Semisolid Medium Research Articles

Contributions of recombination and repair proteins to telomere maintenance in telomerase-positive and negative Ustilago maydis.

Homologous recombination and repair factors are known to promote both telomere replication and recombination-based telomere extension. Herein, we address the diverse contributions of several recombination/repair proteins to telomere maintenance inUstilago maydis, a fungus that bears strong resemblance to mammals with respect to telomere regulation and recombination mechanisms. In telomerase-positive U. maydis, deletion of rad51 and blm separately caused shortened but stably maintained telomeres, whereas deletion of both engendered similar telomere loss, suggesting that the repair proteins help to resolve similar problems in telomere replication. In telomerase-negative cells, the loss of Rad51 or Brh2 caused accelerated senescence and failure to generate survivors on semi-solid medium. However, slow growing survivors can be isolated through continuous liquid culturing, and these survivors exhibit type II-like as well as ALT-like telomere features. In contrast, the trt1Δ blmΔ double mutant gives rise to survivors as readily as the trt1Δ single mutant, and like the single mutant survivors, exhibit almost exclusively type I-like telomere features. In addition, we observed direct physical interactions between Blm and two telomere-binding proteins, which may thus recruit or regulate Blm at telomeres. Our findings provide the basis for further analyzing the interplays between telomerase, telomere replication, and telomere recombination.

Functional Analysis of Erythroid Progenitors by Colony-Forming Assays.

The capacity of erythroid-lineage progenitors to form colonies of maturing red blood cells in semisolid media has provided a functional assay for these progenitors and has greatly contributed to our understanding of erythropoiesis. Studies since the 1970s have led to the development of a model of the erythron, whereby the earliest erythroid-committed progenitor, the immature burst-forming unit erythroid (BFU-E), gives rise sequentially to late-stage BFU-E and to colony-forming units erythroid (CFU-E). CFU-E give rise, in turn, to maturing erythroblast precursors that hemoglobinize. It is these terminal cells that comprise the mature colonies of erythroid cells derived from the progenitors cultured in semisolid media. The in vitro generation of erythroid colonies requires cytokine support, most notably erythropoietin (EPO), which is critical for CFU-E survival and for promoting erythroblast maturation.During mouse embryogenesis, a transient population of primitive erythroid colony-forming progenitors (EryP-CFC) emerges in the yolk sac and gives rise to a wave of maturing primitive erythroblasts in the fetal bloodstream. This wave of EryP-CFC is followed closely by a wave of BFU-E in the yolk sac that enter the bloodstream and seed the fetal liver to generate the first definitive red cells in the fetus. BFU-E in the fetal liver, unlike those in the adult bone marrow, can give rise to colonies in vitro when cultured with EPO alone and also are more sensitive to EPO levels. Here, we describe methods for the in vitro culture of murine embryonic (primitive) and fetal/adult (definitive) erythroid progenitors in semisolid media.

ExVivo Selection of Transduced Hematopoietic Stem Cells for Gene Therapy of β-Hemoglobinopathies.

Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications. Here, we fused codon-optimized puromycin N-acetyltransferase to herpes simplex virus thymidine kinase. When expressed from a ubiquitous promoter within a complex lentiviral vector comprising the βAT87Q-globin gene, viral titers and therapeutic gene expression were maintained at effective levels. Complete selection and preservation of transduced HSCs were achieved after brief exposure to puromycin in the presence of MDR1 blocking agents, suggesting the procedure’s suitability for human clinical applications while affording the additional safety of conditional suicide.

Plating Bacteriophage M13.

A plaque of bacteriophage M13 derives from infection of a single bacterium by a single virus particle. The progeny particles infect neighboring bacteria, which, in turn, release another generation of daughter virus particles. If the bacteria are growing in semisolid medium (e.g., containing agar or agarose), then the diffusion of the progeny particles is limited. Cells infected with bacteriophage M13 are not killed, but have a longer generation time than uninfected Escherichia coli In consequence, plaques appear as areas of slower-growing cells on a faster-growing lawn of bacterial cells. This protocol describes plating of bacteriophage M13 stocks. Plaques are readily detectable on top agar after 4-8 h of incubation at 37°C.

