Complete Loss Of Viability Research Articles

Storage Temperature and Grain Moisture Effects on Market and End Use Properties of Red Lentil

Storing lentil is a strategy used by growers to manage price volatility. However, studies investigating the impact of storage conditions on the market and end use properties of lentil are limited. This study examined the effects of storage temperature (4, 15, 25, and 35 °C) and grain moisture (10 and 14%, w/w) on traits related to market (seed coat colour), viability (germination capacity), and end use properties (hydration capacity, milling efficiency, and cooking quality) in four red lentil cultivars (PBA Bolt, PBA Hallmark, PBA Hurricane, PBA Jumbo2) over 360 days. Storing lentil at 14% moisture content and 35 °C significantly (p = 0.05) darkened seed coat after 30 days, caused complete loss of viability within 180 days and reduced cooking quality (cooked firmness) after 120 days across all tested cultivars. Storing lentil at 10% moisture content and 35 °C reduced hydration capacity after 30 days, and milling efficiency after 120 days across all cultivars tested. PBA Jumbo2 exhibited a higher rate of degradation in hydration capacity and cooking quality, and a lower rate of degradation in the other traits studied. Storing lentil at ≤15 °C prevented degradation of all quality traits. These findings will support improved lentil storage protocols to maintain quality and improve economic outcomes for the pulse industry.

Cyanocobalamin (vitamin B12) as insensitive porphyrinoid to solar LED irradiation

Cobalamins are cobalt corrin complexes (vitamin B12) with typical porphyrinoid structure and absorption which partly overlaps with the solar spectrum of irradiation. This highly conjugated structure is assumed to have not only the well-known curative action as co-factor and vitamin, but also phototoxicity on dysfunctional, and, in a low extent, on normal cells. Moreover, the cobalamin can have properties as a photosensitizer at appropriate light conditions. The present study aims at characterization of the cyanocobalamin (CNCbl) as a synthetic cobalamin under solar-spectrum irradiation. The photo- and dark cytotoxicity studies were carried out on a normal cell line (BALB/c 3T3, A31) showing no effect due to light application. The photodynamic action of CNCbl due to its porphyrinoid-like structure was not observed. The results on two breast cancer cell lines (MDA-MB-231 and MCF-7) versus a non-tumorigenic cell line (MCF-10) showed similar values for cytotoxicity. A complete loss of viability was observed at approx. 10-4 M CNCbl after 24-hs incubation and soft irradiation (10 J/cm2) for the spectral range 360-960 nm of a LED Helios-iO solar simulator. The cobalt corrin complex showed non-photodynamic ability to reduce the viability of both breast tumor cells depending on the nature of the cell line but not on the light application.

Revisiting the pericarp as a barrier restricting water entry/loss from cotyledons and embryonic axis of temperate desiccation-sensitive Quercus acorns.

Fully mature acorns of Quercus variabilis, Q. aliena, Q. mongolica, and Q. glandulifera are desiccation-sensitive. X-ray computer tomography showed that cotyledons shrink during drying, but embryos are protected. Information available on recalcitrant acorns of tropical and sub-tropical species of Quercus suggests that an impermeable pericarp, which limits the entry and loss of water only through the hilum (scar), is the underlying mechanism that prevents drying of the embryo axis following dispersal until the germination season. However, there is a lack of consensus supporting this proposition across species, and it is not well understood if such mechanisms occur in temperate Quercus species. This study investigated the significance of the acorn pericarp for temperate oak species and presents an ecological framework based on the post-dispersal climatic conditions. Using Quercus variabilis, Q. aliena, Q. mongolica, and Q. glandulifera acorns, the relationship between moisture content (MC) and germination was established, and X-ray computed tomography (X-ray CT) was used to understand the internal structural changes of cotyledons and embryonic axis occurring during desiccation. Water entry and exit routes through the scar, pericarp and apex were determined by imbibition and drying experiments. Climatic data and acorn morphological characteristics and germination were subjected to a principal component analysis (PCA). Freshly dispersed acorns of all species had a moisture content (MC) above 35% fresh weight (FW) basis, but drying to 15-10% MC resulted in complete loss of viability, implying recalcitrance behaviour. X-ray CT images suggested that the pericarp offers some protection to cotyledons and embryonic axis during desiccation, but it is contingent on MC. Extensive drying to a low MC with the scar and apex covered with vaseline resulted in internal tissues shrinkage, corresponding with viability loss. Water could enter or exit through the pericarp, albeit at a much slower rate than through the scar. A combination of factors including acorn anatomy, moisture content at the time of dispersal, microhabitat, the position of acorns in the soil prevent embryo desiccation below the critical MC and thus promotes survival of acorns on/in the soil during winter in temperate regions. Pericarp anatomy, to some extent, prevents excessive drying of the embryonic axis by slowing water movement, but prolonged drying or predatory pressure could result in pericarp cracks, favouring the absorption of water during sporadic rain. In the latter case, the survival of acorns possibly depends extensively on the continuous erratic rainfall, i.e. continuous wet-dry cycle, but in-situ experiments are yet to be performed to test this hypothesis.

