sucrose. For PLB regeneration using liquid culture system, 0.5 g callus were inoculated in a temporary immersion bioreactor system (RITA®) containing 150 ml liquid medium with immersion time of 5 minutes in every 125 minutes. SE, SDP and SP explants produced calli as early as 30 days with the percentages of explant forming callus at 77.0% ± 4.5, 94.4% ± 11.0, and 66.7% ± 14.4 respectively after 90 days of culture. The calli regenerated on medium supplimented with 2.27 µM TDZ and 12.0 µM BAP, but with very low percentage (15.0% ± 13.7 callus produced the average of 3 PLBs). PLB regeneration capacity increased to 37.5% ± 13.7 with the average of 5.9 PLBs for callus originating from an induction medium containing 4.54 µM TDZ, when a lower sucrose concentration (15 mM) was used in the regeneration medium. Callus proliferation using RITA® system showed an almost 2-fold increased in fresh weight and 168 PLBs per gram calli were regenerated. In contrast to semi-solid culture, the regeneration capacity in liquid culture system increased to 190 PLBs per gram calli when sucrose concentration in the medium was elevated from 15 mM to 58 mM.

Analysis of the Transcriptome in Aspergillus tamarii During Enzymatic Degradation of Sugarcane Bagasse.

The production of bioethanol from non-food agricultural residues represents an alternative energy source to fossil fuels for incorporation into the world's economy. Within the context of bioconversion of plant biomass into renewable energy using improved enzymatic cocktails, Illumina RNA-seq transcriptome profiling was conducted on a strain of Aspergillus tamarii, efficient in biomass polysaccharide degradation, in order to identify genes encoding proteins involved in plant biomass saccharification. Enzyme production and gene expression was compared following growth in liquid and semi-solid culture with steam-exploded sugarcane bagasse (SB) (1% w/v) and glucose (1% w/v) employed as contrasting sole carbon sources. Enzyme production following growth in liquid minimum medium supplemented with SB resulted in 0.626 and 0.711 UI.mL−1 xylanases after 24 and 48 h incubation, respectively. Transcriptome profiling revealed expression of over 7120 genes, with groups of genes modulated according to solid or semi-solid culture, as well as according to carbon source. Gene ontology analysis of genes expressed following SB hydrolysis revealed enrichment in xyloglucan metabolic process and xylan, pectin and glucan catabolic process, indicating up-regulation of genes involved in xylanase secretion. According to carbohydrate-active enzyme (CAZy) classification, 209 CAZyme-encoding genes were identified with significant differential expression on liquid or semi-solid SB, in comparison to equivalent growth on glucose as carbon source. Up-regulated CAZyme-encoding genes related to cellulases (CelA, CelB, CelC, CelD) and hemicellulases (XynG1, XynG2, XynF1, XylA, AxeA, arabinofuranosidase) showed up to a 10-fold log2FoldChange in expression levels. Five genes from the AA9 (GH61) family, related to lytic polysaccharide monooxygenase (LPMO), were also identified with significant expression up-regulation. The transcription factor gene XlnR, involved in induction of hemicellulases, showed up-regulation on liquid and semi-solid SB culture. Similarly, the gene ClrA, responsible for regulation of cellulases, showed increased expression on liquid SB culture. Over 150 potential transporter genes were also identified with increased expression on liquid and semi-solid SB culture. This first comprehensive analysis of the transcriptome of A. tamarii contributes to our understanding of genes and regulatory systems involved in cellulose and hemicellulose degradation in this fungus, offering potential for application in improved enzymatic cocktail development for plant biomass degradation in biorefinery applications.

Up-regulation of miR-210 induced by a hypoxic microenvironment promotes breast cancer stem cells metastasis, proliferation, and self-renewal by targeting E-cadherin.

