Semisolid Medium Research Articles

Developing Single Cell Live Imaging Strategies to Determine MEP Fate and Predict Potential

The molecular mechanisms underlying lineage commitment of stem and progenitor cells have implications for deriving specific cell types in vitro for regenerative medicine purposes and elucidating the aberrant pathways responsible for pathological conditions. We investigated Megakaryocytic-Erythroid Progenitors (MEP) commitment to the megakaryocytic (Mk) and erythroid (E) lineages as a model of cell fate decisions. Colony forming unit (CFU) assays are used to test the functional output, or lineage potential, of progenitor cell populations. As single progenitor cells proliferate, their progeny remain near each other to form a colony. This potential is deduced from the mature cell types comprising the colony. However, this assay has several limitations: 1) there is an assumption that each colony arose from a single progenitor cell based on the low probability that individual progenitors would form overlapping colonies when plated sparsely; 2) there is a surfeit of kinetic data about the colony forming cells that is not collected with a single end-point; and 3) the outcome may be misleading if there is selective loss of specific cell types of early committed progeny prior to the endpoint. To overcome these limitations, we developed a time-lapse microscopy and lineage tracing approach to visualize single sorted MEP, as well as committed Megakaryocytic progenitors (MkP) and Erythroid progenitors (ErP), as they proliferate, specify and progress down either the Mk or E lineages. We plated primary adult human MEP (Sanada and Xavier-Ferrucio, et al. Blood, 2016) at low density in semisolid media supplemented with cytokines in a MatTek plate under a coverslip to minimize focal range and support colony formation for up to 14 days. The plated cells were imaged with an Olympus VivaView. Fluorescently conjugated anti-CD41 (a marker of Mk) and anti-CD235a (a marker of E) were added to the culture dishes at the end to confirm the lineage of each cell in the colonies. The average time from plating to the first division was 38 h, and not significantly different between MEP, ErP, and MkP. We optimized the image acquisition settings to permit accurate tracking of the plated cells and their progeny for up to 11 days. Average colony forming efficiency was 75%, which was equal to unimaged CFU assays, indicating minimal phototoxicity. Acquired images were stacked into time-lapse videos and automatically tracked with the Baxter Algorithm (Magnusson et al. IEEE Trans Med Imaging, 2015). After manual segmentation and track correction, lineage trees were generated for each colony (Figure 1). We exported quantifiable features of the cells including the time between cell divisions and velocity of each cell from the algorithm. We defined cells that give rise to only CD41-labeled cells as committed MkP, cells that give rise to only CD235-labeled cells as ErP, and cells that give rise to at least one progeny of both as MEP. However, these progenitor definitions are based on an inaccurate assumption that fate reflects potential, since a bipotent cell may stochastically give rise to progeny that are committed to a single lineage. To convert fate to potential, we first need to identify the precise period of commitment to more accurately define MEP, MkP, and ErP in our culture system. To achieve this, we are developing a mathematical model that can predict the potential of cells based on quantifiable features that are measured form the time-lapse images and are significantly different between MEP, MkP, and ErP. Utilizing statistical tools, we have identified that the duration of the cell cycle and the velocity of single progenitor cells along with their lineage history, permit predictions of progenitor potential and provide several novel insights into the process of lineage commitment. We found that ErP have a significantly shortened cell cycle duration and slower velocity compared to MkP, while MEP have the longest cell cycle and an intermediate velocity until undergoing their fate decisions (Figures 2 and 3). With this time-lapse imaging approach, we hope to better investigate molecular mechanisms that direct fate decisions of multipotent progenitor cells. Disclosures No relevant conflicts of interest to declare.

