Radiation response mechanisms of the extremely radioresistant bacterium Deinococcus radiodurans.

Effect of microgravity on recovery of bacterial cells from radiation damage was examined in IML-2, S/MM-4 and S/MM-9 experiments using the extremely radioresistant bacterium Deinococcus radiodurans. The cells were irradiated with gamma rays before the space flight and incubated on board the Space Shuttle. The survival of the wild type cells incubated in space increased compared with the ground controls, suggesting that the recovery of this bacterium from radiation damage was enhanced under the space environment. No difference was observed between the survivals of radiosensitive mutant rec30 cells incubated in space and on the ground. The amount of DNA-repair related RecA protein induced under microgravity was similar to those of ground controls, however, induction of PprA protein, product of a unique radiation-inducible gene (designated pprA) responsible for loss of radiation resistance in repair-deficient mutant, KH311, was enhanced under microgravity compared with ground controls. Recent investigation in vitro showed that PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited Escherichia coli exonuclease III activity, and stimulated the DNA end-joining reaction catalyzed by DNA ligases. These results suggest that D. radiodurans has a radiation-induced non-homologous end-joining (NHEJ) repair mechanism in which PprA plays a critical role.     

The influence of microgravity and spaceflight on columella cell ultrastructure in starch-deficient mutants of Arabidopsis.

The ultrastructure of root cap columella cells was studied by morphometric analysis in wild-type, a reduced-starch mutant, and a starchless mutant of Arabidopsis grown in microgravity (F-microgravity) and compared to ground 1g (G-1g) and flight 1g (F-1g) controls. Seedlings of the wild-type and reduced-starch mutant that developed during an experiment on the Space Shuttle (both the F-microgravity samples and the F-lg control) exhibited a decreased starch content in comparison to the G-1g control. These results suggest that some factor associated with spaceflight (and not microgravity per se) affects starch metabolism. Elevated levels of ethylene were found during the experiments on the Space Shuttle, and analysis of ground controls with added ethylene demonstrated that this gas was responsible for decreased starch levels in the columella cells. This is the first study to use an on-board centrifuge as a control when quantifying starch in spaceflight-grown plants. Furthermore, our results show that ethylene levels must be carefully considered and controlled when designing experiments with plants for the International Space Station.     

Tricyclic isoxazoles are novel inhibitors of the multidrug resistance protein (MRP1)

Tricyclic isoxazoles were identified from a screen as a novel class of selective multidrug resistance protein (MRP1) inhibitors. From a screen lead, SAR efforts resulted in the preparation of LY 402913 (9h), which inhibits MRP1 and reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC(50)=0.90 microM), while showing no inherent cytotoxicity. Additionally, LY 402913 inhibits ATP-dependent, MRP1-mediated LTC(4) uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC(50)=1.8 microM). LY 402913 also shows selectivity ( approximately 22-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, LY 402913 delays the growth of MRP1-overexpressing tumors in vivo.     

Quantification by laser scan microscopy of intracellular doxorubicin distribution.

Changes in intracellular drug localization accompany doxorubicin resistance in multidrug resistant tumor cells. The purpose of this study was to develop a method to quantify these changes and so detect different levels of resistance. Tumor cells were incubated with the fluorescent anthracycline doxorubicin (excitation at 480 nm; emission maximum at 560-590 nm) and were quantified using laser scanning microscopy. The fluorescent mode was used to record the intracellular drug distribution, whereas the absorption mode was used to define the nuclear and cytoplasmic boundaries. The cell compartments were delineated interactively on an image processing system and the ratio nuclear fluorescence/cytoplasmic fluorescence (N/C ratio) was determined. N/C ratios were: 1.8 in the Chinese hamster ovarian cell line AUXB1 and 0.1 in its MDR subline CHRC5; 3.8 in the human squamous lung cancer cell line SW-1573 and 1.8 and 0.4 in its MDR sublines SW-1573/2R120 and SW-1573/2R160, respectively; and 3.6 in the human myeloma cell line 8226/S and 2.1 and 1.0 in its MDR sublines 8226/Dox4 and 8226/Dox40, respectively. The doxorubicin distribution was independent of the doxorubicin concentration within a range from 1-32 microM. Furthermore, the progressive mean of the nuclear/cytoplasmic doxorubicin fluorescence ratio showed that a minimal sample size of 30 cells is necessary for reliable results. The results of two independent assessments showed a high reproducibility (r = 0.97). Thus, with the method described in this paper, it is possible to detect relatively low levels of doxorubicin resistance (factor 8).     