Breast cancer stem cells (BCSCs), a small subset of breast cancer cells with stem cell-like properties, are essential in tumor formation, metastasis, resistance to anticancer therapies, and cancer recurrence. MicroRNAs (miRNAs) are involved in tumorigenicity by regulating specific oncogenes and tumor-suppressor genes, and their roles in BCSCs are becoming apparent. A novel, 3-dimensional (3D), semisolid culture system was established to culture MCF-7 spheroid cells with high percentage of BCSCs. The differences in miRNA expression among the MCF-7 parental cells, BCSC-enriched MCF-7 spheroid cells, and CD44+/CD24- MCF-7 cells were evaluated by miRNA microarray, and the high expression of miR-210 in MCF-7 spheroid cells and CD44+/CD24- MCF-7 cells was verified by quantitative RT-PCR. MCF-7 cells were cultured in a hypoxic chamber to detect the effect of hypoxia on miR-210 expression and the stemness of the cells. The 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide (MTT), transwell, and sphere-formation assays were performed to detect the proliferation, migration, and self-renewal ability of miR-210-overexpressed MCF-7 cells and MCF-7 spheroid cells with miR-210 knocked down. The target of miR-210 was validated with a dual-luciferase reporter assay and Western blotting. In vivo xenograft assay and metastasis assay were performed to study the effects of miR-210 targeting E-cadherin on BCSCs growth and lung metastasis, and the tumors were assessed by immunohistochemistry and immunofluorescence. We developed a novel 3D, semisolid culture system to culture MCF-7 spheroid cells, which are enriched in BCSCs, and found, by performing miRNAs expression profiling, miR-210 was up-regulated in those cells compared with MCF-7 parental cells. High miR-210 expression was also detected in CD44+/CD24- MCF-7 cells and human CD44+/CD24- breast cancer cells, which was demonstrated to be partially due to the hypoxic microenvironment around BCSCs in MCF-7 spheroids or solid tumors. Ectopic expression of miR-210 in MCF-7 cells promoted their migration, invasion, proliferation, and self-renewal in both in vitro and in vivo studies. We further reported that miR-210 suppressed E-cadherin expression by targeting the open reading frame region of E-cadherin mRNA and by up-regulation of E-cadherin transcription repressor, Snail. Accordingly, E-cadherin overexpression compromises the migration, invasion, proliferation, and self-renewal ability of miR-210-overexpressed MCF-7 both in vitro and in vivo. These findings reveal a novel regulatory pathway centered on hypoxia-mediated miR-210 targeting of E-cadherin, which contributes to the properties and breast tumorigenesis of BCSCs.-Tang, T., Yang, Z., Zhu, Q., Wu, Y., Sun, K., Alahdal, M., Zhang, Y., Xing, Y., Shen, Y., Xia, T., Xi, T., Pan, Y., Jin, L. Up-regulation of miR-210 induced by a hypoxic microenvironment promotes breast cancer stem cells metastasis, proliferation, and self-renewal by targeting E-cadherin.

Phytosulfokine-alpha, an enhancer of in vitro regeneration competence in recalcitrant legumes

Oligopeptides have been recognized as signaling molecules playing an important role in plant cell growth and development. Phytosulfokine-alpha (PSK), a plant-specific disulfated pentapeptide, is involved at nanomolar concentrations in initial steps of cellular dedifferentiation, proliferation, and re-differentiation, with a biological function similar to that of plant hormones. On the other hand, legume crops are generally known for their recalcitrance to in vitro regeneration approaches, which has restrained the exploitation of biotechnological tools for their genetic improvement. Against this background, we added PSK at concentrations of 10−10 to 10−6 M, to semisolid MS-based culture media previously shown to permit some regeneration responses with pea (Pisum sativum) and highly recalcitrant faba bean (Vicia faba). Callus, cell suspensions and embryo-derived explants of pea cultivars Frisson and Cameor and a low vicin, zero tannin faba bean genotype (Fevita©) were assessed for their embryogenic and organogenic regeneration competence. For all genotypes, PSK reliably and significantly enhanced the regeneration competence producing somatic embryos and organs that yielded regenerated plants of both pea cultivars, and had a major organogenic effect leading to plant regeneration with faba bean where somatic embryos, although produced, failed to convert into viable plants. This is the first report on the use of PSK with legume species.