An efficient protocol for commercial micropropagation of malanga (Colocasia esculenta L. Schott) using temporary immersion

Temporary immersion systems allow semi-automatization and reduce production costs during micropropagation on a commercial scale. The objective of this study was to evaluate temporary immersion (TI) as a new technique for commercial micropropagation of malanga (Colocasia esculenta). The effect of the semi-solid and liquid medium using the Temporary Immersion Bioreactor (TIB) was compared. For the TIB culture system, the immersion frequency and volume of culture medium per explant on shoot development were evaluated. At 30 d of culture, the number of shoots per explant, shoot length and number of leaves per shoot were scored. In addition, the chlorophyll content during multiplication and survival during acclimatization were evaluated in the different culture systems. The results showed that the highest shoot production, shoot length and number of leaves per shoot were obtained in TI with an immersion frequency of 2 min every 4 h and with a volume of culture medium of 25 and 50 mL per explant. An increase in chlorophyll content in TI was observed with an immersion frequency every 4 h and with a culture medium volume of 50 and 100 mL per explant. Regarding the percentage of survival during acclimatization, no significant differences were observed between culture systems, immersion frequencies and volume of culture medium per explant. In conclusion, the evaluated immersion system is an alternative for commercial micropropagation of C. esculenta.

Cell Culture and Gene Expression Studies in Relation to Biomineralization in the Black-lip Pearl Oyster, Pinctada margaritifera

Nacre is composed of aragonite platelets and organic material formed by molluscs as inner shell layer through biomineralization process, and mantle epithelial cells control nacre formation. A primary culture of granulated epithelial cells was established from mantle tissue of Pinctada margaritifera, through a process of continuous sub-culturing at 28°C in yeast supplemented sea water culture medium. Nuclear beads were placed in culture wells containing semi-solid agar medium and incubated in vitro with cultured granulated epithelial cells in order to evaluate the nacre secretion. On visual observation, a brown coloration was observed on the surface of the bead after 7-10 days. Evaluation of the surface of the nuclear beads by scanning electron microscopy (SEM) after 60 days of incubation revealed a good brick and mortar pattern, characteristic of nacreous layer formation. A lustrous hue was also seen to develop on bead surfaces after this stage. SEM images of a cross section of the nacre-coated bead showed a pattern of arrangement of aragonite tablets similar to that seen in cross sections of the nacre layer of shell of molluscs. The functional ability of cultured granulated epithelial cells was further confirmed by detecting gene expression of two matrix proteins, nacrein and amorphous calcium carbonate binding proteins (ACCBP), which play an important role in formation of the nacreous layer, in both cultured cells and in native mantle tissue. Amplification products for nacrein (480 bp) and ACCBP (500 bp) genes were obtained in both native mantle tissue and in vitro cultured mantle epithelial cells. There was good correlation between the expression patterns of the two genes in in vitro cultured cells and in native mantle tissue, signifying that cultured mantle epithelial cells retain their functional characteristics of biomineralization.

Single-Cell Cloning of Hybridoma Cells by Growth in Soft Agar.

Single-cell cloning during hybridoma production ensures that cells that produce the antibody of interest are truly monoclonal and that the secretion of this antibody can be stably maintained. Cloning of hybridoma cells in semisolid medium is one of the most commonly used methods for producing single-cell clones. The technique is easy, but, because it is performed in two stages, it does take longer than other methods. Not all cells will grow in soft agar, and there may be a bias on the type of colony that appears. However, most of the commonly used myeloma fusion partners have relatively good cloning efficiencies in soft agar, and, consequently, so do most hybridomas. Even though every attempt is made to ensure that the cells are in a single-cell suspension before plating, there is no way to guarantee that the colonies do not arise from two cells that were stuck together. Therefore, single-cell cloning in soft agar should be repeated at least twice before the cells are considered clonal.

PIK3CA Cooperates with KRAS to Promote MYC Activity and Tumorigenesis via the Bromodomain Protein BRD9.

Tumor formation is generally linked to the acquisition of two or more driver genes that cause normal cells to progress from proliferation to abnormal expansion and malignancy. In order to understand genetic alterations involved in this process, we compared the transcriptomes of an isogenic set of breast epithelial cell lines that are non-transformed or contain a single or double knock-in (DKI) of PIK3CA (H1047R) or KRAS (G12V). Gene set enrichment analysis revealed that DKI cells were enriched over single mutant cells for genes that characterize a MYC target gene signature. This gene signature was mediated in part by the bromodomain-containing protein 9 (BRD9) that was found in the SWI-SNF chromatin-remodeling complex, bound to the MYC super-enhancer locus. Small molecule inhibition of BRD9 reduced MYC transcript levels. Critically, only DKI cells had the capacity for anchorage-independent growth in semi-solid medium, and CRISPR-Cas9 manipulations showed that PIK3CA and BRD9 expression were essential for this phenotype. In contrast, KRAS was necessary for DKI cell migration, and BRD9 overexpression induced the growth of KRAS single mutant cells in semi-solid medium. These results provide new insight into the earliest transforming events driven by oncoprotein cooperation and suggest BRD9 is an important mediator of mutant PIK3CA/KRAS-driven oncogenic transformation.