Doxorubicin-transferrin conjugate selectively overcomes multidrug resistance in leukaemia cells

Neoplastic cells frequently have an increased number of transferrin receptors. Coupling transferrin to an anti-neoplastic drug has the potential to overcome multidrug resistance (MDR). The purpose of this study was to examine the distribution and action of doxorubicin-transferrin conjugate (DOXTRF) in a leukaemia cell line (HL60), a multidrug-resistant leukaemia cell line (HL60ADR) and a normal tissue cell line (human fibroblasts). The intracellular accumulation of DOX and DOX-TRF was monitored by direct fluorescence. More DOX-TRF than free DOX was delivered to the tumour cells, and consecutively the levels of DNA double-strand breaks and apoptosis increased even in the multidrug-resistant cell line. In the normal tissue cell line, DOX-TRF did not accumulate, and therefore, the levels of DNA double-strand breaks and apoptosis did not increase. Cell viability was determined using the MTT assay. The IC₅₀ for DOX-TRF was lower than the IC₅₀ value for the free drug in both leukaemia cell lines. The IC₅₀ values for the HL60 cells were 0.08 μM for DOX and 0.02 μM for DOX-TRF. The IC₅₀ values for HL60ADR cells were 7 μM for DOX and 0.035 μM for DOX-TRF. In conclusion, DOX-TRF was able to overcome MDR in the leukaemia cell lines while having only a very limited effect on normal tissue cells.     

Microgravity and clinorotation cause redistribution of free calcium in sweet clover columella cells

In higher plants, calcium redistribution is believed to be crucial for the root to respond to a change in the direction of the gravity vector. To test the effects of clinorotation and microgravity on calcium localization in higher plant roots, sweet clover (Melilotus alba L.) seedlings were germinated and grown for two days on a slow rotating clinostat or in microgravity on the US Space Shuttle flight STS-60. Subsequently, the tissue was treated with a fixative containing antimonate (a calcium precipitating agent) during clinorotation or in microgravity and processed for electron microscopy. In root columella cells of clinorotated plants, antimonate precipitates were localized adjacent to the cell wall in a unilateral manner. Columella cells exposed to microgravity were characterized by precipitates mostly located adjacent to the proximal and lateral cell wall. In all treatments some punctate precipitates were associated with vacuoles, amyloplasts, mitochondria, and euchromatin of the nucleus. A quantitative study revealed a decreased number of precipitates associated with the nucleus and the amyloplasts in columella cells exposed to microgravity as compared to ground controls. These data suggest that roots perceive a change in the gravitational field, as produced by clinorotation or space flights, and respond respectively differently by a redistribution of free calcium.     

The gene encoding vacuolar H(+)-ATPase subunit C is overexpressed in multidrug-resistant HL60 cells.

Previous studies have suggested that vacuolar H(+)-ATPase activity may play a role in modulating drug transport mechanism in multidrug resistant HL60 cells. In the present study we have used a cDNA of human vacuolar H(+)-ATPase subunit C (SC-H(+)-ATPase) to analyze expression of this gene in HL60 cells isolated for resistance to adriamycin or vincristine. The results demonstrate that development of resistance to either agent results in a major increase in the levels of SC-H(+)-ATPase mRNA. Furthermore in resistant cells which have partially reverted to drug sensitivity there is a parallel reduction in SC-H(+)-ATPase mRNA levels. Southern blot analysis shows that the SC-H(+)-ATPase gene is not amplified in the resistant cells. These results therefore demonstrate a correlation between the development of multidrug resistance and enhanced expression of the SC-H(+)-ATPase gene.     

Effect of liposomes on P-glycoprotein function in multidrug resistant cells.