Abstract 4311: Rb may play a key role in green tea-induced growth inhibition of human myeloid leukemia cells

Abstract Although the benefits of green tea to human health including preventing cancer have been widely reported in recent years, the specific effect and action mechanisms of green tea on cancer cells are largely unclear. In this study, we presented evidence that green tea extract and its major chemical component, Epigallocatechin-3-gallate (EGCG), inhibit both suspension and adherence cancer cells through the regulation of pRb-CDK complexes. Addition of green tea extract to the culture of myeloid leukemia TF-1a and MV4-11 cells significantly inhibited their proliferation measured by XTT and BrdU assays. The inhibition is dose dependent with more than 35% inhibition being observed at a concentration of 4% (v/v) of the tea extracts derived from 8% (g/ml) green tealeaf in distilled water. The tea extract also markedly reduced the numbers of stem/progenitors derived from TF-1a, MV4-11, and Hep-G2 cells, determined by counting colony-forming cells in semi-solid clonal culture. EGCG showed a similar inhibitory effect on TF-1a, MV4-11, and Hep-G2 cells and their stem/progenitors. Next, we investigated what mechanism(s) is responsible for the green tea-induced growth inhibition of the cells. Surprised to us the expression of Rb was significantly upregualted with a maximal enhancement being observed when 0.00001% of EGCG (g/ml) or 4% (v/v) of green tea extracts were added to the cell culture. The upregulation of Rb was significantly blocked by pre-treatment with cycloheximade, a protein synthesis inhibitor. In contrast, green tea extract and EGCG downregulated dramatically phosphorylation of pRb. Addition of cycloheximade had no effect on the phosphorylation of Rb in the absence or presence of green tea extract or EGCG analyzed by Western blotting. We also found that green tea extract and EGCG had no significant effect on the expression of CDC2, CDK2, CDK4, CDK6, and CDK inhibitors, p21 and p27, but the formation of CDK4-pRb and CDK6-pRb complexes were significantly downregulated detected by immunoprecipitation. Taken together, our data suggest that green tea (the extract and EGCG) regulate Rb protein at both translation and post-translation levels. The upregulation of Rb synthesis, downregulation of Rb phosphorylation and CDK-Rb complexes all promote the binding of Rb to E2F transcription factors and inhibit the E2F-dependent transcription, leading to cell growth inhibition (supported by Barry Faculty Stimulating Grant 2017-2018). Citation Format: Darrell Henry, Sebastien Brumaire, Xiaotang Hu. Rb may play a key role in green tea-induced growth inhibition of human myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4311.

Negative regulation of microRNA-9 by ecotropic viral integration site1 involved in the pathogenesis of acute myeloid leukemia

Objective To investigate the regulation mechanism of microRNA-9(miR-9)by ecotropic viral integration site1(EVI1)its impact on proliferation of AML cells and its role in the pathogenesis of myelogenous leukemia. Methods EVI1 was forced to express in Uocm1 cell lines by murine stem cell virus-EVI1(MSCV-EVI1) plasmid infection.EVI1 overexpressed Uocm1 cells were then treated with 0.1 μmol/L 5-aza-2′-deoxycytidine (5-AZA) dissolved in dimethyl sulfoxide (DMSO). The methylation level of miR-9 promoter was tested by DNA bisulfite sequencing technology.The cell cycle was observed by flow cytometry (FCM). The proliferation ability of the cells was detected by the colony forming assay in semi-solid Methylcellulose medium culture. Results EVI1 level was dramatically increased after being infected by MSCV-EVI1 plasmid.Forced expression of EVI1 in Uocm1 significantly downregulated miR-9 by inducing hypermethylation of miR-9 promoter.Relative expression level of miR-9 was lower in EVI1 overexpressed group(0.004±0.000)than that of the control group(0.006±0.001)(t=4.09, P<0.05). When EVI1 was overexpressed in Uocm1, the rate of G0/G1 cells decreased markedly(P<0.05), while rates of S phage and G2 phage increased significantly(all P<0.05). Seven days after 500 cells plated in semi-solid medium, EVI1 overexpressed Uocm1 cells gave rise to more colony (122.3±7.8) than Uocm1 cells infected with vector(45.7±6.1)(t=-13.44, P<0.01).0.1 μmol/L 5-AZA recovered miR-9 expression(P<0.01)by decreasing EVI1 induced hypermethylation of miR-9 promoter.G0/G1 phase cell proportion was(48.25±2.19)% in control group, while (65.90±2.90)% in 5-AZA group(t=-6.85, P<0.05).5-AZA group formed less colony (51.00±10.01)than the control group (123.40±8.12) (t=9.59, P<0.01), which indicated that 5-AZA inhibited cell proliferation by G0/G1 cell cycle retardation in EVI1 overexpressed uocm1 cells. Conclusions EVI1 may enhance proliferation ability of myeloid leukemia cells by downregulating miR-9 through inducing hypermethylation of miR-9 promotor, which plays a crucial role in the pathogenesis of AML.5-AZA may be an effective hypomethylating agent in the therapy of EVI1 high acute myeloid leukemia. Key words: Ecotropic viral integration site 1; MicroRNA-9; Acute myeloid leukemia; Pathogenesis; 5-aza-2′-deoxycytidine