Human HepaRG liver spheroids: cold storage protocol and study on pyridinium oxime-induced hepatotoxicity in vitro

Oximes play an essential role in the therapy of organophosphorus compound (OP) poisoning by reactivating inhibited acetylcholinesterase. Impairment of liver function was observed in OP poisoning and associated with obidoxime treatment by some reports. In this study human three-dimensional HepaRG spheroids were used as complex in vitro model to investigate oxime-induced liver toxicity. In this context, cold storage of liver spheroids at 4 °C in standard culture medium and in optimized tissue preservation solutions of up to 72 h was assessed. Cold storage in standard culture medium resulted in a complete loss of viability whereas an optimized tissue preservation solution preserved viability. Separately from that liver spheroids were exposed to the four oximes pralidoxime, obidoxime, HI-6, MMB-4 and cytotoxicity (effective concentration, EC50) was determined with an ATP-based assay at several time points. The release of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin secretion was measured in supernatants. The same parameters were assessed with diclofenac as positive hepatotoxic control and with the OP pesticides malathion and malaoxon alone or in the presence of obidoxime. All individual tested oximes and OP showed a low cytotoxicity with effective concentrations mostly >2,000 μM. In contrast, the exposure to malaoxon in the presence of 1,000 μM obidoxime resulted in a marked decrease of viability and an increased release of AST indicating risk of liver injury only if oxime antidotes are strongly overdosed.

Photodynamic inactivation of Leishmania braziliensis doubly sensitized with uroporphyrin and diamino-phthalocyanine activates effector functions of macrophages in vitro

Photodynamic inactivation of Leishmania has been shown to render them non-viable, but retain their immunological activities. Installation of dual photodynamic mechanisms ensures complete inactivation of species in the Leishmania subgenus, raising the prospect of their safe and effective application as whole-cell vaccines against leishmaniasis. Here, we report the successful extension of this approach to L. braziliensis in the Viannia subgenus, viz. genetic engineering of promastigotes for cytosolic accumulation of UV-sensitive uroporphyrin (URO) and their loading with red light excitable phthalocyanines (PC) that was cationized by chemical engineering. The transgenic strategy used previously produced L. braziliensis transfectants, which gave the same phenotype of aminolevulinate (ALA)-inducible uroporphyria as found in Leishmania subgenus, indicative of pre-subgenus evolutionary origin for similar genetic deficiencies in porphyrin/heme biosynthesis. In the present study, 12 independent clones were obtained and were invariably ALA-responsive, albeit to different extent for uroporphyrinogenesis and UV-inactivation. In a separate study, L. braziliensis was also found, like other Leishmania spp., to take up diamino-PC (PC2) for red light inactivation. In vitro interactions of a highly uroporphyrinogenic clone with primary macrophages were examined with the intervention of URO/PC2-medated double-photodynamic inactivation to ascertain its complete loss of viability. Doubly sensitized L. braziliensis transfectants were photo-inactivated before (Strategy #1) or after (Strategy #2) loading of macrophages. In both cases, macrophages were found to take up L. braziliensis and degrade them rapidly in contrast to live Leishmania infection. The effector functions of macrophages became upregulated following their loading with L. braziliensis photodynamically inactivated by both strategies, including CD86 expression, and IL6 and NO production. This was in contrast to the immunosuppressive infection of macrophages with live parasites, marked by IL10 production. The results provide evidence that photodynamically inactivated L. braziliensis are susceptible to the degradative pathway of macrophages with upregulation of immunity relevant cytokine and co-stimulatory markers. The relative merits of the two loading strategies with reference to previous experimental vaccination were discussed in light of the present findings with L. braziliensis.