Surface sterilization and micropropagation of Ludisia discolor

Abstract The jewel orchid Ludisia discolor, found in many parts of Asia, is valued due to its appearance and medicinal benefits. Existing surface sterilization protocols were found to be not successful in initiating in vitro cultures of L. discolor in this study. Hence, a surface sterilization protocol, incorporating the use of 0.4% (w/v) mercury chloride, was developed to address this issue. This method yielded the lowest contamination (7.7%) and best growth at 22.5%. Nodal segments of L. discolor produced the best growth when placed in half-strength semi-solid MS medium with 0.2% (w/v) activated charcoal, 8% (w/v) Mas banana cultivar homogenate, 3% (w/v) sucrose, 3.5 g L−1 Gelrite, 1.0 mg L−1 1-naphthaleneacetic acid, and 0.1 mg L−1 thidiazuron, a confirmation of the protocol promoted by Shiau et al. (2005). All in vitro cultures of L. discolor were successfully acclimatised under low light conditions using coconut coir, coconut husk and peat moss.

Development of a novel in vitro rooting culture system for the micropropagation of highbush blueberry (Vaccinium corymbosum) seedlings

Here, we established a novel, efficient, and cost-effective rooting culture system for highbush blueberry (Vaccinium corymbosum). This method overcomes the inefficient, expensive, and time-consuming nature of traditional methods and provides a theoretical basis for the industrial rooting of micropropagated blueberry seedlings. Semisolid half-strength woody plant medium (1/2WPM), supplemented with carrageenan as fixing material, was packed into high molecular polypropylene plastic bags. Optimum response of ‘Ozarkblue’ was observed in 1/2WPM supplemented with 0.1 mg/l indole-3-butyric acid (IBA); 97.7% rooting rate was achieved at 45 days after culture, with the average root number and length reaching 6.5 ± 0.39 and 3.5 ± 0.16 cm, respectively. The highest rooting rate of ‘Weiwa Kuahuat’ (84.0%) was achieved with 0.5 mg/l IBA, resulting in a favorable average root number (3.0 ± 0.43) and length (3.0 ± 0.43 cm). ‘Sierra’ showed the highest rooting rate (100.0%) in the presence of 1.0 mg/l IBA, with an average root number and length of 5.9 ± 0.9 and 3.9 ± 0.62 cm, respectively. The earliest and fastest rooting was recorded when shoots were transplanted into bags for 13 days. A space utilization experiment showed that the density of well-grown blueberry seedlings achieved in bags (1600 seedlings/m2) was superior to that obtained using traditional in vitro and ex vitro rooting methods (270 and 420 seedlings/m2, respectively). This is the first report of a complete rooting system that could serve as a feasible blueberry rooting method with superior rooting time, rooting rate, medium consumption, space requirement, and portability. We established a novel, efficient, and cost-effective rooting culture system for the micro-propagation of highbush blueberry seedlings that overcomes many of the drawbacks of traditional methods of industrial rooting.

Feasibility of the new method for orchid in vitro rooting using liquid and chemical sterilized culture medium under different sucrose concentration

Abstract The in vitro propagation of orchids is the only commercial large scale technique to obtain healthy and high quality plantlets with clonal origin. The use of new technologies in plant tissue culture systems could lead to efficiency increases and costs reduction of micropropagation systems. The main actual micropropagation system is based on semi-solid culture media solidified using agar, followed by sterilization using autoclaving, and cultivation under photomixotrophic conditions using sucrose as main source of energy to plant in vitro culture. We proposed in this study the use of new micropropagation system using chemical sterilized liquid medium using polyurethane foam as support and LED source of light in rooting stage of Miltassia ‘Shelobie Tolkien’. Thus, the objective of this research was to test different concentrations of sucrose, comparing the conventional semi-solid agar-based culture medium (control) and the use of liquid medium with polyurethane foam support. The following sucrose concentrations were used: 0, 7.5, 15, 22.5 and 30 g L-1. The experiment was conducted in a 2 × 5 factorial, in a completely randomized design with ten replications each over a total period of 105 days of cultivation. The chemical sterilization using ClO2 showed 100% of decontamination in all treatments. The use of liquid media with polyurethane foam showed better results than plants cultivated in agar medium, and can be used for replace agar-based for orchid in vitro rooting.