Presentation of doxorubicin in liposomes has shown to enhance the sensitivity of multidrug resistant CH LZ cells to the drug (Thierry et al. Cancer Commun. 1:311-316, 1989). We confirmed that liposomally encapsulated doxorubicin may partially overcome multidrug resistance in the human ovarian carcinoma SKVLB cell line and that this effect is, at least in part, due to an increase of cellular drug accumulation. When used at high concentration, empty liposomes appear to be specifically cytotoxic in the MDR SKVLB and CH LZ cells. As observed with certain multidrug resistance modulators, empty liposomes inhibited the specific [3H]-vincristine binding to P-glycoprotein-enriched membranes isolated from CH LZ cells (60% at 0.2 mg lipid/ml). Our data suggest that liposomes may alter the P-glycoprotein function by direct interaction.     

Behavior and reproduction of invertebrate animals during and after a long-term microgravity: space experiments using an Autonomous Biological System (ABS).

Aquatic invertebrate animals such as Amphipods, Gastropods (pond snails), Ostracods and Daphnia (water flea) were placed in water-filled cylindrical vessels together with water plant (hornwort). The vessels were sealed completely and illuminated with a fluorescent lamp to activate the photosynthesis of the plant for providing oxygen within the vessels. Such ecosystem vessels, specially termed as Autonomous Biological System or ABS units, were exposed to microgravity conditions, and the behavior of the animals and their reproduction capacity were studied. Three space experiments were carried out. The first experiment used a Space shuttle only and it was a 10-day flight. The other two space experiments were carried out in the Space station Mir (Shuttle/Mir mission), and the flight units had been kept in microgravity for 4 months. Daphnia produced their offspring during a 10-day Shuttle flight. In the first Mir experiment, no Daphnia were detected when recovered to the ground. However, they were alive in the second Mir experiment. Daphnia were the most fragile species among the invertebrate animals employed in the present experiments. All the animals, i.e., Amphipods, pond snails, Ostracods and Daphnia had survived for 4 months in space, i.e., they had produced their offspring or repeated their life-cycles under microgravity. For the two Mir experiments, in both the flight and ground control ecosystem units, an inverse relationship was noted between the number of Amphipods and pond snails in each unit. Amphipods at 10 hours after the recovery to the ground frequently exhibited a movement of dropping straight-downward to the bottom of the units. Several Amphipods had their legs bent abnormally, which probably resulted from some physiological alterations during their embryonic development under microgravity. From the analysis of the video tape recorded in space, for Ostracods and Daphnia, a half of their population were looping under microgravity. Such looping animals could be observed still at the end of the 4 month stay in space. No looping behavior was noted for Amphipods and pond snails.     

Growth in microgravity increases susceptibility of soybean to a fungal pathogen.

The influence of microgravity on the susceptibility of soybean roots to Phytophthora sojae was studied during the Space Shuttle Mission STS-87. Seedlings of soybean cultivar Williams 82 grown in spaceflight or at unit gravity were untreated or inoculated with the soybean root rot pathogen P. sojae. At 3, 6 and 7 d after launch while still in microgravity, seedlings were photographed and then fixed for subsequent microscopic analysis. Post-landing analysis of the seedlings revealed that at harvest day 7 the length of untreated roots did not differ between flight and ground samples. However, the flight-grown roots infected with P. sojae showed more disease symptoms (percentage of brown and macerated areas) and the root tissues were more extensively colonized relative to the ground controls exposed to the fungus. Ethylene levels were higher in spaceflight when compared to ground samples. These data suggest that soybean seedlings grown in microgravity are more susceptible to colonization by a fungal pathogen relative to ground controls.     

Gravitropism and development of wild-type and starch-deficient mutants of Arabidopsis during spaceflight

The "starch‐statolith" hypothesis has been used by plant physiologists to explain the gravity perception mechanism in higher plants. In order to help resolve some of the controversy associated with ground‐based research that has supported this theory, we performed a spaceflight experiment during the January 1997 mission of the Space Shuttle STS‐81. Seedlings of wild‐type (WT) Arabidopsis , two reduced‐starch strains, and a starchless mutant were grown in microgravity and then given a gravity stimulus on a centrifuge. In terms of development in space, germination was greater than 90% for seeds in microgravity, and flight seedlings were smaller (60% in total length) compared to control plants grown on the ground and to control plants on a rotating clinostat. Seedlings grown in space had two structural features that distinguished them from the controls: a greater density of root hairs and an anomalous hypocotyl hook structure. However, the slower growth and morphological changes observed in the flight seedlings may be due to the effects of ethylene present in the spacecraft. Nevertheless, during the flight, hypocotyls of WT seedlings responded to a unilateral 60‐min stimulus provided by a 1‐ g centrifuge while those of the starch‐deficient strains did not. Thus, the strain with the greatest amount of starch responded to the stimulus given in‐flight, and, therefore, these data support the starch‐statolith model for gravity sensing.     