Influence of freeze-drying and spray-drying preservation methods on survivability rate of different types of protectants encapsulated Lactobacillus acidophilus FTDC 3081.

The aims of this study were to compare the effectiveness of different drying methods and to investigate the effects of adding a series of individual protectant such as skim milk, sucrose, maltodextrin, and corn starch for preserving Lactobacillus acidophilus FTDC 3081 cells during spray and freeze-drying and storage at different temperatures. Results showed a remarkable high survival rate of 70-80% immediately after spray- and freeze-drying in which the cell viability retained at the range of 109 to 1010 CFU/mL. After a month of storage, maltodextrin showed higher protective ability on both spray- and freeze-dried cells as compared to other protective agents at 4°C, 25°C, and 40°C. A complete loss in viability of spray-dried L. acidophilus FTDC 3081 was observed after a month at 40°C in the absence of protective agent.

The varying effects of a range of preservatives on Myoviridae and Siphoviridae bacteriophages formulated in a semi-solid cream preparation.

Bacteriophages may be formulated into semi-solid bases for therapeutic delivery. This work investigated the effects of a range of preservatives on the viability of Myoviridae and Siphoviridae bacteriophages when these were formulated into a standard semi-solid cream base. The six preservatives tested included: benzoic acid (0·1%), chlorocresol (0·1%), combination hydroxybenzoates (propyl 4-hydroxybenzoates with methyl 4-hydroxybenzoates) (0·1%), methyl 4-hydroxybenzoate (0·08%), 2-phenoxyethanol (1%) and propyl 4-hydroxybenzoate (0·02%). These were each formulated into cetomacrogol cream aqueous to generate six individual semi-solid bases into which Myoviridae and Siphoviridae bacteriophages were added and tested for stability. Optimal bacteriophage stability was seen when the preservative chlorocresol was used. Bacteriophage in the acidic benzoic acid were the least stable, resulting in complete loss of viability after 4-5weeks. Of the bacteriophages tested, the Myoviridae KOX1 was significantly more stable than the Siphoviridae PAC1 after 91days in formulations with each of the preservatives. Our results suggest the need for individual testing of specific bacteriophages in pharmaceutical formulations, as their efficacy when exposed to preservatives and excipients in these delivery forms may vary. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages are being increasingly investigated as alternatives to antibiotics. While bacteriophages can be formulated in diverse ways for therapeutic delivery, there has been scant work on how excipients and preservatives in these formulations affect stability of different bacteriophages. We demonstrate that the nature of preservatives in formulations will affect bacteriophage stability, and that in these formulations, viability of bacteriophage differs according to their morphology. Our work highlights the need for individual testing of specific bacteriophages in pharmaceutical formulations, as efficacy when exposed to preservatives and excipients in these delivery forms may vary.

Influence of protein damage and proteasome gene expression on the longevity of recalcitrantMadhuca latifoliaRoxb. seeds