Study protocol: characterising the clinical, epidemiological and aetiological aspects of leptospirosis in Sri Lanka: a hospital based clinico-epidemiological study

IntroductionSri Lanka has one of the highest incidences of leptospirosis worldwide. We hypothesised that different geographical locations and patient context will have a distinct molecular epidemiology of leptospirosis, based on...

Effect of additives on enhanced in-vitro shoot multiplication and their functional group identification of Chlorophytum borivilianum Sant. Et Fernand

The present study was commenced with the aim to optimize the proficient in vitro regeneration protocol and phytochemical constituents and their functional groups identification through FT-IR spectrum analysis. As per of our knowledge, very few or no reports are available on the effects of additives on the nodal explant of C. borivilianum. Surface sterilized explants viz. nodal segments were inoculated on MS basal medium supplemented with 19 different concentrations of cytokinins (BAP, Kinetin and zeatin). BAP at a concentration of 2 mg ml−1 was found superior out of three cytokinins for shoot induction. This study confirmed that the inclusion of additives (Adenine sulphate, casein hydrolysate and putrescine) had a promoting effect on multiple shoot induction and shoot proliferation. Best response was achieved on MS medium supplemented with 2.0 mg l−1 BAP + 1.0 mg l−1 NAA + 20.0 mg l−1 AdSO4 from nodal explant on 45th day of culture. For in vitro rooting, the semi solid rooting medium with IBA (2.0 mg l−1) + putrescine (50.0 mg l−1) was found to be best with 90% maximum field survival rate. The proposed protocol can be used for the conservation of germplasm and propagation of these endangered plant species. FT-IR spectroscopic analysis revealed the presence of alcohols, phenols, alkanes, alpha and beta-unsaturated aldehydes, ketones, aromatic amines, alkyl halides, aliphatic amines, ether linkage and alkenes might be responsible for the various medicinal properties of test plant.

Suppression of lactate production by using sucrose as a carbon source in lactic acid bacteria

Lactic acid bacteria (LAB) grow by producing lactate from sugar. However, the accumulation of lactate inhibits their growth. Here, the lactate productivity per cell in a semi-solid medium prepared with a chlorella powder in several LAB strains was much lower than that in the conventional MRS medium. Furthermore, the lactate production was suppressed not only in semi-solid medium, but also in chlorella liquid medium. The lactate productivity by Lactococcus lactis subsp. lactis NBRC 12007 in the chlorella liquid medium and MRS medium was 3.0 and 6.9g-lactate·g-cell-1, respectively. The productivity of lactate in the chlorella liquid medium decreased to 44% of that in MRS medium. Gas chromatography/mass spectrometry (GC/MS) analysis of the culture supernatants revealed that the utilization of sucrose in the chlorella powder led to the suppression of lactate production. Comparison of the metabolites extracted from the cells indicated that the two ATP generating pathways, the arginine deiminase pathway and the decarboxylation reaction of glutamate and GABA, which are usually repressed by glucose, are activated in chlorella medium. It was considered that these pathways which do not require NAD+ for generation of ATP are not repressed when sucrose is used as a carbon source. Thus, the utilization of these pathways results in the suppression of the lactate production.

Assessment of different temporary immersion systems in the micropropagation of anthurium (Anthurium andreanum).