Bacterias diazotróficas con actividad promotora del crecimiento vegetal en Daucus carota L.

Este trabajo se propuso aislar bacterias diazotróficas con actividad promotora del crecimiento vegetal, asociadas al cultivo de zanahoria. Se realizaron 3 muestreos, a los 30, 60 y 115 días, en una granja ubicada en una zona rural del municipio de Manizales (Caldas). El aislamiento de las bacterias diazotróficas se efectuó en medios de cultivo semisólidos libres de nitrógeno. A los aislamientos obtenidos se les realizó descripción macro y microscópica, identificación bioquímica y molecular. Se evaluaron características como determinación de compuestos indólicos, actividad nitrogenasa y solubilización de fosfatos. Como cepa patrón se empleó Gluconacetobacter diazotrophicus ATCC 49037. Se recuperaron 20 aislamientos asociados a la rizosfera y 12 al rizoplano. La identificación molecular mostró cinco géneros presentes: Rhizobium, Achromobacter, Bacillus, Enterobacter y Stenotrophomonas. La producción de compuestos indólicos presentó concentraciones entre 9,73 y 112,8 µg/mL. La cepa patrón presentó una actividad mayor, con una producción de compuestos indólicos de 172,5 µg/mL. En la actividad nitrogenasa los aislamientos GIBI411, 394 y 399 tuvieron una actividad mayor o similar a la cepa patrón. Los aislamientos más eficientes en la solubilización de fosfato tricálcico fueron GIBI378 y 385. La solubilización de fosfato de aluminio se valoró por el índice de producción de ácido, siendo los aislamientos GIBI378, 391, 387 y 388 los de mejor comportamiento en esta variable. Los aislamientos encontrados son candidatos potenciales para desarrollar nuevos procesos biotecnológicos para la producción de nuevos biofertilizantes alternativos, considerando las importantes propiedades de promoción del crecimiento vegetal determinadas en este trabajo.

Removal of Cd from Soil by Aspergillus fumigatus in a Semi-solid Culture

A method was proposed to remove Cd from contaminated soils by a semi-solid culture containing Aspergillus fumigatus that have a strong resistance to Cd. The removal efficiencies of Cd in different simulated Cd pollution levels were studied and the changes in Cd adsorption and the enrichment in Aspergillus fumigatus were measured. The results showed that Aspergillus fumigatus could remove some Cd from the soil in the semi-solid culture system. When the concentration of Cd was 10 mg·kg-1, the total removal rate of Cd was up to 31%. Meanwhile, the dry weight of Aspergillus fumigatus and the pH of the system were studied during the culture process. The results showed that the dry weight decreased with the increase in culture time and Cd concentration, the maximum decrease rate of dry weight was 64%. The removal efficiency was the best when the pH was varied from 5.6 to 6.0. The changes in different extraction fractions for Cd showed that the main fractions of Cd removed by Aspergillus fumigatus were the acid-soluble fraction and the reducible fraction and the oxidizable fraction of Cd remained essentially unchanged before and after the culturing. The proposed method would provide valuable information for the remediation of heavy metal-contaminated soil by fungi.