Enhanced cellular damage during desiccation is considered to be one of the key factors limiting vigour and viability of seeds. The uncontrolled accumulation of reactive oxygen species and resultant damaging reactions such as the oxidation of lipids and DNA in desiccating seeds of Madhuca latifolia (Roxb.) J. F. Macbr. has already been well characterized. However, hydrolytic and (or) oxidative damage to proteins requires further study. This study investigated the desiccation-induced oxidative damage to proteins and proteolytic systems in recalcitrant M. latifolia seeds during ambient storage. Seeds experienced a significant drop in seed water content [ca. 1.32 to ca. 0.23 g·(g dry mass)−1] during storage resulting in complete loss of viability after 35 days of storage. A considerable decline in total protein content (3.0–3.6 fold) and activity (4.8–13.8 fold) in the gene expressions of proteasome subunits (α, β, and E2) were recorded in the embryonic axis of desiccating M. latifolia seeds. In contrast, increases in the level of protein carbonyls (2.46 fold), hydroperoxides (2.31 fold), malondialdehyde- and 4-hydroxy-2-nonenal-protein adducts (1.8 and 3.9 fold), and Amadori and Maillard reaction products, along with proteases (14.5–30.4 fold) were observed in desiccating M. latifolia seeds. This study revealed that increased oxidation/modification of proteins and proteasome dysfunction are involved in the deterioration of desiccating M. latifolia seeds.

Optimal long-term seed storage conditions for the endangered seagrass Zostera japonica: implications for habitat conservation and restoration

BackgroundSeagrass meadows are recognized as critical and among the most vulnerable habitats on the planet. The alarming rates of decline in seagrass meadows have attracted the attention globally. There is an urgent need to develop techniques to restore and preserve these vital coastal ecosystems. So far little work has been done to develop effective long-term storage method for seagrass seeds. The seagrass Zostera japonica Asch. & Graebn is an endangered species in its native range. Here we utilized combinations of different storage times, salinities, and temperature to determine the most appropriate conditions for optimal seed storage.ResultsZostera japonica seeds were strongly desiccation sensitive, with a complete loss of viability after 24 h of desiccation. Therefore, long periods of exposure to air should be avoided to minimize seed mortality. In addition, Z. japonica seeds could not endure freezing conditions such as – 5 °C. However, our results indicated that reduced storage temperature to 0 °C could effectively prolong the duration of dormancy of Z. japonica seeds. Seeds stored at 0 °C under a salinity of 40–60 psu showed relatively low seed loss, high seed vigor and fast seed germination, suggesting these to be optimal seed storage conditions. For example, after storage for 540 days (ca. 600 days since the seed collection from reproductive shoots in early October, 2016) at 0 °C under a salinity of 50 psu, seeds still had a considerable vigor, i.e. 57.8 ± 16.8%.ConclusionOur experiments demonstrated that seeds stored at 0 °C under a salinity of 40–60 psu could effectively prolong the duration of dormancy of Z. japonica seeds. The proposed technique is a simple and effective long-term storage method for Z. japonica seeds, which can then be used to aid future conservation, restoration and management of these sensitive and ecologically important habitat formers. The findings may also serve as useful reference for seed storage of other threatened seagrass species and facilitate their ex situ conservation and habitat restoration.

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult.

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.

Abstract 1236: Subclinical doses and choice of replication stress inducer in combination with CHK1 inhibitors for optimal tumor control and immune responses in vitro and in vivo

Abstract Background: Lung cancer and melanoma are responsible for almost 25% of cancer deaths in Australia. Despite recent progress with targeted and immuno-therapies, the 5 year survival rate in late stage patients is still<20%. CHK1 inhibitors are being investigated as chemosensitizing agents with agents that increase replication stress, primarily Gemcitiabine. Clinical trials of this combination have shown some good responses but high levels of normal tissue toxicity. Materials and methods: Here we have investigated the molecular basis of sensitivity to CHK1 inhibitors as combinations with subclinical doses of hydroxyurea (HU) in melanoma and non-small cell lung cancer (NSCLC) tumor spheres and cell lines in 2% O2 conditions and xenograft models in immune compromised mice and with humanized immune systems. Results: We report that low dose HU increased the sensitivity of >70% of both melanoma and NSCLC cell lines to CHK1 inhibitor (GDC-0575) triggered apoptosis, with complete loss of viability found with clinically achievable doses of this combination. Similar sensitivity was observed in xenograft models of both melanoma and NSCLC. We also demonstrate that a low dose of Gemcitabine combination with CHK1 inhibitor results in complete loss of proliferative potential in normal tissue, whereas normal tissue retaining proliferative potential after treatment with even high doses of hydroxyurea in combination with CHK1 inhibitor. In vivo, this translates to minimal effect on lymphocyte populations in the blood. The combination also triggers an inflammatory response involving the recruitment of macrophages, associated with increased HMGB1 nuclear staining. Immune responses were also assessed in humanized mouse models. Discussion: These data indicate that the combination of low dose HU and CHK1 inhibitor have strong anti-cancer activity in the setting of melanoma and NSCLC cancer, and triggers an inflammatory response, and is likely to be better tolerated than current combinations with Gemcitabine. Our data suggest a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination, and this appears to include an immune response to the tumor. Citation Format: Zay Yar Oo, Martina Proctor, Alex Stevenson, Deborah Nazareth, Jill Larsen, Nikolas Haass, Brian G. Gabrielli. Subclinical doses and choice of replication stress inducer in combination with CHK1 inhibitors for optimal tumor control and immune responses in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1236.