Anthurium has been micropropagated mainly through conventional techniques in semisolid culture medium. However, this culture system involves constraints due to the low number of shoots produced and the high costs of the gelling agent and labor. Temporary immersion systems (TIS) are an alternative for increasing biological performance, reducing costs, and facilitating a semi-automated micropropagation process. The objective of this study was to compare the efficiency of different types of TIS during the in vitro propagation of anthurium. We used 2-cm-long nodal segments from in vitro plants. Explants were cultured in different TIS: temporary immersion bioreactors (TIB®), Ebb-and-Flow bioreactor, and recipient for automated temporary immersion (RITA®), with a 2-min immersion frequency at 12-h intervals. We used Murashige and Skoog (MS) medium supplemented with 3% (w/v) of sucrose and 8.88µM benzylaminopurine. After 60days of culture, we evaluated various physiological variables and the percent survival in the different TIS. The largest numbers of shoots per explant were observed in TIB® and Ebb-and-Flow, with 50.83 and 43.16 shoots per explant, respectively; the lowest number of shoots per explant was observed in RITA®, with 30.66. TIB® yielded the highest content of photosynthetic pigments (chlorophyll a, b, and total chlorophyll), stomatal index, and percentage of closed stomata relative to both Ebb-and-Flow and RITA®. The TIB® and RITA® systems showed a 99% shoot survival, while Ebb-and-Flow yielded 86% survival. In conclusion, TIS design and type affect a number of physiological processes and in vitro development, with TIB® as a feasible option for the commercial micropropagation of anthurium.

COMMUNITY-ACQUIRED PNEUMONIA IN HIV-INFECTED SUBJECTS: MICROFLORA, ANTIBIOTIC RESISTANCE: DEPENDENCE ON THE LEVELS OF CD4 LYMPHOCYTES

Purulent inflammatory diseases of various types and etiology comprise major causes of death among the HIV-infected individuals. The purpose of this work was to determine a variety of communityacquired pathogens causing pneumonia, their antibiotic resistance profiles, and dependence on the CD4 lymphocyte levels, as well as identification of methicillin-resistant Staphylococcus aureus species and their molecular genetic characteristics in HIV-infected patients from the Krasnoyarsk City. Over the period of 2012 to 2016, we have examined 152 HIV-infected patients at the Clinical Pulmonology Department with a verified diagnosis of community-acquired pneumonia. Sputum specimens, bronchoalveolar lavage, pleural fluid, washings, pleural pus, as well as nasal and pharyngeal smears were studied for microflora, and blood tests for sterility were performed in these patients, by means of bacteriological techniques. Antibiotic sensitivity was determined by the disc diffusion method; drug sensitivity of staphylococci was performed by screening, PCR technique, serial dilution in semi-solid medium, according to the CLSI and EUCAST recommendations. PCR, M-PCR, and gene sequencing were applied for genotyping and determination of their genetic features. The results were processed with WHONET digital program (WHO). The significance level was p < 0.05. During the entire study period, the yeast-like fungi of Candida genus (30.4 and 35.6%) were consistently isolated from HIV-infected patients. These microorganisms were isolated in a pure cultures at etiologically significant amounts from one-third of the HIV-infected cohort. At the same time, they formed active associations, mostly with Enterobacteriaceae family members. At the same time, Candida fungi were most frequently detected in the lower respiratory tract of those HIV-infected persons who showed severe immunodeficiency (CD4 cell levels < 200 cells/µl). We have also isolated non-fermenting Gram-negative bacteria (12.9%), staphylococci (8.9%), and Enterobacteriaceae (4.4%). The microorganisms were characterized by polyresistance to antimicrobial agents. The MRSA clone circulating in the HIV-infected cohort was characterized as ST239/spa3(t037) /agr1/SCCmecIII.1.1.2 (IIIA)/coaIV/tst+ with high virulence and multiresistance levels. Hence, we have found a number of poly-resistant microorganisms playing a role for development of community-acquired pneumonia in HIV-infected patients, i.e., Candida spp, Gram-negative microorganisms, MRSA, often presenting a component of microbial associations. Candida fungi were detected most often in the HIV-infected individuals with severe immunodeficiency, at the CD4 level of < 200 cells/µl. High detection frequency of such microflora requires some modifications of antimicrobial therapy in HIV-infected subjects affected by the community-acquired pneumonia.

