Generation Doubling Time Research Articles

Cultivation, cryopreservation and resuscitation of Theileria annulata transformed cells in serum-free media.

Tropical theileriosis is a protozoan disease caused by Theileria annulata that affects cattle in Northern Africa, the Middle East and Asia where vector ticks of the genus Hyalomma occur. Various measures are applied to control the disease, including vaccination with attenuated T. annulata schizonts. Cultivation of T. annulata schizonts is mainly conducted in media containing Fetal Bovine Serum (FBS), which has some disadvantages such as costs, batch- to-batch variation and ethical concerns. In this study, we conducted three experiments to evaluate the ability of (1) T. annulata strains grown in RPMI with 10% FBS (RPMI-FBS) to adapt and grow in serum-free media (i.e., HL-1, RPMI without FBS supplementation, ISF-1, and M199), (2) a T. annulata strain grown in ISF-1 and subsequently frozen in this medium to grow in ISF-1 again after long-term storage in liquid nitrogen, and (3) a T. annulata strain freshly isolated from infected bovine lymphocytes to growin ISF-1, also after cryopreservation. Cell numbers, schizont index, the viability and generation doubling time were calculated in all experiments. In the first experiment, the Hessiene and Beja cell lines from Tunisia previously cultivated in RPMI-FBS and adapted to serum-free media continued to grow significantly better in RPMI-FBS compared to the serum-freemedia. In the second experiment, a Tunisian cell line (Hessiene) cryopreserved in ISF-1 with 5%[v/v] dimethylsulfoxide (DMSO) grewbetter after thawing in RPMI-FBS compared to ISF-1 with a highly significant difference in cell growth (p < 0.001), whereas the third experiment showed that the Ankara cell line had similar growth characteristics in both RPMI-FBS and ISF-1 before and after thawing, with a shorter generation doubling time in ISF-1 than in RPMI-FBS (p = 0.23). Our findings suggest that freshly isolated cells can be propagated, frozen and thawed in serum-free media such as ISF-1, but once cells are adapted to cultivation in the presence of FBS or resuscitated from frozen storage, propagation in serum-free media may not perform as well as cultivation in RPMI-FBS.

Serum-free in vitro cultivation of Theileria annulata and Theileria parva schizont-infected lymphocytes.

Theileriosis is a tick-borne disease caused by intracellular protozoa of the genus Theileria. The most important species in cattle are Theileria annulata and Theileria parva. Both species transform leucocyte host cells, resulting in their uncontrolled proliferation and immortalization. Vaccination with attenuated T. annulata-infected cell lines is currently the only practical means of inducing immunity in cattle. Culture media for Theileria spp. typically contain 10%-20% foetal bovine serum (FBS). The use of FBS is associated with several disadvantages, such as batch-to-batch variation, safety and ethical concerns. In this study, the suitability of serum-free media for the cultivation of Theileria-transformed cell lines was examined. Three commercial serum-free media (HL-1, ISF-1 and Hybridomed DIF 1000) were evaluated for their ability to support growth of the T. annulata A288 cell line. The generation doubling times were recorded for each medium and compared with those obtained with conventional FBS-containing RPMI-1640 medium. ISF-1 gave the shortest generation doubling time, averaging 35.4±2.8hr, significantly shorter than the 52.2±14.9hr recorded for the conventional medium (p=.0011). ISF-1 was subsequently tested with additional T.annulata strains. The doubling time of a Moroccan strain was significantly increased (65.4±15.9hr) compared with the control (47.7±7.5hr, p=.0004), whereas an Egyptian strain grew significantly faster in ISF-1 medium (43.4±6.5hr vs. 89.3±24.8hr, p=.0001). The latter strain also showed an improved generation doubling time of 73.7±21.9hr in an animal origin-free, serum-free, protein-free medium (PFHM II) compared with the control. Out of four South African T. parva strains and a Theileria strain isolated from roan antelope (Hippotragus equinus), only one T. parva strain could be propagated in ISF-1 medium. The use of serum-free medium may thus be suitable for some Theileria cell cultures and needs to be evaluated on a case-by-case basis. The relevance of Theileria cultivation in serum-free media for applications such as vaccine development requires further examination.

