Rapid Loss Of Viability Research Articles

EFFECT OF CRYO-STORAGE ON STORABILITY OF PAPAYA SEEDS

A fruit crop of significant economic importance, papayas are often propagated from seeds. Based on their freezing injury even at low moisture content, papaya seeds are classified as "intermediate seeds"; and rapid loss of viability in storage is assumed to be the primary cause of poor germination; thus, the development of appropriate technology is necessary to prolong the storability of papaya seeds. One such effective technology for prolonged seed storability is cryo-storage. The purpose of the current study was to investigate how cryostorage affects papaya seed storability. For this investigation, TNAU Papaya variety CO 8 seeds were utilised. Five distinct treatments were applied to the seeds, which were then kept for approximately six months at liquid nitrogen's vapour phase (-140°C). The biochemical parameters such as protein content, free amino acid, total carbohydrates, free sugars, total lipids, and dehydrogenase activity were measured on a monthly basis along with seed germination and seedling vigour in the cryopreserved papaya seeds. Results indicated that papaya seeds that had been vitrified (seeds with a 10% moisture content that had been soaked in loading solution for 20 minutes, then immersed in Plant Vitrification Solution 2 (PVS2) for another 20 minutes, packed in cryovials and kept in cryo-storage at -140°C) compared to papaya seeds that were packed in cloth bags and stored in ambient conditions had higher seed germination, dry matter production and vigour index at six months after storage. In comparison to fresh seeds, the results also showed that, after six months of cryo-storage, biochemical proximate, protein content, total carbohydrate content, total lipid content, and dehydrogenase activity were maintained in the aforementioned treatment with minimum reduction, while electrical conductivity, free amino acid content, and free sugar content gradually increased with the storage period at minimum rate. In crux, vitrified papaya seeds packaged in cryovials and kept in cryo-storage at -140°C preserved their quality for six months, meeting Indian Minimum Seed Certification Standards. This could be useful for storing papaya seeds for longer periods of time. Keywords: Papaya, vitrification, differential scanning calorimetry, liquid nitrogen, cryo-storage

302 Targeted Inhibition of the Epigenetically Activated miR-483-5p IGF-2 Pathway as a Novel Therapeutic Strategy for Meningioma

INTRODUCTION: Meningioma is the most common primary central nervous system tumor. These are usually treated by surgical resection with curative intent, however gross total resection is not always feasible and there are many cases of recurrent and refractory disease despite adjuvant radiotherapy. Presently, there is no effective pharmacotherapeutic options for these tumors. METHODS: Small RNA sequencing was performed on meningioma tumor samples to study grade-dependent changes in microRNA expression. Gene expression was analyzed by chromatin marks, qRT-PCR and western blot. miRNA modulation, anti-IGF-2 neutralizing antibodies, and inhibitors against IGF1R were evaluated in a tumor-derived primary cultures of meningioma cells. RESULTS: Meningioma tumor samples showed high, grade-dependent expression of miR-483-5p, associated with high mRNA and protein expression of its host gene IGF-2. Inhibition of miR-483-5p reduced the growth of cultured meningioma cells, whereas a miR-483 mimic increased cell proliferation. Similarly, inhibition of this pathway with anti-IGF-2 neutralizing antibodies reduced meningioma cell proliferation. Small molecule tyrosine kinase inhibitor blockade of the IGF-2 receptor (IGF1R) resulted in rapid loss of viability of cultured meningioma tumor-derived cells, suggesting that autocrine IGF-2 feedback is obligatory for meningioma tumor cell survival and growth. The observed IGF1R-inhibitory IC50 for GSK1838705A and Ceritinib in cell-based assays along with the available pharmacokinetics data predicted that effective drug concentration could be achieved in vivo as a new medical treatment of meningioma. CONCLUSIONS: Meningioma cell growth is critically dependent on autocrine miR-483/IGF-2 stimulation and the IGF-2 pathway provides a new feasible meningioma treatment target.

Physiological quality of seeds of arabic coffee cultivars stored for a period of two years.