In vitro establishment of Monstera acuminata Koch and Monstera deliciosa Liebm

In this paper we present the bases for the aseptic establishment of Monstera acuminata Koch and Monstera deliciosa Liebm (Araceae) from leaves and the induction of in vitro organogenesis of M. acuminata K. from discs of stems of young shoots. Different disinfection protocols were applied to mature leaves and young shoots, from which foliar explants of 1 cm2 of both species were extracted, and stem disks approximately 1 mm of thick in M. acuminata K. The explants were established in semi-solid media with different hormonal treatments during the stage of aseptic establishment and induction of organogenesis. Disinfection with 3 % of sodium hypochlorite (NaClO) for 20 min and 50 % Murashige y Skoog medium (1962) with PPM, led to less oxidation in the leaf explants of both species. All the explants in the two treatments belonging to M. deliciosa, grown in medium added with PPM and different disinfection protocols, survived, did not present contamination and more than 80 % maintained answer’s capacity until 49 days of culture. After 35 days of culture, with disinfection in Tween 20 + Ethanol 20% + 2.5% NaClO, and sowing of the explants in the MS medium added with 1 mg/l of BAP, 0.5 mg/l of AIA and 0.1 mg/l of ANA were indueces seven new shoots of stem disks. M. deliciosa had better ability to adapt to in vitro conditions. Advances in the establishment and induction of organogenesis in native araceas for wicker production are the basis for ex situ conservation of local populations.

Validation of a semi automatic device to standardize quantification of Colony-Forming Unit (CFU) on hematopoietic stem cell products

Accurate characterization of hematopoietic stem cells (HSC) products is needed to better anticipate the hematopoietic reconstitution and the outcome in patients. Although CD34+ viable cells enumeration is a key predictor of time to correction of aplasia, it does not fully inform about functionality of cells contained in the graft. CFU assay is the gold standard in vitro potency assay to assess clonogenicity of HSC and consists on the count and identification of colonies several days after culture in a semi solid media. Manual count of colonies with optic microscope is the most commonly used method but its important variability and subjectivity hinders the universal implementation of this potency assay. The aim of this study is to validate a standardized method using the STEMvision™ system, the first semi-automated instrument for imaging and scoring hematopoietic colonies, according to French and European recommendations. Results obtained highlight better performance criteria with STEMvision™ system than the manual method. This semi-automatic device tends to reduce the coefficients of variation of repeatability, inter-operator variability and intermediate precision. This newly available platform could represent an interesting option, significantly improving performances of CFU assays used for the characterization of hematopoietic progenitors.

Paraburkholderia tropica as a plant-growth–promoting bacterium in barley: characterization of tissues colonization by culture-dependent and -independent techniques for use as an agronomic bioinput

Characterization of barley-tissue–colonization efficiency (Hordeum vulgare L.) by Paraburkholderia tropica MTo-293 after seed inoculation was studied in two plant-growth systems: (1) in flasks with semisolid agar-containing sterile medium, as a suitable environment to study plant-bacteria interaction and also optimize molecular-biology techniques, and (2) in a pot-substrate system as an approach to understand their potential behavior as bioinput. Culture-dependent techniques were implemented to quantify surface and endophytic bacterial populations in plant tissues. Culture-independent techniques were employed to detect and localize the inoculated bacteria in tissue samples along with evaluating the biofilm-forming capability by epifluorescent, confocal-laser-scanning, and scanning-electron microscopy. Plant-growth parameters were measured to evaluate the effects of the inoculated bacteria on the development of the barley plants. Paraburkholderia tropica grew as a biofilm on both abiotic and biotic surfaces and efficiently colonized barley roots and stems in plants grown in flasks in presence of other microorganisms. The bacteria was localized on root surfaces, hairs, and central-cylinder areas. Paraburkholderia tropica also colonized the roots and stems in plants grown in the pot-substrate system. Although no endophytic root colonization occurred, the presence of the inoculated bacteria improved the aerial weight. A nested PCR detected P. tropica in the tissue samples. Paraburkholderia tropica MTo-293 was characterized as an efficient biofilm-forming and barley-tissue–colonizing bacterium despite the presence of other microorganisms, but root endophytic colonization resulted dependent on the plant-growth system. Molecular-biology techniques were optimized, and also, its presence was correlated with plant-growth–promoting activity.

Sub-MIC effects of a proline-rich antibacterial peptide on clinical isolates of Acinetobacter baumannii.