Abstract A069: Increasing replication stress by reducing nucleoside levels sensitizes melanomas and non-small cell lung cancers to CHK1 inhibitor in vitro and in vivo

Abstract CHK1 inhibitors are being investigated as chemosensitizing agents with agents that increase replication stress. Here we have investigated the molecular basis of sensitivity to CHK1 inhibitors as single agents in melanoma and as combinations with subclinical doses of hydroxyurea in melanoma and non-small cell lung cancer (NSCLC). We have found that sensitivity in vitro and in vivo to single agent CHK1 inhibitor is associated with defective S phase cell cycle checkpoint response to low dNTP levels. Loss of checkpoint response by overexpressing components of the checkpoint or inhibition of Wee1, convert CHK1 inhibitor insensitive cells to sensitive, and similarly depletion of CDC25A reduces CHK1 inhibitor sensitivity in sensitive lines. The increased DNA damage found with CHK1 inhibitor treatment is not sufficient to induce cell death, and cell death was triggered without entry into mitosis. We have found that about 20% of melanomas are sensitive to CHK1 inhibitor as a single agent, but we show that >70% of melanomas and NSCLCs are sensitive to combination with subclinical doses of hydroxyurea. The mechanism of cell death in the combination appears identical to CHK1 inhibitor alone in inhibitor-sensitive melanomas, with cells dying without entering mitosis. In half the cell melanomas tested, the combination produced a complete loss of viability; in the other half, the viable population after treatment was incapable of further proliferation in the absence in drug. The sensitivity to the combination is independent of other known risk factors, suggesting a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination. Citation Format: Zay Yar Oo, Martina Proctor, Alexander Stevenson, Jill Larsen, Brian G. Gabrielli. Increasing replication stress by reducing nucleoside levels sensitizes melanomas and non-small cell lung cancers to CHK1 inhibitor in vitro and in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A069.

Improved stability of live attenuated vaccine gdhA derivative Pasteurella multocida B:2 by freeze drying method for use as animal vaccine

The efficacy of attenuated strain of gdhA derivative Pasteurella multocida B:2 mutant as a live vaccine to control haemorrhagic septicaemia (HS) disease in cattle and buffaloes has been demonstrated. In order to use P. multocida B:2 mutant as a commercial product, it is essential to optimise its formulation for high viability and stability of the live cells. The effectiveness of freeze-drying process using different protective agent formulations for improving cells viability was explored. Sugar and nitrogen compounds were used as protective agents in freeze-drying and the capability of these compounds in maintaining the viability of mutant P. multocida B:2 during subsequent storage was investigated. A complete loss in viability of freeze-dried mutant P. multocida B:2 was monthly observed until 6–12 months of storage at −30 °C, 4 °C and 27 °C when nitrogen compound or no protective agent was added. Trehalose and sucrose showed significantly high survival rate of 93–95% immediately after freeze-drying and the viability was retained during the subsequent storage at −30 °C and 4 °C. A smooth cell surface without any cell-wall damage was observed for the cells formulated with trehalose under scanning electron micrograph. This study presented a freeze-drying process generating a dried live attenuated vaccine formulation with high stability for commercial applications.