Clustering of endocytic organelles in parental and drug-resistant myeloid leukaemia cell lines lacking centrosomally organised microtubule arrays

Spatial organisation and trafficking of endocytic organelles in mammalian cells is tightly regulated and dependent on cytoskeletal networks. The dynamics of endocytic pathways is modified in a number of diseases, including cancer, and notably in multidrug resistant (MDR) cells that are refractory to the effects of several anti-cancer agents. These cells often upregulate expression of drug-efflux pumps but this may be synergistic with alternative resistance mechanisms including increased acidification of endocytic organelles that enhances vesicular sequestration of weak-base anti-cancer drugs such as daunorubicin away from their nuclear target. Here, we characterised the distribution of sequestered daunorubicin in commonly used leukaemia cell lines, HL-60, K562, KG1a and the multidrug resistant HL-60/ADR line, and related this to the spatial distribution of their endocytic organelles and microtubule networks. HL-60 and KG1a cells contained microtubule arrays emanating from organising centres, and their endocytic organelles and daunorubicin labelled vesicles were scattered throughout the cytoplasm. HL-60/ADR and K562 cells showed extensive clustering of early and recycling endosomes, late endosomes, lysosomes and daunorubicin to a juxtanuclear region but these cells lacked microtubule arrays. Microtubular organisation within these clustered regions was however, required for spatial tethering of endocytic organelles and the Golgi, as treatment with nocodazole and paclitaxel had major effects on their distribution. HL-60 and HL-60/ADR cells had similar lysosomal pH of <5.0 and overall these findings suggests a general relationship between the absence of microtubule arrays and the propensity of leukaemia cell lines to cluster endocytic organelles and daunorubicin into the juxtanuclear region.     

Role of oxygen free radical formation in the mechanism of menogaril resistance in multidrug resistant tumor cells

The mechanisms of action and resistance to menogaril, a clinically active anthracycline antitumor drug, were evaluated in sensitive and doxorubicin-selected multidrug resistant human breast tumor (MCF-7) cell lines. While MCF-7/ADRR cells were highly resistant (250-500-fold) to doxorubicin, they displayed only marginal resistance (10-fold) to menogaril. In contrast to doxorubicin, the mechanism of resistance to menogaril in these cells does not involve differential inhibition of DNA synthesis as measured by thymidine incorporation. P-170-glycoprotein-dependent drug transport did not contribute to resistance as there was no difference in the accumulation and retention of menogaril by sensitive and resistant cell lines. However, there was a 2-fold decrease in oxygen free radical formation in the resistant cells, compared to sensitive cells, in the presence of menogaril. Since resistant cells contain 12-fold higher glutathione peroxidase activity than the parental sensitive cells, the detoxification of hydrogen peroxide may be responsible for the decreased free radical formation and thus, may play a role in the resistance to menogaril.     

Dibenzocyclooctadiene lignans: a class of novel inhibitors of multidrug resistance-associated protein 1

We recently reported that dibenzocyclooctadiene lignans were a novel class of P-glycoprotein (P-gp) inhibitors. In this study, we demonstrated that the lignans of this class were also effective inhibitors of multidrug resistance-associated protein 1 (MRP1). The activities of 5 dibenzocyclooctadiene lignans (schisandrin A, schisandrin B, schisantherin A, schisandrol A, and schisandrol B) to reverse MRP1-mediated drug resistance were tested using HL60/Adriamycin (ADR) and HL60/Multidrug resistance-associated protein (MRP), two human promyelocytic leukemia cell lines with overexpression of MRP1 but not P-gp. The five lignans could effectively reverse drug resistance of the two cell lines to vincristine, daunorubicin, and VP-16. This study, together with our previous reports, proves that dibenzocyclooctadiene lignans have multiple activities against cancer multidrug resistance, including inhibition of P-gp and MRP1, and enhancement of apoptosis. Considering that cancer multidrug resistance (MDR) is multifactorial, agents with broad activities are preferable to the use of combination of several specific modulators to prevent drug-drug interaction and cumulative toxicity.     