Auxin-cytokinin synergism in vitro for producing genetically stable plants of Ruta graveolens using shoot tip meristems.

An efficient micropropagation protocol was developed for Ruta graveolens Linn. using shoot tip meristems derived from a 4-month-old field grown plant. In vitro shoot regeneration and proliferation was accomplished on Murashige and Skoogs (MS) semi-solid medium in addition to different doses of cytokinins viz.6- benzyl adenine (BA), Kinetin (Kn) or 2-isopetynyl adenine (2iP), singly or in combination with auxins viz. indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Highest regeneration frequency (27.6%) was obtained on (MS) medium composed of BA (10 µM) with maximum number (9.4) of shoots and 4.3 cm shoot length after 4 weeks of incubation. Among various combinations tried best regeneration frequency (71%) of multiple shoot formation with highest number (12.6) of shoots per shoot tip explants were achieved in MS medium augmented with a combination BA (10.0 µM) and NAA (2.5 µM) after 4 weeks of incubation. The optimum frequency (97%) of rhizogenesis was achieved on half-strength MS medium having 0.5 µM IBA after 4 weeks of incubation. Tissue culture raised plantlets with 5–7 fully opened leaves with healthy root system were successfully acclimatized off in Soilrite™ with 80% survival rate followed by transportation to normal soil under natural light. Genetic stability among in vitro raised progeny was evaluated by ISSR and RAPD markers. The entire banding pattern revealed from in vitro regenerated plants was monomorphic to the donor. The present protocol provides an alternative option for commercial propagation and fruitful setting up of genetically uniform progeny for sustainable utilization and germplasm preservation.

Non-aerated liquid culture promotes shoot organogenesis in Eucalyptus globulus Labill

Eucalyptus is very recalcitrant to in vitro culture. In this research, an efficient shoot organogenesis system was developed using 60-day-old plants of Eucalyptus globulus grown in vitro and non-aerated liquid medium to improve shoot proliferation. Cultures were initiated with hypocotyls and leaf segments from plantlets cultivated on semisolid ½ MS modified medium supplemented with 4.44 µM 6-Benzyladenine (BA) and 16.1 µM 1-Naphthaleneacetic acid (NAA). Calli were transferred to shoot induction medium, with either 0.5 or 2.7 µM NAA. Shoot multiplication was carried out on 4.44 µM BA + 0.5 µM NAA medium, and semisolid and non-aerated liquid systems were compared for improving shoot proliferation. Rooting of adventitious shoots was evaluated on medium containing NAA or Indole-3-butyric acid -IBA (5 and 16 µM). Callogenesis was obtained from both types of explants, although shoot formation was only obtained from leaf-derived calli. Shoot proliferation on 4.44 µM BA + 0.5 µM NAA resulted in the most shoots/callus. Non-aerated liquid medium was more efficient in promoting shoot multiplication (53.5 shoots/callus) than was semisolid medium (28.5 shoots/callus). Levels of phenolic compounds were significantly reduced in the shoots cultivated in liquid medium. Efficient rooting (76%) was obtained using 16 µM IBA.

Micropropagation of axillary shoots of Salix viminalis using a temporary immersion system

Key message Culturing axillary shoot explants of willow in liquid medium in plantform™ and RITA ® vessels increased the multiplication coefficient relative to that obtained by culturing the explants in semisolid medium in jars.

Assessing the influence of subcultures and liquid medium during somatic embryogenesis and plant regeneration in oil palm (Elaeis guineensis Jacq.)