Comparison of fertility of liquid or frozen semen when varying the interval from CIDR removal to insemination

Cryopreservation allows for long-term storage of semen; however, it leads to damage of sperm that may result in complete loss of viability or changes that possibly decrease sperm functionality. Liquid semen is not exposed to these stressors and may result in a longer lifespan in the female reproductive tract, thus increasing the range in timing of insemination without affecting fertility. The objective of this study was to compare fertility of liquid and frozen semen when varying the interval from CIDR removal to AI using the 7-day CO-Synch+CIDR protocol for synchronization of time of estrus. Within age group, crossbred cows (n=389) were randomly assigned to insemination at 36 or 60h after CIDR removal with either liquid or frozen semen (36L, 60L, 36F, and 60F) from one of two Angus bulls. Cows were monitored for estrous activity from CIDR removal until 60h thereafter. Cows that failed to exhibit estrus received GnRH (100μg, i.m.), and a blood sample was collected for analysis of estradiol concentration. There was no difference in pregnancy rates when liquid or frozen semen (53% and 52%) was used, but cows inseminated at 60h had a greater (P<0.01) pregnancy rate than those inseminated at 36h (72% and 31%). There was no time of AI by semen type interaction (P=0.57). Estrus was detected in 63%, 61%, 56%, and 62% of 36F, 36L, 60F, and 60L, respectively (only 5% and 1% of 36F and 36L were detected in estrus before insemination). Overall cows that exhibited estrus had a greater pregnancy rate compared with cows that did not (P<0.01; 79% compared with 24%). Among cows that did not exhibit estrus, those inseminated with liquid semen tended to have greater pregnancy rates than those inseminated with frozen semen (P=0.06). Cows that became pregnant had greater (P<0.01) concentrations of estradiol at 60h than those that did not (10.7±0.55 compared with 7.9±0.26pg/mL). In summary, there was no difference in pregnancy success between liquid and frozen semen. However, cows that exhibited estrus and were inseminated at 60h after CIDR removal had greater pregnancy success compared to cows that did not exhibit estrus.

Seed longevity, germination and seedling vigour of Rheum australe D. Don: A step towards conservation and cultivation

A 60 months study was carried out to assess the seed longevity and effect of duration of storage period on viability, germination and seedling vigour in Rheum australe D. Don. (rubarb), a highly valued medicinal herb. The species is in high demand and has endangered status therefore, required conservation/cultivation interventions. The study was carried out using seeds of same lot after storage of every six months (0, 6, 12, 18 and so on) till complete loss of seed viability. Freshly harvested seeds exhibited high seed viability (94%) and were non-dormant; above 80% germination occurred within a week. The above status was completely retained till 12 months storage. Beyond this period both seed viability and germination declined consistently with the progression of storage period. A complete loss of viability was observed beyond 60 months storage. Seed pre-treatments tested namely, GA3, KNO3 and chilling neither affected significantly the final germination of fresh seeds (86, 80 and 78%, respectively) nor differentially stored seeds. However, GA3 (1mM) and chilling significantly reduced the time required for germination. The seed longevity (time taken for 50% decline in seed viability) of R. australe was about 45 months, but a storage period of no longer than 24 months can be recommended. During 24 months storage period the seed viability and germination were reasonably high and resulted in the production of healthy seedlings with high survival rate. The outcome was favourable for the conservation/cultivation of the species.

In situ or ex situ seed conservation: which is the more effective way to maintain seed longevity of an endangered cactus?