The rapid loss of viability, coupled with the difficulties and limitations in conserving coffee seeds, are some of the challenges that nurseries are currently facing. Thus, the objective of this work was to analyze the physiological quality of stored Arabica coffee seeds from cultivars recommended for planting in Brazilian mountainous regions. Seeds from 10 Arabica coffee cultivars were used: Catucaí-785/15, Catucaí-2SL, Catucaí-24/137, Japan, Arara, Acauã, Catuaí IAC-81, Mundo Novo IAC-379/19, Catuaí IAC-62, and Caturra IAC-479. The seeds were collected in the Arabica coffee seed production field, in the municipality of Marechal Floriano-ES. They were subsequently processed and dried in the shade, at room temperature, until reaching a humidity of 35 ± 1%, analyzed and stored in a natural laboratory environment (25 ± 2 °C), for 24 months. The following were analyzed: seed water content, germination, germination speed index, electrical conductivity and potassium leaching. The experimental design used was completely randomized, with ten cultivars, two storage times, with four replications of 25 seeds. Storing arabica coffee seeds for 24 months results in a drop in the germination percentage. Stored seeds of the Catucai 24/137 and Arara cultivars showed germination percentages similar to those of newly harvested seeds. Seeds of the Catuai IAC-62 cultivar maintain vigor during storage. The stored seeds of the Caturra IAC-476 and Japi cultivars showed a reduction in physiological quality. Electrical conductivity and potassium leaching tests are efficient in identifying seeds in an advanced state of deterioration. Seeds of Arabica coffee cultivars stored for 24 months, under the conditions of the present study, produce abnormal seedlings.

An essential Trypanosoma brucei protein kinase: a functional analysis of regulation and the identification of inhibitors

IntroductionThe protein serine/threonine kinase AEK1 is essential in the pathogenic stage of Trypanosoma brucei, the causative agent of African trypanosomiasis. AEK1 is a member of the AGC protein kinase family, although it is not closely related to a specific human AGC kinase. Our previous chemical genetic studies showed that targeted inhibition of AEK1 in parasites expressing analog-sensitive AEK1 blocked parasite growth and enhanced survival of infected mice.MethodsTo further validate AEK1 as a drug target, we used the chemical genetic system to determine the effect of a 24 hour loss of AEK1 activity on cell viability at the clonal level. A panel of 429 protein kinase inhibitors were screened against the wild-type protein for binding, using time-resolved fluorescence energy transfer (TR-FRET). The role of phosphorylation sites and motifs was probed by determining whether expression of proteins harboring mutations in these sequences could rescue AEK1 conditional knockout parasites. To determine the effect that mutations in the phosphosites have on the kinase activity of cellular AEK1 we compared the in vitro kinase activity of mutant and wild-type proteins immunoprecipitated from parasite lysates using the exogenous substrate MBP. Finally, the tagged AEK1 protein was localized by deconvolution microscopy.ResultsAfter a 24 hour exposure to an AEK1 inhibitory analog in the chemical genetic system, less than five percent of the remaining live cells can clonally expand, further validating AEK1 as a drug target. In the AEK1 inhibitor screening assay, we identified 17 hit compounds. Complementation studies showed that of the two known phosphorylation sites in the activation loop; mutation of one abolished function while mutation of the other had no discernable effect. Mutation of the other two AEK1 phosphosites gave intermediate phenotypes. Mutations in either the hydrophobic motif at the C-terminus of the protein or in the region of AEK1 predicted to bind the hydrophobic motif were also required for function. All parasites with defective AEK1 showed reduced proliferation and defects in cytokinesis, although the tested mutations differed in terms of the extent of cell death. Kinase activity of immunoprecipitated AEK1 phosphosite mutants largely paralleled the effects seen in complementation studies, although the mutation of the phosphosite adjacent to the hydrophobic motif had a greater impact on activity than predicted by the complementation studies. AEK1 was localized to cytoplasmic puncta distinct from glycosomes and acidocalcisomes.DiscussionThe rapid loss of viability of cells inhibited for AEK1 supports the idea that a short course of treatment that target AEK1 may be sufficient for treatment of people or animals infected with T. brucei. Key regulatory elements between AEK1 and its closest mammalian homolog appear to be largely conserved despite the vast evolutionary distance between mammals and T. brucei. The presence of AEK1 in cytoplasmic puncta raises the possibility that its localization may also play a role in functional activity.