ABSTRACTThe current analysis describes an improved protocol for somatic embryogenesis and plant regeneration in oil palm (Elaeis guineensis) through liquid medium, and assesses the influence of successive subcultures during induction of calluses in three Brazilian oil palm varieties. Calluses were induced in a Murashige and Skoog (MS) medium with 450 Picloram, 0.5 g L−1 glutamine, 2.5 g L−1 activated charcoal, 30 g L−1 sucrose, and solidified with 2.5 g L−1 Phytagel. In a first experiment, the effect of continued subculture of explants every 30 days to fresh culture medium was determined. During a second experiment, part of the embryogenic calluses obtained were transferred to a liquid medium under agitation, consisting of MS with 5 µM picloram or 2,4-dichlorophenoxyacetic acid (2,4-D). After 210 days, the calluses were transferred to semi-solid media for differentiating somatic embryos. It was observed that continued subculture of explants monthly was a determinant in stimulating and improving the formation of embryogenic calluses. Embryogenic calluses in liquid medium with 2,4-D significantly improved the percentage of differentiated somatic embryos (up to 80.2%), with the largest amount of torpedo embryos (8.3 per callus). Regenerated plants with roots were individualised and transferred to a greenhouse, with close to 95% survival.

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.

Exploring in vitro germplasm conservation options for sugarcane (Saccharum spp. hybrids) in South Africa

In vitro plantlets of sugarcane cultivar NCo310 were maintained in slow growth conditions at both 18 and 24°C and on four semi-solid media: SG1—Murashige and Skoog (MS) salts and vitamins with 20 g L−1 sucrose, SG2—½ MS with 10 g L−1 sucrose, SG3—MS with 20 g L−1 sucrose and 1 mg L−1 abscisic acid (ABA), and SG4—½ MS with 10 g L−1 sucrose and 1 mg L−1 ABA. After 8, 12, 24, 36, and 48 mo shoot multiplication rates were recorded, shoots were removed from storage and subcultured every 2 wk on SG1 with 0.015 mg L−1 kinetin and 0.1 mg L−1 benzyl aminopurine for 2 mo. At 18°C, all media supported storage for 48 mo with subculturing every 12 mo. Shoot multiplication post-retrieval was significantly higher on the SG2 medium compared with the non-stored control (362 ± 84 and 126 ± 26 shoots per recovered shoot after 2 mo, respectively). In addition, shoots could be maintained for 48 mo on SG2 medium with one subculture without compromising post-storage multiplication ability. At 24°C, storage on all four media supported recovery and multiplication of shoots for 8 mo and only SG2 medium facilitated survival for 12 mo. There was no advantage to incorporating ABA into the storage media, regardless of the temperature and storage time. Cryopreservation of cultivar NCo376 in vitro-derived shoot meristems using the V-cryo-plate method demonstrated that the sucrose concentration in the loading solution (0.8–1.8 M) had no significant effect on survival of the meristems, which ranged from 41.7 ± 4.8 to 69.4 ± 10%.

Steps on How a Phantom can be 3D Printed and Embedded within a Medium Suitable for Training of Ultrasound-Guided Procedures

Practice is required for acquiring procedural proficiency and this can be obtained from clinical practice, or more sensibly, having novices first practice the skill on an inanimate phantom or trainer. Commercially produced phantoms are expensive and 3D printing is becoming more cost effective. In our example, we have used a 3D-printed thoracic spine. This allows practitioners to improve their ultrasound-guided regional anesthesia needle imaging skills through deliberate practice. In this report, we describe in detail the requirements for producing a 3D-printed phantom comprising a 3D segment of thoracic spine set within a semi-solid medium.

Comparison of different in vitro micropropagation methods of Stevia rebaudiana B. including temporary immersion bioreactor (BIT®)

Stevia rebaudiana is beneficial to treat diabetes because of its low-calorie glucoside sweeteners. Natural and vegetative propagation are inefficient. In vitro techniques are an attractive alternative but low propagation and reproducibility rates have been reported. Therefore, different ways to increase natural sweetener production in vitro, such as BIT®, are required. We compared semisolid medium, liquid medium and BIT® in terms of Stevia biomass and steviol glycosides production. At 21 days of culture, morphological quality of BIT®-derived shoots was best and coupled with shoot fresh and dry weight that were more than seven times higher in BIT® compared with micropropagation in liquid or semisolid media. In turn, the total content of steviol glycosides produced was also higher in bioreactors. The usefulness of BIT® to produce plant metabolites in vitro is again demonstrated, even if additional experiments are required to increase the economic efficiency of the process.

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.