With restricted populations and a small number of individuals, Discocactus bahiensis Britton &amp; Rose (Cactaceae) is an endangered species in Brazil and its capacity for the formation of seed banks in the soil and the maintenance of seed viability remains unknown. Thus, the aim of the present study was to determine the most efficient way to maintain viability during storage of seeds of D. bahiensis . Seeds were stored in paper bags and either kept in a cold chamber (7 ± 2°C) in the dark (ex situ conservation) or buried in the soil to a depth of 5 cm in an area of natural occurrence of the species (in situ conservation). Germinability of the seed banks was evaluated monthly for 20 months. During the first 10 months of storage, germinability of the seeds conserved in situ and ex situ was similar to that of recently collected seeds. After this period, a 70% reduction in germinability was found for the seeds maintained in situ and there was nearly complete loss of viability after 12 months of storage in the field (germinability &lt; 10% in the last 8 months of the experiment), indicating the ability to form persistent soil seed banks. In contrast, the seeds stored in the cold chamber maintained greater than 70% germinability throughout the entire analysis period, demonstrating that ex situ conservation is the most efficient way to maintain the viability of the seeds of this endangered species.

Efficacy of organic solvents for medium term storage of oil palm (&lt;italic&gt;Elaeis guineensis&lt;/italic&gt; Jacq.) pollen

The oil palm is the richest source of vegetable oil in the world. The hybrid nature of crop necessitates artificial hybridization between proven parent palms which in turn require the pollen from male parent to be collected and stored without loss of viability for sufficiently long period. Reports suggested that pollen grains of various taxa could be successfully preserved in different organic solvents. In the present study, attempt was made to evaluate the suitability of organic solvents to store the oil palm pollen at 0 to -5°C temperature. Among the five solvents used, there was significant variation for viability and germination percentages. Viability was the maximum for pollen stored in Diethyl ether (90.45 %) as well as in n-Hexane (88.41%) after 200 days of storage. Viability of pollen stored in Chloroform and Acetone were moderate and in case of Methyl alcohol, complete loss of viability was observed. With respect to pollen germination &lt;italic&gt;in vitro&lt;/italic&gt;, similar trend was observed. The trend was same at different intervals/period of storage. This was confirmed through &lt;italic&gt;in vivo&lt;/italic&gt; pollination and fruits set studies. Thus, present method indicated that it is feasible to store oil palm pollen in non polar solvents beyond 200 days without considerable loss of viability or germination.

Abstract 3787: Effects of hyperthermia on DNA repair capacity and long term survival in ovarian and colon carcinoma cells

Abstract Background: Gynecological and gastrointestinal malignancies have the potential to spread into the peritoneal cavity. Peritoneal carcinomatosis is associated with rapid disease progression and poor prognosis. Aggressive cytoreductive surgery combined with intraperitoneal hyperthermic chemotherapy (HIPEC) is actually considered as promising treatment option. However, it is unknown if all cytotoxic drugs currently used in the clinics can be efficiently potentiated by hyperthermia. Furthermore, the mechanisms of hyperthermia induced sensitization of cancer cells to chemotherapy are poorly understood. Methods: Two ovarian (Ovcar 3, Ovcar 8) and 2 colon carcinoma cell lines (HCT116, LOVO3) were pulse treated for one hour with clinically relevant dosages of cisplatin, oxaliplatin, doxorubicin, mytomycin C, paclitaxel, and 5-FU at 37°C or 42°C. Long term survival was assessed using colony forming assay. Single and double strand breaks were quantified using alkaline COMET immediately after treatment and in a time course for up to 8 h. Efficacy of DNA repair was determined by γH2AX, Rad51, and P53BP1 foci formation. Results: Hyperthermia significantly potentiated cytotoxicity of cisplatin, oxaliplatin, mitomycin C, and doxorubicin in all cell lines investigated. The most prominent effects were observed with doxorubicin leading to an almost complete loss of viability. Of note, hyperthermia did not affect cytotoxicity of paclitaxel and 5-FU. The molecular mechanism of hyperthermia induced sensitization to doxorubicin was further investigated. Hyperthermia significantly influenced doxorubicin transport since intracellular concentration was approximately doubled at 42°C compared to 37°C. However, despite adjusting the concentration of doxorubicin at 42°C to that observed with 37°C, hyperthermia still potentiated cytotoxicity indicating that other mechanisms are also involved. We detected no differences in the amount of DNA strand breaks immediately after treatment between 37°C and 42°C. However, time course experiments revealed a significant delay in DNA repair at 42°C. Whereas DNA strand breaks were almost completely repaired after 8 h at 37°C, there was hardly any repair detectable at 42°C. This was also mirrored by an enhanced number of γH2AX foci in nuclei of cells treated with doxorubicin at 42°C 3 h after treatment. Interestingly, we observed no differences in the number of Rad51 but a significantly enhanced number of P53BP1 foci at 42°C indicating a shift towards unfaithful non homologous end joining (NHEJ) at higher temperature. Conclusion: Hyperthermia significantly potentiated effects of DNA damaging agents such as doxorubicin via two different mechanisms. (1) Enhanced temperature leads to an enhanced uptake or reduced efflux of doxorubicin. (2) Hyperthermia reduces DNA repair capacity and potentially shifts DNA double strand repair to an error-prone NHEJ. Citation Format: Lea Schaaf, Heiko Van Der Kuip, Thomas E. Mürdter, Christoph Ulmer, Walter E. Aulitzky. Effects of hyperthermia on DNA repair capacity and long term survival in ovarian and colon carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3787. doi:10.1158/1538-7445.AM2014-3787