TCP1 increases drug resistance in acute myeloid leukemia by suppressing autophagy via activating AKT/mTOR signaling

T-complex protein 1 (TCP1) is one of the subunits of chaperonin-containing T complex (CCT), which is involved in protein folding, cell proliferation, apoptosis, cell cycle regulation, and drug resistance. Investigations have demonstrated that TCP1 is a factor being responsible for drug resistance in breast and ovarian cancer. However, the TCP1 role in acute myeloid leukemia (AML) remains elusive. In the present study, we discovered that the TCP1 expression was elevated in AML patients and high TCP1 expression was associated with low complete response rate along with poor overall survival. TCP1 showed higher expression in the adriamycin-resistant leukemia cell line HL60/A and K562/A, comparing to their respective parent cells HL60 and K562 cells. TCP1 inhibition suppressed drug resistance in HL60/A and K562/A cells, whereas TCP1 overexpression in HL60 cells incremented drug resistance, both in vitro and in vivo. Mechanistic investigations revealed that TCP1 inhibited autophagy and adriamycin-induced cell apoptosis, and TCP1-mediated autophagy inhibition conferred resistance to adriamycin-induced cell apoptosis. Furthermore, TCP1 interacted with AKT and mTOR to activate AKT/mTOR signaling, which negatively regulates apoptosis and autophagy. Pharmacological inhibition of AKT/mTOR signal particularly activated autophagy and resensitized TCP1-overexpressing HL60 cells to adriamycin. These findings identify a novel role of TCP1 regarding drug resistance in AML, which advise a new strategy for overcoming drug resistance in AML through targeting TCP1/AKT/mTOR signaling pathway.Subject terms: Prognostic markers, Acute myeloid leukaemia     

Discovery of thalicthuberine as a novel antimitotic agent from nature that disrupts microtubule dynamics and induces apoptosis in prostate cancer cells

We report for the first time the mechanism of action of the natural product thalicthuberine (TH) in prostate and cervical cancer cells. TH induced a strong accumulation of LNCaP cells in mitosis, severe mitotic spindle defects, and asymmetric cell divisions, ultimately leading to mitotic catastrophe accompanied by cell death through apoptosis. However, unlike microtubule-binding drugs (vinblastine and paclitaxel), TH did not directly inhibit tubulin polymerization when tested in a cell-free system, whereas it reduced cellular microtubule polymer mass in LNCaP cells. This suggests that TH indirectly targets microtubule dynamics through inhibition of a critical regulator or tubulin-associated protein. Furthermore, TH is not a major substrate for P-glycoprotein (Pgp), which is responsible for multidrug resistance in numerous cancers, providing a rationale to further study TH in cancers with Pgp-mediated treatment resistance. The identification of TH's molecular target in future studies will be of great value to the development of TH as potential treatment of multidrug-resistant tumors.     

Intracellular distribution of anthracyclines in drug resistant cells

The unresponsiveness of multidrug resistant tumor cells to antineoplastic chemotherapy is often associated with reduced cellular drug accumulation accomplished by overexpressed transport molecules. Moreover, intracellular drug distribution in resistant cells appears to be remarkably different when compared to their wild type counterparts. In the present paper, we report observations on the intracellular accumulation and distribution of doxorubicin, an antitumoral agent widely employed in chemotherapy, in sensitive and resistant cultured tumor cells. The inherent fluorescence of doxorubicin allowed us to follow its fate in living cells by laser scanning confocal microscopy. This study included flow cytometric analysis of drug uptake and efflux and analysis of the presence of the well known drug transporter P-glycoprotein. Morphological, immunocytochemical and functional data evidentiated the Golgi apparatus as the preferential intracytoplasmic site of drug accumulation in resistant cells, capable of sequestering doxorubicin away from the nuclear target. Moreover, P-glycoprotein has been found located in the Golgi apparatus in drug induced resistant cells and in intrinsic resistant cells, such as melanoma cells. Thus, this organelle seems to play a pivotal role in the intracellular distribution of doxorubicin. This revised version was published online in August 2006 with corrections to the Cover Date.     