EXTH-57. THE IGF-2 SIGNALING PATHWAY IS A PROMISING MENINGIOMA TREATMENT TARGET

Abstract Meningioma is the most common primary central nervous system tumor often causing serious complications while there is no effective medical treatment. We developed a tumor-derived primary culture model featuring tumor cells with retained PDG2S lineage marker as well as non-arachnoid cells mimicking the tumor cell microenvironment. We used this model to test the relevance of miRNA differentially expressed in meningioma tumor samples identified by small RNA sequencing. We found that in all meningiomas studied, there was activation of the IGF2/miR-483 locus with high expression of both miR-483-5p and IGF2. Inhibition of miR-483-5p reduced the growth of cultured meningioma cells, whereas a miR-483 mimic increased cell proliferation. Anti-IGF2 neutralizing antibodies reduced meningioma cell proliferation. Small molecule tyrosine kinase inhibitor blockade of the IGF2 receptor (IGF1R) resulted in rapid loss of viability of cultured meningioma tumor-derived cells. Autocrine IGF2 feed-back appears to be obligatory for meningioma tumor cell survival and growth. Inhibition of this circuit with IGF1R small molecule tyrosine kinase inhibitors or receptor-binding antibodies as well as anti-IGF2 neutralizing antibodies may have therapeutic potential for meningioma.

The fadXDEBA locus of Staphylococcus aureus is required for metabolism of exogenous palmitic acid and in vivo growth.

In Staphylococcus aureus, genes that should confer the capacity to metabolize fatty acids by β-oxidation occur in the fadXDEBA locus, but their function has not been elucidated. Previously, incorporation into phospholipid through the fatty acid kinase FakA pathway was thought to be the only option available for S. aureus to metabolize exogenous saturated fatty acids. We now find that in S. aureus USA300, a fadX::lux reporter was repressed by glucose and induced by palmitic acid but not stearic acid, while in USA300ΔfakA basal expression was significantly elevated, and enhanced in response to both fatty acids. When cultures were supplemented with palmitic acid, palmitoyl-CoA representing the first metabolite in the β-oxidation pathway was detected in USA300, but not in a fadXDEBA deletion mutant USA300Δfad, which relative to USA300 exhibited increased incorporation of palmitic acid into phospholipid accompanied by a rapid loss of viability. USA300Δfad also exhibited significantly reduced viability in a murine tissue abscess infection model. Our data are consistent with FakA-mediated incorporation of fatty acids into phospholipid as a preferred pathway for metabolism of exogenous fatty acids, while the fad locus is critical for metabolism of palmitic acid, which is the most abundant free fatty acid in human plasma.

Effect of different storage methods on germination and seedling emergence of six pawpaw (Carica Papaya L.) varieties planted in Ogbomoso, Oyo state, Nigeria

Pawpaw is known as a fruit that contains nutrients and vitamins ranging from green ripe to over-ripe and unripe fruits of various varieties. The poor and delayed germination, rapid loss of viability in seeds due to the presence of sarcotesta, and condition of storage of seeds could be addressed through the storage in various containers. This paper focused on the investigation of the best storage methods that aid germination, seedling emergence and seedling performance of six pawpaw varieties. The experiment was set up in the Crop and Soil Science Laboratory Department and Nursery Site of the Ladoke Akintola University of Technology, Ogbomoso (Oyo-State). It was a 6 x 5 factorial experiment that consisted of 6 varieties of pawpaw; Pink Solo, Green Solo, Ghana Solo, Sunrise Solo, Ajawa Local, and Berry likewise the 5 storage methods are desiccator, refrigerator, plastic container, aluminum foil paper, and earthen pot. Data were collected by counting the number of germinated seeds manually from 4 – 16 days after sowing inside Petri dishes, emergence rate was also counted after seeds have been sown inside polythene bags and nursed for 12 weeks and the number of seeds germinated and seedling emergence was transformed into percentages. Seedling emergence rates were also counted manually and analysed by using Analysis of Variance (ANOVA), means were compared using the least significant difference at a 5% probability level. Significant differences were observed among storage methods and pawpaw varieties in germination rate, seedling emergence, and performance. The highest plant height was observed in green solo at 12WAS in the nursery (25.63 cm), the highest stem girth was recorded in Ajawa local 3.86 cm while the highest number of leaves was recorded in sunrise 9.9. The total emergence percentage was recorded in Green Solo at 93.8%. The highest germination percentage was observed in Ghana Solo 59.4%. The highest germination, seedling emergence, plant height, and number of leaves were recorded in the desiccator as 44.96%, 84.4%, 23.32 cm, and 9.38. Ajawa Local and Green Solo are recommended to be kept inside a desiccator or plastic container.

