Experimental Adaptation of a strain of the plague microbe to lyophilization process

The use of lyophilization as a means of preserving commercial properties of the dried live plague vaccine is closely linked to a number ofresistant microbial cells surviving in the preparation after microbial population exposure to such stress action. Lyophilized live vaccine efficiency, even without violation of storage rules at low temperatures (4 ± 2 – 6 ± 2 оС), decreases gradually due to death of live cells of microorganisms forming the base of a vaccine. Aim: The aim of this study was to enhance resistance of the reference vaccine strain Yersinia pestis EV of NIIEG lineage to freeze-drying in vacuum (lyophilization) by different techniques: the use of lyophilization process per se as a selection factor, resistant clone selection from populations of strains which underwent single, double and triple lyophiliation, strain culturing at low temperatures (4 ± 2 – 6 ± 2 °С). Summary and conclusion: It was demonstrated that after double and triple lyophilization the Y. pestis EV strain resistance to the process increased by 3–3.5 times. Clonal selection of twice and three times lyophilized variant facilitated detection of resistant clones and stabilization of this property.The clones selected were characterized by increased immunogenicity, high heat stability, as well as by increased duration of vaccine efficiency (by 2.3 times). A psychrophilic variant of Y. pestis EV strain was obtained in vitro acquiring higher resistance to lyophilization (in 2 times or more) in comparison with the reference strain. The number of psychrophilic variant cells surviving post-liophilization was higher in comparison with the commercial strain. Thus the methods used in this study for selection of strains and clones with the highest resistance to lyophilization from Y. pestis EV reference strain population showed a significant potential for quality improvement of dried live plague vaccine. So, the possibility of receiving of a vaccine of more high quality by means of the ways of selection explained in our work is experimentally confirmed. Effectiveness of these ways creates prerequisites for their use in production of a live plague vaccine.

Download Full-text

Insights in Behavior of Variably Formulated Alginate-Based Microcapsules for Cell Transplantation

BioMed Research International ◽

10.1155/2015/965804 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 11

Author(s):

Pia Montanucci ◽

Silvia Terenzi ◽

Claudio Santi ◽

Ilaria Pennoni ◽

Vittorio Bini ◽

...

Keyword(s):

Divalent Cations ◽

Chemical Properties ◽

Live Cells ◽

High Performing ◽

Physical Chemical ◽

Degenerative Disorders ◽

Selection Of ◽

Better Than

Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M) enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation,in vitroandin vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules’ basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others.

Download Full-text

HPRT As a Selectable Safe Harbor for Transgenesis

Blood ◽

10.1182/blood.v124.21.4796.4796 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4796-4796

Author(s):

Anthony Conway ◽

Josée Laganière ◽

David E Paschon ◽

Katrin Hacke ◽

Noriyuki Kasahara ◽

...

Keyword(s):

Fold Increase ◽

Selective Growth ◽

Live Cells ◽

Zinc Finger Nucleases ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Targeted Integration ◽

Selection Of

Abstract A current limitation in gene therapy is obtaining a sufficient number of modified cells to produce a therapeutic effect in vivo. In several diseases, correction of a mutant allele confers a selective growth advantage to the modified cells, thus enhancing efficacy with moderate initial modification. For most diseases, however, there is no selective advantage to the corrected cells. One potential strategy to address this limitation is in vivo selection of modified cells using pharmacological agents. It has previously been shown that 6-thioguanine (6-TG), an FDA-approved chemotherapeutic small molecule, is cytotoxic to cells expressing the enzyme HPRT, allowing for selective growth of HPRT knockout cells. Knockout of HPRT can be achieved by creating a nonsense mutation in an upstream exon, or by terminating splicing by introducing a large transgene into an intron. To allow for selectable transgenesis of only cells which have undergone targeted integration (TI), engineered zinc-finger nucleases (ZFNs) were used to insert a virally-delivered transgene into an HPRT intron. After two weeks of in vitro 6-TG selection following genome modification, a 95-fold increase in TI was observed in pooled K562 cell populations to a final level of 72% TI, whereas a 30-fold increase in transgene-expressing live cells was seen in peripheral blood-mobilized primary CD34+ cells resulting in 90% transgene-positive live cells. Furthermore, a 72-fold increase in transgene mRNA transcript was observed after two weeks of erythroid differentiation and 6-TG selection of CD34+ cells compared to unselected genome-modified controls. These results represent an important step in developing hematopoietic stem cell (HSC)-based gene therapies, as well as a platform technology for creating gene-modified HSC populations with high proportions of therapeutic transgene expression via precise, targeted integration of a transgene of interest. Disclosures Conway: Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment. Cost:Sangamo Biosciences: Employment.