Effect of Two Egyptian Cotton Varieties on Development and Life Table of the Two-Spotted Spider Mite, Tetranychus urticae Koch (Acari: Tetranychidae) in Relation to Leaf Chemical Contents

The development and life table of the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) were studied when reared on two Egyptian cotton varieties: Giza 86 and Giza 90 at laboratory conditions of 27±2°C and 60±5% R.H. Results cleared that the development of T. urticae female was significantly affected by cotton variety. Immatures developmental time was shorter on Giza 90 (3.82 days) than Giza 86 (9.86 days). The adult female longevity averaged 12.29 and 16.65 days on Giza 90 and Giza 86, respectively. In addition, the values of Net reproductive rate (Ro), Intrinsic rate of natural increase (rm) and Finite rate of increase per day (λ) of T. urticae were (65.04, 0.407 and 1.502 days) and (31.95, 0.184 and 1.202 days) on Giza 90 and Giza 86, respectively. The female fecundity recorded 98.63 and 71.00 eggs / female on Giza 90 and Giza 86, respectively. The mean generation time (T) and generation doubling time (DT) values were the lowest on Giza 90 (10.27, 1.702 days) than Giza 86 (18.82, 3.766 days), respectively. A positive significant correlation was recorded between development, fecundity of T. urticae female and availability leaf nitrogen content.

BIOLOGICAL ASPECTS AND LIFE TABLE PARAMETERS OF THE EGGPLANT STEM BORER, Euzophera osseatella Treit. (Lep.: Pyralidae)

Biological aspects and life table parameters of the eggplant stem borer, Euzophera osseatella Treit. (Lep.: Pyralidae) were studied under laboratory conditions at 27℃ and 65% R.H. The egg, larval and pupal stages of E. osseatella lasted 5.8, 33.2 and 12.9 days, respectively. The mortality percentage in the corresponding stages reached 14, 37 and 13%, respectively. The sex ratio in E. osseatella was in favour of females (1:1.1). The preoviposition, oviposition and postoviposition periods of the pest averaged 1.8, 3.1 and 1.2 days, respectively. The daily and total numbers of eggs/female of E. osseatella were 46.6 and 147.2, respectively. The male and female of the insect lived 4.4 and 6.2 days, respectively. The mean generation time (T) of E. osseatella averaged 54.22 days. The net reproductive rate (Ro), the intrinsic rate of increase (rm) and the finite rate of increase (exp.rm/ λ) in the pest were 28.09, 0.062 and 1.063, respectively. The generation doubling time (Dt) of E. osseatella reached 14.01 days. The fecundity curve (Mx) of E. osseatella showed three peaks of 14.14, 20.14 and 12.32 female progeny/female/day at the female age (X) of 50.5, 61.5 and 63.5 days, respectively. The survival curve (Lx) of the insect revealed that most death of females occurred after an extended oviposition period to compensate failure of most immature stages to reach maturity (64%).

Acquired irinotecan resistance is accompanied by stable modifications of cell cycle dynamics independent of MSI status

Irinotecan is a major anticancer agent specifically targeting DNA topoisomerase I. Its cytotoxicity is mediated via a two-step process involving accumulation of reversible DNA‑topoisomerase I complexes associated with transient DNA single-strand breaks which subsequently are converted into permanent DNA double-strand breaks by the replication fork during S phase. Irinotecan may be selectively active for treatment of colorectal cancers that show microsatellite instability (MSI) due to deficiencies in mismatch repair enzymes, compared to tumors that are microsatellite stable but show chromosome instability (CIN). Although the clinical activity of irinotecan is principally limited by acquired drug resistance, surprisingly little is known about the influence of prolonged irinotecan exposure on the cell cycle dynamics. We have developed two colon cancer cell lines resistant to SN-38, the active metabolite of irinotecan, one derived from HT-29 (CIN), the other from HCT-116 (MSI). We here show that besides classical resistance mechanisms, SN-38 resistance is accompanied by an increased generation doubling time, a decreased S phase fraction and an increased G2 fraction in vitro as in tumor xenografts for both CIN and MSI models. As a consequence, SN-38-resistant cells and tumors show cross-resistance to the S-phase selective agent 5-fluorouracil. The resistance is accompanied by increased basal levels of γ-H2AX and phospho-Chk2 without notable changes in the levels of phospho-Chk1. Taken together, our results show that prolonged irinotecan exposure is accompanied by stable modifications of cell cycle dynamics which could have profound impact on tumor sensitivity to a wide range of antitumor agents and may influence tumor progression in patients.