Inhibition of the epigenetically activated miR-483-5p/IGF-2 pathway results in rapid loss of meningioma tumor cell viability

PurposeMeningioma is the most common primary central nervous system tumor often causing serious complications, and presently no medical treatment is available. The goal of this study was to discover miRNAs dysregulated in meningioma, and explore miRNA-associated pathways amenable for therapeutic interventions.MethodsSmall RNA sequencing was performed on meningioma tumor samples to study grade-dependent changes in microRNA expression. Gene expression was analyzed by chromatin marks, qRT-PCR and western blot. miRNA modulation, anti-IGF-2 neutralizing antibodies, and inhibitors against IGF1R were evaluated in a tumor-derived primary cultures of meningioma cells.ResultsMeningioma tumor samples showed high, grade-dependent expression of miR-483-5p, associated with high mRNA and protein expression of its host gene IGF-2. Inhibition of miR-483-5p reduced the growth of cultured meningioma cells, whereas a miR-483 mimic increased cell proliferation. Similarly, inhibition of this pathway with anti-IGF-2 neutralizing antibodies reduced meningioma cell proliferation. Small molecule tyrosine kinase inhibitor blockade of the IGF-2 receptor (IGF1R) resulted in rapid loss of viability of cultured meningioma tumor-derived cells, suggesting that autocrine IGF-2 feedback is obligatory for meningioma tumor cell survival and growth. The observed IGF1R-inhibitory IC50 for GSK1838705A and ceritinib in cell-based assays along with the available pharmacokinetics data predicted that effective drug concentration could be achieved in vivo as a new medical treatment of meningioma.ConclusionMeningioma cell growth is critically dependent on autocrine miR-483/IGF-2 stimulation and the IGF-2 pathway provides a feasible meningioma treatment target.

Method optimisation for prolonged laboratory storage of Ascaridia galli eggs

Eggs in the infective stage of the chicken nematode Ascaridia galli are often required for in vivo and in vitro studies on this parasite. The reliability of any artificial A. galli infection depends on the viability and embryonation capacity of A. galli eggs. The aim of this study was to determine ideal storage conditions for maximising the viability of A. galli eggs and maintaining viability for the longest period. A 2 × 2 × 3 × 5 factorial experimental design was employed to investigate the effects of storage temperature (4°C or 26°C), storage condition (aerobic or anaerobic), storage medium (water, 0.1 N H2SO4 or 2% formalin) and storage period (4, 8, 12, 16 and 20 weeks). The viability of eggs was assessed after eggs in all treatment groups were held aerobically at 26°C for 2 weeks after the storage period to test embryonation capacity. Based on morphological characteristics, they were categorised as undeveloped, developing, vermiform, embryonated or dead. The maintenance of viability during storage at 4°C was optimal under anaerobic conditions while at 26°C it was optimal under aerobic conditions. Anaerobic conditions at 26°C led to a rapid loss of viability while aerobic conditions at 4°C had a less severe negative effect on maintenance of viability. Egg storage in 0.1 N H2SO4 resulted in a significantly higher viability overall (54.7%) than storage in 2% formalin (49.2%) or water (37.3%) (P < 0.0001). Untreated water was the least favourable storage medium when eggs were stored at 26°C while it was a medium of intermediate quality at 4°C. The viability of A. galli eggs decreased significantly with storage time (P < 0.0001) depending on the other factors. The lowest rate of decline was seen with storage of eggs under anaerobic conditions at 4°C or aerobic conditions at 26°C in 0.1 N H2SO4. Eggs in these treatments retained up to 72% of overall viability at 20 weeks with a decline rate of approximately 2% per week with no significant difference between the two. Therefore, this study has clearly revealed opposing aerobic conditions required for prolonged storage of A. galli eggs in the pre-embryonated state at 4°C. It has also identified that 0.1 N H2SO4 provides the best preservation against degradation during storage, particularly at 26°C under aerobic conditions. Achieving strictly anaerobic conditions can be difficult to achieve so storage aerobically at 26°C may be preferred for simplicity.