Abstract 2371: Oncogenic transformation of FLT3-ITD is partially dependent on tyrosine 842 in the activation loop

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease of the blood. About 30 % of AML patients carry an oncogenic mutant of the type III receptor tyrosine kinase FLT3. Among the various mutations in FLT3, the internal tandem duplication (ITD) mutations in the juxtamembrane domain are the most common type of mutation, while point mutations in the kinase domain also occur. Although oncogenic FLT3 mutants are constitutively active, the wild-type receptor needs its ligand (FLT3 ligand, FL) for activation. Ligand binding to the receptor induces dimerization, auto-phosphorylation, and activation of the receptor. Tyrosine phosphorylation sites on FLT3 constitute docking sites for interacting proteins that transduce pro-survival and proliferative signals. The so-called activation loop tyrosine residue (in FLT3, Y842) is well conserved in all receptor tyrosine kinases and long been known essential for the activity of some but not all receptor tyrosine kinases. Recently we have shown that activation loop tyrosine residue in KIT is not essential for its activation but plays an important role in receptor stabilization and downstream signaling. However, so far the role of FLT3 activation loop tyrosine residue remains unknown. We generated 32D cell lines carrying a Y-to-F mutant (Y842F) of FLT3 in either the wild-type or ITD background to study the role of this tyrosine residue. We show that the Y842 residue is not necessary for FLT3 activation or stability but is required for oncogenic transformation. We observed that Y842F mutation results in reduced cell viability, proliferation and in vitro colony formation in semi-solid medium. In addition, cells expressing the Y842F mutant in FLT3-ITD background display a significant reduction in tumor volume and weight in a xenograft model in nude mice. Gene set enrichment analysis (GESA) shows that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we demonstrated that phosphorylated Y842 is one of the binding sites of the protein tyrosine phosphataseSHP2, which is required for activation of signaling through the RAS/ERK pathway. Taken together, our data suggest that activation loop tyrosine residue in FLT3 plays an important role in the cellular transformation which is regulated by RAS/ERK signaling. Citation Format: Julhash U. Kazi, Lars Rönnstrand. Oncogenic transformation of FLT3-ITD is partially dependent on tyrosine 842 in the activation loop [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2371. doi:10.1158/1538-7445.AM2017-2371

Respuesta de cultivares de Musa spp. al estrés hídrico in vitro inducido con polietilenglicol 6000

Los plátanos y bananos son cultivos sensibles al déficit hídrico. Las sequías cada vez más prolongadas sugieren la necesidad de obtener plantas tolerantes a este factor; la selección temprana de estas plantas, comparada con la selección en campo, permite ahorrar tiempo y trabajar con mayores volúmenes de individuos. Para ello es conveniente contar con cultivares patrones cuya respuesta al déficit hídrico in vitro sea favorable. El objetivo del presente trabajo fue determinar la respuesta de cultivares de Musa spp. con diferente composición genómica al estrés hídrico inducido in vitro con polietilenglicol 6000 (PEG-6000). Se estudiaron los cultivares ‘Pelipita’ (ABB), ‘Manzano’ (AAB) y ‘Grande naine’ (AAA). El estrés se indujo con 30 g/L de PEG-6000 en medio de cultivo semisólido de multiplicación. A los 30 días se evaluaron variables indicadoras de estrés morfológicas (altura y número de brotes por explante), fisiológicas (masa fresca y masa seca) y bioquímicas (contenido prolina, peróxido de hidrógeno y malondialdehido). En el cultivar ‘Pelipita’ se afectó solamente la altura de las plantas, mientras que en los demás se afectaron todas las variables excepto la masa seca en el ‘Manzano’. En este último y en el ‘Grande naine’ se incrementó la prolina, el peróxido de hidrógeno y el malondialdehido, lo que evidenció un mayor estrés oxidativo y daño en las membranas celulares. Los cultivares estudiados, pudieran emplearse como controles de tolerancia (‘Pelipita’) y sensibilidad (‘Grande naine’ y ‘Manzano’) en la selección in vitro de plantas tolerantes a la sequía, en futuros programas de mejoramiento genético.