Epigenetic loss of CDH1 correlates with multidrug resistance in human hepatocellular carcinoma cells

Promoter CpG hypermethylation of tumor suppressor genes is an essential step in cancer progression but little is known about its effect on cancer multidrug resistance. In this study, we showed that CDH1 promoter was hypermethylated in drug resistance of a doxorubicin-induced multidrug resistant hepatocellular carcinoma cell line R-HepG2. Transfection of CDH1 cDNA into R-HepG2 cells led to increased amount of doxorubicin uptake, decreased cell viability, decreased P-glycoprotein expression and increased apoptotic population of cells exposed to doxorubicin. Proto-oncogene tyrosine-protein kinase FYN was over-expressed in R-HepG2 cells which displayed a negative correlation with the expression of CDH1. FYN was knocked down in R-HepG2 cells, leading to less drug resistance by increased cell viability, increased doxorubicin uptake and attenuated P-glycoprotein expression. Our findings identified epigenetic silencing of CDH1 in cancer cells might be a new molecular event of multidrug resistance.     

Embryonic development of the freshwater snail Biomphalaria glabrata under microgravity conditions (STS-89 mission)

The embryonic development of the fresh-water snail Biomphalaria glabrata was examined under microgravity-conditions and compared with the ground control and standard embryos, putting special emphasis on the shell formation. The process of shell formation may be particularly sensitive to the change of gravitational forces. The project aimed at determining whether the processes of mineralization during the formation of the exoskeleton in the growing snail embryo take place normally under microgravity conditions. Twenty-four adult individuals of the tropical freshwater snail B. glabrata were maintained 9 days in the Closed Equilibrated Biological Aquatic System (CEBAS Minimodule) on Space Shuttle flight STS-89. The animals produced spawning packs throughout the duration of the mission so that embryos of all developmental stages were achieved. The embryos developed slightly slower in the CEBAS than under standard conditions, and in older embryos a decreased mineralization of the shell was detected. These phenomena, however, were observed in the flight module as well as in the ground control specimens and was not an effect caused by the microgravity conditions. Embryos of B. glabrata showed a correct morphogenesis under microgravity, no teratological effects were noticed, and the shell formation proceeded normally.     

The effect of 8 days of microgravity on regeneration of intact plants from protoplasts

The growth and development of protoplasts of rapeseed (Brassica napus L. cv Line) and carrot (Daucus carota L. cv. Navona) were studied onboard the Space Shuttle‘Discovery’during an 8-day International Microgravity Laboratory [IML-l) mission in January 1992. The Flight experiments were carried out in‘Biorack'. a fully controlled cell biological experimental facility. under microgravity conditions and in a l-g centrifuge. Parallel experiments were performed in a‘Biorack’module on the ground. After retrieval, some samples were subcultured on appropriate media and analysed for callus growth and regeneration to intact plants. The remainder were used for biochemical analysis. Samples fixed on board the Space Shuttle were kept in l% glutaraldehyde fixative at 4°C for 3–7 days for microscopy analysis after retrieval. Protoplasts exposed to microgravity conditions showed a delay in cell wall synthesis. Cells were swollen in appearance and formed cell aggregates with only few cells. Callus were obtained from protoplasts cultured under microgravity (Fogl). on the l-g centrifuge on board the shuttle (Flg), under normal l-g conditions on the ground (G1g) and on a centrifuge on the ground giving 1.4 g (Gl.4g). Regeneration of intact rapeseed plants was obtained from Flg. Glg and G1.4g. However, no plants were regenerated from protoplasts exposed to microgravity (Fog). Biochemical analysis indicated that the microgravity samples (Fog displayed a reduced packed cell volume, an increased concentration of soluble proteins per cell, and a reduced specific activity of peroxidase in the cytoplasm. Morphometric analysis of fixed samples demonstrated that 3-day old protoplasts under microgravity conditions were significantly larger than protoplasts kept on the l-g centrifuge in space. UItrastructural analysis by transmission electron microscopy showed that protoplasts exposed to microgravity conditions for 3 days had larger vacuoles and a slightly reduced starch content compared to Flg cells. Cell aggregates formed under microgravity conditions (Fog) had an average of 2–I cells per aggregate while aggregates formed under Flg had 8–12 cells.