201-OR: Evaluation of the Effects of Harmine on ß-Cell Function and Proliferation in Standardized Human Islets Using 3D High-Content Confocal Imaging and Automated Analysis

Restoration of β-cell mass by inducing proliferation of residual β-cells represents an attractive therapeutic approach for diabetes treatment. Native and dispersed primary human islets undergo rapid loss of viability and function ex vivo, complicating their use in proliferation studies. Here, we establish a novel method for evaluating compound effects on β-cell proliferation and count using reaggregated primary human islet microtissues (MTs) , of homogeneous size and architecture and that maintain stable viability and function for 4 weeks in culture. We utilized this platform to evaluate the effects of short- and long-term harmine treatment on β-cell proliferation and function in a dose-dependent manner. Following harmine treatment and EdU incorporation, islet MTs were stained in 3D for DAPI (nuclear marker) , NKX6.1 (β-cell marker) , and EdU (proliferation marker) , imaged on a high-content confocal microscope, and subjected to automated 3D analysis of numbers of total cells, β-cells, and proliferating β- and non-β-cells per islet MT. In parallel, assessment of insulin secretion, intracellular insulin and ATP contents, and Caspase 3/7 activity was performed for a comprehensive overview of islet MT function and viability. We observed that 4 days of harmine treatment increased β- and non-β-cell proliferation, NKX6.1 expression, and basal and stimulated insulin secretion in a dose-dependent manner, whereas fold-stimulation of secretion peaked at intermediate doses. Interestingly, 15-day harmine treatment led to a general reduction in proliferative effects as well as altered dose-dependent trends. The methodology presented here thus constitutes a unique tool for in vitro high-throughput evaluation of short- and long-term changes in human β-cell proliferation, count and fraction in parallel with a variety of functional parameters, in a representative and standardized 3D human islet microtissue model. Disclosure A.C.Title: None. B.Yesildag: None. M.Karsai: None. J.Mir-coll: None. O.Yavas grining: None. C.Rufer: None. S.Sonntag: None. F.Forschler: None. S.Jawurek: None. T.Klein: Employee; Boehringer Ingelheim International GmbH.

Impact of pre-storage seed invigoration in ash gourd [Benincasa hispida (Thunb.) Cogn.

Ash gourd is widely cultivated as a vegetable crop in Kerala. In the state, prolonging seed longevity is a major concern as the prevailing hot humid conditions accelerates deterioration of seeds leading to rapid loss of viability during storage. Seed deterioration due toageing is indeed inevitable and irreversible phenomenon, the best that can be done is to lower its rate (Coolbear 1995). It is reported that seed priming technology helps in rapid and uniform germination and emergence of seeds and impart a great tolerance to adverse environmental conditions (Heydecker et al. 1973).

Wheat pollen storage leads to metabolic changes contributing to rapid viability loss

Wheat pollen storage leads to metabolic changes contributing to rapid viability loss

Synthesis and characterization of cecropin peptide-based silver nanocomposites: Its antibacterial activity and mode of action.

Novel antimicrobial agents with a low propensity to develop resistance by microorganisms have contemporary relevance. In this perspective, the present study reports the green synthesis and characterization of cecropins peptides (D2A21, D2A10, and D4E1) based silver nanocomposites. The effect of pH and concentration of peptides on the formation of nanocomposite material was studied using UV-Vis spectroscopy. The particle size was determined by transmission electron microscopy, which indicated the size in the range of 3 ± 0.4 to 20 ± 5 nm. Fourier-transform infrared spectroscopy studies suggested the involvement of peptides as a capping and reducing agent. Zeta potential analysis suggested that nanocomposite material was more cationic in nature than its native peptides. Nanocomposite material exhibited significantly enhanced antibacterial activity as compared to native peptides and silver nanoparticles with minimum inhibitory concentration (MIC) ranging from 1 to 3 μg mL-1 against both gram-positive and negative test bacteria; whereas the MICs of native peptides were found to be in the range of 4-6 μg mL-1. The mode of action of P-AgNPs was evaluated using scanning electron microscopy, membrane potential, and membrane integrity studies; wherein the nanocomposite material was found to act at the cell membrane level, causing complete loss of membrane potential and resulting in compromised membrane integrity with irreversible damage to the cell as shown by the rapid loss of viability due to membrane disruption, resulting in lysis. Among the three peptides tested, D2A21-silver nanocomposite had maximal antibacterial activity. Taken together; our experimental findings suggested that the peptide-based-silver nanocomposites can be considered as potential antibacterial agents for various biomedical applications.