Abstract 4051: Combination efficacy of HDAC inhibitor vorinostat and CDK-4/6 dual inhibitor palbociclib against therapy-resistant mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma accounting for about 7% of all non-Hodgkins lymphoma. While multiple therapy regimens are available to treat MCL patients, ultimately relapse from therapy-resistant MCL, making MCL carry the worst prognosis of all B cell lymphomas. Emerging evidence suggest that, dysregulated histone deacetylases (HDACs) and the key molecules of cell cycle regulator cyclin-dependent kinases (CDKs) have been shown to be commonly associated with many lymphomas including MCL, and are considered as promising targets for relapsed lymphoma therapy. Several inhibitors of these target pathways/molecules are in clinical trials as monotherapies or in combination with other anticancer agents. Therefore, in this study, we investigated the single agent and combination efficacies of the HDACs inhibitor vorinostat, and CDK-4/6 dual inhibitor palbociclib on MCL cell growth/survival and underlying molecular mechanism(s) using different MCL cell lines (Granta 519, Jeko-1 and JVM-2) including therapy-resistant MCL cell lines derived from Granta-519 (GRL, GRK and GRR). We used MTT assay to measure the survival and proliferation, agar colony formation assay to determine the effects of these inhibitors on the anchorage independent growth, Annexin-V analyses to examine the cells undergoing apoptosis following treatments and western blot analyses to determine the expression levels of target molecules including pRb, histone acetylation, cyclin-D1 and Bcl-2. The concentration of these inhibitors used in this study were similar to the published literature including sub-IC50 levels determined in our laboratory. Our results showed that both inhibitors as single agents or combined, significantly suppressed the cell growth, and induced apoptosis in therapy-resistant and other MCL lines in vitro. The single agent and combined anti-lymphoma efficacies of these inhibitors were further confirmed with the colony formation ability of the MCL cells grown in 0.3% agar semisolid media. In addition, combination of vorinostat and palbociclib treatment significantly inhibited the activation of phosphorylated-retinoblastoma (pRb) protein, a key molecule of cell cycle and increased the expression of acetylated-Histone H3 (H3-Ac) as determined by western blot analyses. Subsequently, the expression of Cyclin D1 (proliferation, MCL hallmark) and anti-apoptotic Bcl-2 proteins were also downregulated by these inhibitors. Together, our findings suggest that combination of vorinostat and palbociclib showed a significant synergistic anti-MCL activity by targeting associated pathways/molecules. This targeted approach warrants further preclinical evaluation for translation to the clinic. Citation Format: Nagendra K. Chaturvedi, Nathan D. Hatch, Garrett L. Sutton, Matthew J. Kling, Julie M. Vose, Shantaram S. Joshi. Combination efficacy of HDAC inhibitor vorinostat and CDK-4/6 dual inhibitor palbociclib against therapy-resistant mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4051. doi:10.1158/1538-7445.AM2017-4051

Dermatopathy Caused by Enterobacter aerogenes and Pseudomonas aeruginosa in Boa constrictor amarali