Biology and Life Table Parameters of the Date Palm Dust Mite, Oligonychus afrasiaticus (McGregor) (Acari: Tetranychidae) as affected by Host and Controlled conditions

Developmental times and reproduction rates of the date palm dust mite Oligonychus afrasiaticus (McG.) were studied on three different date Palm tree parts (i.e. kamry fruit stage (characterized by the green color of fruits), yellow khelal stage and inner frond) of Khalas variety at laboratory conditions of 15, 25 & 35 ± 2°C; 30 & 70 ± 5% R.H. and 16:8 L:D. The mite failed to develop at 15°C. Immatures developmental time was fastest on kamry stage (10.60 days) followed by yellow khelal (12.35 days) then inner fronds (12.71 days). Adult female longevity averaged 15.08, 14.62 and 13.83 days on kamry and khelal yellow stages and inner frond, respectively. The shortest generation time was 9.5, 11.32 and 16.04 days at 35°C & 30% R.H., while the longest was 22.74, 26.74 and 26.68 days at 25°C & 70% R.H. on kamry, yellow khelal stage and inner frond; respectively. Sex ratio was affected by temperature as proportion of females increased with temperature increase. In addition, the values of rm, R0 and λ increased with temperature increase and decreased with R.H. increase. The mean generation time (T) and generation doubling time (DT) values decreased with temperature increase and R.H. decrease; thus the reproduction of mite increase. This mite favored high temperature and low R.H. Also immature date (kamry stage) was the favorable followed by yellow khelal then inner frond.

Effect of Temperature Degrees on the Biology and Life Table Parameters of Tetranychus urticae Koch on Two Pear varieties

Developmental times and reproduction rates of the two-spotted spider mite Tetranychus urticae Koch were studied when fed on leaves of two pear varieties (i.e. Lacont and Hood varieties) under laboratory conditions of 15, 20, 25 & 30 ± 2°C, 70% ± 5% RH. The longest developmental stages reached 30.6 and 30.1 days for female and male at 15°C when the T. urticae was fed on Lacont pear variety, while when reared on Hood pear variety it was 34 and 32.1 days at the same temperature. The highest total mean fecundity rate was recorded at 30oC (156.8 and 143.6 eggs /female). Sex ratio was affected by temperature as proportion of female increased. In addition, the values of rm and λ increased with temperature increase, while mean generation time (T) and generation doubling time (DT) values decreased with temperature increase, thus the reproduction of mites increased. This mite favored high temperature. The Lacont pear variety was more favored to this mite.

Amniotic-Fluid Stem Cells: Growth Dynamics and Differentiation Potential after a CD-117-Based Selection Procedure

Amniotic fluid (AF) has become an interesting source of fetal stem cells. However, AF contains heterogeneous and multiple, partially differentiated cell types. After isolation from the amniotic fluid, cells were characterized regarding their morphology and growth dynamics. They were sorted by magnetic associated cell sorting using the surface marker CD 117. In order to show stem cell characteristics such as pluripotency and to evaluate a possible therapeutic application of these cells, AF fluid-derived stem cells were differentiated along the adipogenic, osteogenic, and chondrogenic as well as the neuronal lineage under hypoxic conditions. Our findings reveal that magnetic associated cell sorting (MACS) does not markedly influence growth characteristics as demonstrated by the generation doubling time. There was, however, an effect regarding an altered adipogenic, osteogenic, and chondrogenic differentiation capacity in the selected cell fraction. In contrast, in the unselected cell population neuronal differentiation is enhanced.