Acinetobacter baumannii is one of the most important nosocomial pathogens, mainly due to its ability to accumulate antibiotic-resistances and to persist in the hospital environment - characteristics related to biofilm production. It is well-known that A. baumannii is inhibited by the proline-rich peptide Bac7(1-35), but its putative effects at sub-MICs were never considered. We examined the sub-MIC effect of Bac7(1-35) on the growth rate, resistance induction and some A. baumannii features linked to virulence. Growth kinetics in the presence of sub-MICs of Bac7(1-35) were evaluated spectrophotometrically. Peptide uptake was quantified by cytometric analysis. The ability of Bac7(1-35) to interfere with biofilm production was investigated by the crystal violet method and confocal microscopy. Bacterial motility was observed at the interphase between a layer of a semi-solid medium and the polystyrene bottom of a Petri dish. The induction of resistance was evaluated after serial passages with sub-MICs of the peptide. Although the MIC of Bac7(1-35) was between 2-4 µM for all tested strains, its effect on the growth rate at sub-MICs was strain-dependent and correlated with the amount of peptide internalized by each strain. Sub-MICs of Bac7(1-35) induced a strongly strain-dependent effect on biofilm formation and reduced motility in almost all strains, but interestingly the peptide did not induce resistance. Bac7(1-35) is internalized into A. baumannii and is able to inhibit biofilm formation and bacterial motility, without inducing resistance. This study stresses the importance of considering possible effects that antimicrobials could have at sub-MICs, mimicking a common condition during antibiotic treatment.

Biofilm formation by Aquaspirillum spp. and saprophytic Leptospira spp. isolated from environmental source of Argentine

Leptospirosis is a zoonotic disease of global distribution, caused by bacteria of the genus Leptospira. These spirochetes are living organisms free of mud and water; pathogenic leptospires can survive several days in fresh water when pH and temperature are adequate. During 2016, water samples were collected from Callvú Leovú stream (Azul, Buenos Aires); samples were inoculated in liquid EMJH medium and incubated at 28° C for 90 days. Six isolates of saprophytic leptospires and six of spirils (Aquaspirillum spp.) were obtained. The isolates were inoculated in EMJH (liquid and semi-solid) medium and sterile stream water at 4-10° C and 28-30° C; development was observed periodically using dark field microscopy. Both bacteria (alone or together) grew exponentially in first three weeks in all media incubated at 28-30° C; the semi-solid medium was the most efficient at 28-30° C of incubation, and the bacteria remained viable after 16 weeks. At 4-8° C both bacteria remained undetectable but viable in media incubated at 4-8° C for three weeks until the temperature was optimal (thermal stimulation). Leptospires developed in all media used and remained viable for 112 to 168 days (at 4-8° C incubation) in liquid media. The formation of cellular aggregate between Leptospira spp. and Aquaspirillum spp. was independent at the incubation temperature. These results suggest that Aquaspirillum spp. coexists with the genus Leptospira in surface waters, and their presence may indicate possible circulation of leptospires.

PF239 COMBINED TARGETING OF BCL-2 AND SET-PP2A INTERACTION INDUCES POTENT AND SYNERGISTIC ANTI-LEUKEMIC EFFECTS ON ACUTE MYELOID LEUKEMIA