Background: Bacterial diseases are the main cause of the high mortality rates of snakes, especially those caused by gramnegative agents. However, studies on dermatopathy caused by these bacterial agents in snakes are scarce; and no reports have been found on Enterobacter aerogenes as causative agent of dermatopathy in snake species. Thus, the objective of this study was to describe the clinical signs, and lesion evolution of a dermatopathy in a male snake (Boa constrictor amarali) specimen of approximately seven years old; and to describe the isolation and identification of the Enterobacter aerogenes and Pseudomonas aeruginosa agents involved in the cause of this disease.Case: The Boa constrictor amarali evaluated presented blackened cutaneous lesions in the dorsal, snout-vent and tail regions; and well-defined subcutaneous nodules of 2.0-3.0 cm diameter, with soft consistency, reddish color, cutaneous flaccidity, and areas of scale ulceration in the dorsolateral region. The clinical evaluation of the animal showed dehydration signs and pale mucous membranes. The blackened lesions were subjected to mycological analysis - after procedure of deep scale scraping - which showed presence of septate hyphae. The nodule was punctured for microbiological and biochemical analysis. The sample was collected with a sterilized alginate cotton tip swab, and was stored in a plastic tube containing a semi-solid Stuart transport medium, for microbiological analysis. Then, this sample was incubated in a bacteriological oven at 37°C for 24 h. Typical colonies of Pseudomonas and Enterobacter grew on MacConkey agar medium; these bacteria were identified by the colony morphology and their typical odor. The colonies grown in MacConkey agar were also identified through biochemical tests in the mediums: Phenol red, Lysine, Phenylalanine, Citrate, Urea and SIM (Sulfide, Indole, Motility). The results of these tests were able to confirm and identify the P. aeruginosa and E. aerogenes species. The animal died within 36 h, before the identification of the causative agents of the disease, thus, no pharmacological interference was possible.Discussion: Immunodepression, malnutrition, and temperatures and humidity outside the animal thermal comfort zone, are predisposing factors for the development of bacterial diseases in reptiles. Little information about pathogen agents affecting Boa constrictor specimens in their native area is available; however, captive snakes are subject to a wide variety of diseases - most of which caused or intensified by the captivity conditions. Among the bacteria involved in reptile diseases, few are primary causative agents. In general, clinical bacterial infections tend to be secondary to viral infections. The bacterial agents found in this study are commonly described in scientific literature with location in the oral cavity, differently from the results found in this study. Moreover, the bacterium E. aerogenes has not yet been described in other studies as a causative agent of dermatopathy. Reptiles are considered reservoirs of important zoonotic microorganisms, such as P. aeruginosa, which can be transmitted by fecal contact, bites and wounds. However, the little information on P. aeruginosa in captive reptiles indicates the need for further studies to establish its zoonotic potential. A most adequate management conditions for the snake species could have decreased the severity of the lesions. The occurrence of P. aeruginosa and E. aerogenes found in this work may alert professionals for future clinical suspicions and adequate therapeutic management.

Evaluación de los sistemas de cultivo semisólido y BIT en la multiplicación in vitro de Juglans neotrópica

The purpose of this study was to evaluate two culture systems, semi-solid and temporary immersion in bioreactors (BIT), for the micropropagation of Juglans neotropica, using zygotic embryos. Additionally, in the BIT culture system also the effect of the frequency of immersion and forced aeration on the micropropagation was examined. The culture growth of the shoots was measured by the following characteristics: number and size of the shoots, number of leaves, and the wet and dry weight. No statistical differences were detected. In general, the BIT immersion system produced an equivalent number of plants of similar characteristics as the embryos propagated in the traditional semi-solid medium method. However, BITs produced more shoots, forced ventilation resulted in less hyperhydric shoots and the BIT system was considerable cheaper, resulting in an average production cost of $0.21 per plant versus $0.40 in the semi-solid propagation method.

Measuring the aggregation of CHO cells prior to single cell cloning allows a more accurate determination of the probability of clonality.

The manufacturing process for biotherapeutics is closely regulated by the Food and Drug Administration (FDA), European Medicines Agency (EMA) and other regulatory agencies worldwide. To ensure consistency of the product of a manufacturing cell line, International Committee on Harmonization guidelines (Q5D, 1997) state that the cell substrate should be derived from a single cell progenitor, i.e., clonal.Cell lines in suspension culture may naturally revert to cell adhesion in the form of doublets, triplets and higher order structures of clustered cells. We can show evidence of a single colony from limiting dilution cloning or in semi-solid media, but we cannot determine the number of cells from which the colony originated. To address this, we have used the ViCELL® XR (Beckman Coulter, High Wycombe, UK) cell viability analyzer to determine the proportion of clusters of two or more cells in a sample of the cell suspension immediately prior to cloning. Here, we show data to define the accuracy of the ViCELL for characterizing a cell suspension and summarize the statistical model combining two or more rounds of cloning to derive the probability of clonality. The resulting statistical model is applied to cloning in semi-solid medium, but could equally be applied to a limiting dilution cloning process. We also describe approaches to reduce cell clusters to generate a cell line with a high probability of clonality from a CHO host lineage. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:593-601, 2018.