Download Full-text

In Vitro Development of Resistance to Six and Four Fluoroquinolones in Mycoplasma pneumoniae and Mycoplasma hominis, Respectively

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.3.1190-1193.2005 ◽

2005 ◽

Vol 49 (3) ◽

pp. 1190-1193 ◽

Cited By ~ 55

Author(s):

D. Gruson ◽

S. Pereyre ◽

H. Renaudin ◽

A. Charron ◽

C. Bébéar ◽

...

Keyword(s):

Mycoplasma Pneumoniae ◽

Reference Strain ◽

Mycoplasma Hominis ◽

Quinolone Resistance ◽

In Vitro Development ◽

Development Of Resistance ◽

Resistant Mutants ◽

Vitro Development ◽

Selection Of

ABSTRACT Selection of resistant mutants in sequential subcultures with increasing concentrations of six and four different fluoroquinolones was studied for one reference strain each of Mycoplasma pneumoniae and Mycoplasma hominis, respectively. All fluoroquinolones tested selected for resistance, with alterations affecting the quinolone resistance-determining regions of the four target topoisomerase genes.

Download Full-text

Complex in vitro selection of cell lines of mangel beet resistant to a toxin of bacteriosis pathogene and low temperatures

Biopolymers and Cell ◽

10.7124/bc.000560 ◽

2000 ◽

Vol 16 (2) ◽

pp. 138-144 ◽

Cited By ~ 1

Author(s):

N. Ya. Gubanova ◽

O. V. Dubrovnaya ◽

T. V. Chugunkova

Keyword(s):

Cell Lines ◽

Low Temperatures ◽

In Vitro Selection ◽

Selection Of

Download Full-text

Evaluation the resistance of cocoa clones (Theobroma cacaoL.)

Pelita Perkebunan (a Coffee and Cocoa Research Journal) ◽

10.22302/iccri.jur.pelitaperkebunan.v30i1.192 ◽

2014 ◽

Vol 30 (1) ◽

Author(s):

Agung Wahyu Susilo ◽

Indah Anita Sari

Keyword(s):

Plant Pathology ◽

Clonal Selection ◽

Field Evaluation ◽

Lesion Size ◽

Phytophthora Palmivora ◽

Early Evaluation ◽

In Vitro Inoculation ◽

The Stability ◽

Selection Of

Acceleration on clonal selection of cocoa resistance to pod rot (Phytophthora palmivora)was carried out by early evaluation of the resistance using laboratory test. This research has objective to select the promising clone resistance to P. palmivora for field evaluation. Trials were carried out at the Laboratory of Plant Pathology at the Indonesian Coffee and Cocoa Research Institute using in-vitro inoculation. Isolate of P. palmivorawere collected from the infected pods at Jatirono Estate, Banyuwangi then inoculated to three mature pods of each tested clones. Trials were carried out in two steps to confirm the stability of performance of the resistance. A total of 41 clones were tested in these trials. However due to the availability of pod sample was limited then at 1st trial 31 clones were tested and at 2nd trial 37 clones were tested, both of the test using 27 same clones. The assessed variables were lesion size on pod surface due to Phytophthorainfection at 1st to 7th day after inoculation. The lesion sizes were significantly different among tested clones that performing any variability of the resistance. The variability were grouped into five groups as the resistant classification by which three clones were identified, namely Jano/IV/4/13 (TSH 858 x ICS 13), Jano next to I/7 and Kate/I/10/18 (Sulawesi 01 x TSH 858) consistently performing lowest size of the lesion compared to Sca 6 that could be selected as the resistant clones for field evaluation. Key words: cocoa clones, resistance, pod inoculation, Phytophthora palmivora

Download Full-text

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166117 ◽

1996 ◽

Vol 54 ◽

pp. 730-731

Author(s):

E. D. Salmon ◽

J. C. Waters ◽

C. Waterman-Storer

Keyword(s):

Imaging System ◽

Ccd Camera ◽

Transmitted Light ◽

Microtubule Assembly ◽

Live Cells ◽

Optical Efficiency ◽

Multiple Band ◽

Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

Download Full-text