Complete germination of papaya (Carica papaya L. cv. `Maradol Roja´) somatic embryos using temporary immersion system type RITA® and phloroglucinol in semi-solid culture medium

A critical factor in somatic embryogenesis protocols in papaya (Carica papaya L.) has been incomplete germination of somatic embryos due to formation of a basal callus, which prevents the emission of the radicle. This work aims to achieve complete germination of somatic embryos in liquid and semi-solid culture media. The effect of the culture conditions on germination of somatic embryos using the RITA® temporary immersion system were evaluated as well as the effect of phloroglucinol on germination of somatic embryos in semi-solid culture medium. The results of using the RITA® culture medium with a combination of 0.02 μM BAP and 2.90 μM gibberellic acid had a good response for total germination (100%) but somatic embryos had only partial germination with 400 mg fresh mass. However, the optimum inoculum density was 200 mg fresh mass of somatic embryos which produced 100% total germination and 95% somatic embryos with complete germination. Also, it was possible to achieve complete germination of somatic embryos with low callus formation (13%) using phloroglucinol at a concentration of 475.8 μM on semi-solid culture medium. This is the first report of two biotechnological strategies for complete germination of plants from somatic embryos in the papaya cultivar `Maradol Roja´.

Human Umbilical Cord Mesenchymal Stromal Cells Support Viability of Umbilical Cord Blood Hematopoietic Stem Cells but not the "Stemness" of Their Progeny in Co-Culture.

Cell-cell interactions and the ability of mesenchymal stromal cells to support the expansion of hematopoietic progenitor cells were studied in co-culture of human umbilical cord tissue-derived mesenchymal stromal cells and nucleated umbilical cord blood cells. It was found that hematopoietic stem cells from the umbilical cord blood are capable to adhere to mesenchymal stromal cells and proliferate during 3-4 weeks in co-culture. However, despite the formation of hematopoietic foci and accumulation of CD34+ and CD133+ cells in the adherent cell fraction, the ability of newly generated blood cells to form colonies in semi-solid culture medium was appreciably reduced. These findings suggest that human umbilical cord tissue-derived mesenchymal stromal cells display a weak capability to support the "stemness" of hematopoietic stem cell progeny despite long-term maintenance of their viability and proliferation.

Growth and enzymatic activity of Leucoagaricus gongylophorus, a mutualistic fungus isolated from the leaf-cutting ant Atta mexicana, on cellulose and lignocellulosic biomass.

According to the best of our knowledge, this is the first report about the growth of Leucoagaricus gongylophorus, isolated from the colony of the ant Atta mexicana, on semisolid medium with cellulose and solid-state cultures with lignocellulosic materials. The maximum CO2 production rate on grass was three times higher than on sugarcane bagasse. Endoglucanase activity was detected and it was possible to recover glucose from the fungal gongylidia. The cellulolytic activity could be used to process lignocellulosic residues and obtain sugar or valuable products, but more work is needed in this direction.

Characterization, regulation, and targeting of erythroid progenitors in normal and disordered human erythropoiesis.

The erythroid progenitors burst-forming unit-erythroid and colony-forming unit-erythroid have a critical role in erythropoiesis. These cells represent a heterogeneous and poorly characterized population with modifiable self-renewal, proliferation and differentiation capabilities. This review focuses on the current state of erythroid progenitor biology with regard to immunophenotypic identification and regulatory programs. In addition, we will discuss the therapeutic implications of using these erythroid progenitors as pharmacologic targets. Erythroid progenitors are classically characterized by the appearance of morphologically defined colonies in semisolid cultures. However, these prior systems preclude a more thorough understanding of the composite nature of progenitor populations. Recent studies employing novel flow cytometric and cell-based assays have helped to redefine hematopoiesis, and suggest that erythroid progenitors may arise from different levels of the hematopoietic tree. Moreover, the identification of cell surface marker patterns in human burst-forming unit-erythroid and colony-forming unit-erythroid enhance our ability to perform downstream functional and molecular analyses at the population and single cell level. Advances in these techniques have already revealed novel subpopulations with increased self-renewing capacity, roles for erythroid progenitors in globin gene expression, and insights into pharmacologic mechanisms of glucocorticoids and pomalidomide. Immunophenotypic and molecular characterization resolves the diversity of erythroid progenitors, and may ultimately lead to the ability to target these progenitors to ameliorate diseases of dyserythropoiesis.