P13: Vitamin D3 therapy increases cryoablation efficacy: A novel strategy for the treatment of prostate cancer

Introduction Adjuvant therapies contribute to the successful treatment of cancer. Our previous reports have shown that combining cryoablation with cytotoxic agents enhances prostate cancer cell death. Vitamin D 3 (VD 3 ) is a natural steroidal hormone that has been shown to exhibit cytotoxic properties preferentially inducing apoptosis in a variety of human cancer cells. Human prostate cancer cells are known to be resistant to many cytodestructive agents, including cryoablation and VD 3 . Here, we evaluated the efficacy of VD 3 combined with cryoablation on androgen insensitive human prostate cancer (PC-3 and LNCaP-HP) cell death. Methods Freezing and VD 3 exposure to PC-3 and LNCaP-HP cells were performed using both 2D and 3D culture systems and efficacy was determined by cell viability assays. Resultant cell death and specific signaling components were determined using apoptotic inhibitors, fluorescence microscopy, protease activity assays and immunoblotting. Results Exposure of LNCaP-HP cells to freezing (-15°C) or VD 3 (50 μM) results in minimal cell death (15% and 10% respectively 3 days post treatment), while a complete loss of viability was observed with the combination. The synergistic effect was found to be due to a marked increase in apoptosis. Western blot analysis revealed a decrease in pro-caspase-9 and -3 between 6 and 18 h post-exposure. Caspase activation assays confirmed the reduction in pro-caspase levels was a result of caspase activation. Protease inhibitors were incorporated into the combination protocol to determine the overall contribution of caspase activity in cell death. Inhibition of caspase-9 significantly blocked the combination induced cell death compared to cells that did not receive the inhibitor (55% vs. 21% viable, respectively). The addition of the caspase-8 inhibitor resulted in only minimal protection, indicating a specific mitochondrial-mediated event. Importantly, the combination was not effective when applied to normal prostate cells. Conclusions VD 3 sensitizes CaP cells to cryoablation. The significant increase in cell death was attributed to the activation of apoptosis, specifically through mitochondrial -mediated events. The results describe a novel therapeutic model for the treatment of prostate cancer and provide support for future in vivo studies.

Seed desiccation tolerance and germination of a potentially threatened Chinese species, Fosbergia shweliensis

Fosbergia shweliensis is a potentially critically endangered Chinese tree species. However, there is little known about the seed biology of this species. Here we performed germination and desiccation trials of its seeds. Fresh seeds of F. shweliensis were not dormant and germinated optimally at 25 degrees C (100% germination). There was 92% germination when seed moisture content (MC) was reduced to 16% (fresh weight basis). Further drying at 15 degrees C and 15% relative humidity, reduced germination to 73% when seed MC was 12% and drying to 7% MC resulted in complete loss in viability. These results indicate that F. shweliensis seeds are partly desiccation-tolerant (i.e. intermediate in storage behaviour). The present research supplied seed germination information and guidance about seed storage for the genetic conservation and restoration of this species.