Background: Acute myeloid leukemia (AML) is an aggressive malignancy that arises from the transformation of hematopoietic stem cells. Most AML patients are older than 60 years, and in this group only 5–15% of cases are cured (Döhner et al., 2015). Therefore, it is necessary to explore new therapeutic options. Overexpression of the anti-apoptotic BCL-2 protein is a common finding in AML. ABT-199 (Venetoclax), a potent and selective inhibitor of BCL-2, has been recently developed. However, clinical activity of ABT-199 monotherapy is modest in AML (Konopleva et al., 2016), which could be explained by the genetic and epigenetic heterogeneity of AML. Our group has shown that inactivation of PP2A is present in 78% of AML patients, and that FTY720 treatment restores PP2A activity by disrupting the SET-PP2A interaction, thereby inhibiting proliferation of AML cells (Pippa et al., 2014). Since PP2A promotes apoptosis by affecting the phosphorylation state of a variety of pro- and anti-apoptotic factors, we hypothesized that BCL-2 targeting could synergize with PP2A re-activation and induce synergistic anti-leukemic effects. Aims: To investigate whether the combination of ABT-199 with FTY720 could induce synergistic anti-leukemic effects in vitro and in vivo. Methods: A panel of six human AML cell lines was used to perform cell culture experiments. To generate zebrafish AML xenograft models, AML cell lines were treated for 24 h with ABT-199, FTY720 or their combination. Subsequently, cells were stained with a fluorescent cell tracker and injected into the yolk sac of 48 hours-post-fecundation wild type zebrafish embryos. After injection, cell proliferation and colonization index were measured. Colony-Forming-Unit (CFU) assays in semi-solid medium were performed on patient-derived AML cells isolated from three patients with AML at diagnosis. Results: GI50 values for ABT-199 and FTY720 showed that HL-60, MV4–11 and MOLM-13 were the most sensitive cell lines to ABT-199, while OCIAML-3 and HEL cells were resistant to BCL-2 specific inhibition (GI50 value greater than 10 μM). Regarding FTY720 treatment, we observed that OCIAML-3 and MV4–11 were the most sensitive cell lines, while HEL cells were FTY720-resistant. By western-blot analyses, we ascertained that sensitivity to ABT-199 was dependent on BCL-2, MCL-1 and BCL-XL expression levels, and sensitivity to FTY720 was dependent on PP2A and SET expression. Next, survival of AML cells was analyzed after exposure to ABT-199, FTY720, or their combination at different concentrations (accordingly on the above GI50 calculated values). Analyses of the combination index values showed that combination of ABT-199 and FTY720 had a potent and synergistic anti-leukemic effect, even in OCI-AML3 cells, which were ABT-199-resistant cells. Mechanistically, combination of ABT-199 and FTY720 induced a synergistic pro-apoptotic activity by increasing significantly the number of Annexin/IP positive cells, the activity of Caspases 3/7, and PARP cleavage. Compared to single treatments, treatment with ABT-199 plus FTY720 also reduced significantly cell proliferation and/or cell invasive potential of HL-60, MOLM-13, and MV4–11 cells in zebrafish xenograft models. Furthermore, we demonstrated in primary-AML cells that the combined treatment significantly decreased the number as well as the size of CFUs. Summary/Conclusion: Combined targeting of BCL-2 and the SET-PP2A interaction has potent and synergistic anti-leukemic effects in vitro and in vivo. Our findings provide a rational basis for further testing of novel combined therapies with ABT-199 and FTY720 in AML patients.

Cell Suspension Culture of Plumbago europaea L. Towards Production of Plumbagin.

BackgroundPlumbagin is as an important bioactive secondary metabolite found in the roots of Plumbago spp. The only one species, Plumbago europaea L., grows wild in Iran. The therapeutic use of plumbagin is limited due to its insufficient supply from the natural sources as the plants grow slowly and take several years to produce quality roots.ObjectivesTo develop an efficient protocol for the establishment of callus and cell suspension cultures of P. europaea and to evaluate production of plumbagin in callus and cell suspension cultures of P. europaea for the first time.Material and MethodsStems and leaves explants were cultured on agar solidified (7% w/v) MS media, supplemented with different combination of 2, 4-D and Kin or 6-Benzylaminopurin (BA) for callus induction. The rapid growing calli were cultured in liquid Murashige and Skoog (MS) media in agitated condition for establishing cell suspension cultures of P. europaea. Moreover, the effects of light and dark conditions on the cell growth, cell viability and plumbagin production in cell suspension cultures of P. europaea were assessed.ResultsFriable calli were successfully induced using stem segments of P. europaea in semisolid MS medium supplemented with 1 mg.L-1 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 0.5 mg.L-1of kinetin (Kin). Optimal cell growth was obtained when the cells were grown in MS liquid media supplemented with 1 mg.L-1 2, 4-D and 0.5 mg.L-1 kinetin with an initial cell density of ~3×105 cells per ml incubated in the dark at 25 ± 1 °C. Growth curve revealed that the maximum cell growth rate (14.83×105 cells per ml) achieved on the day 18 and the highest plumbagin content (0.9 mg.g-1 Dry Cell Weight (DCW)) in the cells was obtained at the late exponential phase under dark condition which determined by High Performance Liquid Chromatography (HPLC) technique. Based on the obtained results, cell viability remained around 82.73% during the 18 days of cell culture in darkness. These suspension cultures showed continuous and stable production of plumbagin.ConclusionsOur study suggests that cell suspension cultures of P. europaea represent an effective system for biosynthesis and production of plumbagin as a valuable bioactive compound.