The toxicity of Roundup ® 360 SL formulation and its main constituents: Glyphosate and isopropylamine towards non-target water photoautotrophs

The toxicity of commercial formulation of Roundup ® 360 SL, widely used, nonselective herbicide and its main constituents, glyphosate (PMG), equimolar (1:1) isopropylamine salt of glyphosate (GIPA) and isopropylamine (IPA) was examined towards eight aquatic microphotoautotrophs; seven cyanobacterial strains representing either saline or freshwater communities, and common eukaryotic algae Chlorella vulgaris Beijerinck. Autotrophs were cultured 21 days in their appropriate standard media supplemented with various amounts of Roundup ®, glyphosate, GIPA and IPA. The determination of the growth of examined photoautotrophs was performed by time-course measurements of total chlorophyll content in experimental cultures. The growth rates related to corresponding concentrations of chemicals, the EC 50 values and generation doubling time were determined in order to present the toxicity Roundup ® 360 SL formulation and its main constituents. Market available formulation of Roundup ® was found to possess toxicity significantly higher than this, attributed to its main constituents; however both these compounds, isopropylamine and glyphosate, also inhibited the growth of examined strains in a dose-dependent manner. Notably, the interpretation of toxicity of the examined substances was found to be significantly dependent on the method of EC 50 calculation. The choice of molar or weight concentration of substances tested separately and in specific formulation was found to be essential in this matter. Due to these findings the EC 50 values were calculated based either on molar or on weight concentrations. Considering Roundup ® 360 SL formulation, these values ranged from 10 −3 up to 10 −1 mM and they were one order of magnitude lower than those found for isopropylamine. Quite surprisingly the minimum EC 50 values found for glyphosate did not reach micromolar concentrations, whereas most of the EC 50 values revealed to IPA did not exceed this range. Notably, in all the cases except for Synechocystis aquatilis Sauvageau, isopropylamine alone was indicated as more toxic than glyphosate.

Production of Phenols and Alkyl Gallate Esters by Rhodobacter sphaeroides OU5

Rhodobacter sphaeroides OU5 grows phototrophically with generation doubling time of 18 h on L-phenylalanine when used as sole source of nitrogen. Phenols accumulated in the medium and gallate (0.5 mM) was identified as one of the major product. The others namely protocatechuate (0.2 mM) and caffeate (0.1 mM) and also three putative phenol alkyl esters were identified using Liquid chromatography-mass spectroscopy (LC-MS) analysis from the culture supernatant. Rhodobacter sphaeroides OU5 strain could explain its capability to produce the bioactive compounds during the growth. This study sheds production of bioactive and their biological exploring molecules.

Bloodstream forms of Trypanosoma brucei require only small amounts of iron for growth.

Bloodstream forms of Trypanosoma brucei require transferrin (Tf) for growth. Uptake of Tf is mediated by a heterodimeric, glycosylphosphatidylinositol-anchored receptor. Competition studies of the uptake of iron-loaded Tf (holo-Tf) versus iron-free Tf (apo-Tf) showed that bloodstream-form trypanosomes required only small amounts of iron for growth. The competition experiments were possible as the trypanosome Tf receptor shows a similarly high affinity for both holo-Tf and apo-Tf. Calculations revealed that accumulation of about 40,000 iron atoms during 1 generation-doubling time was sufficient for parasite multiplication.

A novel cultivation technique for long-term maintenance of bloodstream form trypanosomes in vitro

We used an axenic cultivation system to grow African trypanosomes in vitro. Long-term cultivation for more than 60 days has been achieved by replacing the culture medium at regular intervals between 6 and 48 h. In contrast to a control culture without medium replacement, increasing amounts of maximum cell concentrations have been obtained, ranging from 5 × 10 6 to 2 × 10 7 trypanosomes ml −1, whereas the generation doubling time remained constant (about 6 h). Higher cell concentrations have only been obtained by total medium replacement; neither addition of fresh medium nor serum led to a higher cell yield, suggesting that a trypanosome-derived factor or metabolite accumulated in the medium rather than medium was depleted of an essential nutrient. Most interestingly, however, successive waves have been obtained which eventually led to a damped oscillation curve with a constant high population density after about 40 days of cultivation. Cultures were started with a homogeneous population of the long-slender form. As judged by light microscopy, cells showed a stumpy morphology during the declining phase and became slender again in the following growth phase. At later time points, when cells remained in a stationary phase at high population density, many different morphological stages have been observed, similar to those described by early authors as intermediate forms [Ormerod, W.E. (1979) In: Biology of the Kinetoplastida, Vol. 2, pp. 340–393], although many dividing forms are still present at that time. In contrast, identically treated procyclic cultures were unable to produce cyclic growth waves. Based on these results, a novel concept considering a possible differentiation mechanism is discussed.