Chromomycin, an antibiotic produced by Streptomyces flaviscleroticus might play a role in the resistance to oxidative stress and is essential for viability in stationary phase.

The well-known role of antibiotics in killing sensitive organisms has been challenged by the effects they exert at subinhibitory concentrations. Unfortunately, there are very few published reports on the advantages these molecules may confer to their producers. This study describes the construction of a genetically verified deletion mutant of Streptomyces flaviscleroticus unable to synthesize chromomycin. This mutant was characterized by a rapid loss of viability in stationary phase that was correlated with high oxidative stress and altered antioxidant defences. Altered levels of key metabolites in the mutant signalled a redistribution of the glycolytic flux toward the PPP to generate NADPH to fight oxidative stress as well as reduction of ATP-phosphofructokinase and Krebs cycle enzymes activities. These changes were correlated with a shift in the preference for carbon utilization from glucose to amino acids. Remarkably, chromomycin at subinhibitory concentration increased longevity of the non-producer and restored most of the phenotypic features' characteristic of the wild type strain. Altogether these observations suggest that chromomycin may have antioxidant properties that would explain, at least in part, some of the phenotypes of the mutant. Our observations warrant reconsideration of the secondary metabolite definition and raise the possibility of crucial roles for their producers.

Effects of environmental factors and ageing on germination of golden crownbeard (Verbesina encelioides)-A wide spread weed of Northern India

Golden crownbeard (Verbesina encelioides), which is abundant along roadsides in Northern India, has started to invade field borders in the South West of Punjab. This study was conducted to find the effect of environmental factors and accelerated ageing on germination of this weed. It germinated over a wide range of temperatures (15/5–35/25°C) with optimum germination at 25/15°C. Light was not a pre-requisite for germination. Germination was completely inhibited at −0.6 MPa. The seeds germinated at 160 mM sodium chloride (13.3%), but no germination was observed at 180 mM NaCl. Germination of seeds was very low at pH less than 5 or more than 8. Germination was 95% when seeds were placed on the soil surface. No emergence was observed when seeds were buried to 6 cm or greater depth. Accelerated ageing of seeds for 20 or more days completely inhibited germination. Results indicate that this weed can emerge in multiple germination flushes. It also has the ability to invade drought affected areas, and can grow in soils that are moderately saline, slightly acidic, or alkaline. However, this weed is not expected to buildup persistent soil seed banks due to rapid loss of viability (time to 50% persistence = 4.11 days) under accelerated ageing.

Evaluation of 2 ex vivo Bovine Cornea Storage Protocols for Drug Delivery Applications

The use of corneal tissue for ex vivo therapeutic evaluations is limited due to its rapid loss of viability after excision. Optimization of storage conditions may allow prolonged retention of physical tissue properties. In this study, we evaluated how storage in optimized organ culture (OC) medium at 37°C or phosphate-buffered saline (PBS) at 2–8°C impacted physical properties of bovine corneas. Tissue hydration, permeability and histology were monitored at baseline and following 1, 4 and 7 days of storage. Corneas stored in OC demonstrated significantly higher hydration and permeability when compared to those stored in PBS. Histology revealed that storage in OC consistently caused detachment of the epithelial layer by day 4 of storage, whereas both storage conditions caused a significant increase in stromal thickness and tissue vacuolation. This study highlights the limitations of currently available corneal tissue storage approaches for ex vivo drug permeation studies.

Changes in lipid metabolism convey acid tolerance in Saccharomyces cerevisiae

BackgroundThe yeast Saccharomyces cerevisiae plays an essential role in the fermentation of lignocellulosic hydrolysates. Weak organic acids in lignocellulosic hydrolysate can hamper the use of this renewable resource for fuel and chemical production. Plasma-membrane remodeling has recently been found to be involved in acquiring tolerance to organic acids, but the mechanisms responsible remain largely unknown. Therefore, it is essential to understand the underlying mechanisms of acid tolerance of S. cerevisiae for developing robust industrial strains.ResultsWe have performed a comparative analysis of lipids and fatty acids in S. cerevisiae grown in the presence of four different weak acids. The general response of the yeast to acid stress was found to be the accumulation of triacylglycerols and the degradation of steryl esters. In addition, a decrease in phosphatidic acid, phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine, and an increase in phosphatidylinositol were observed. Loss of cardiolipin in the mitochondria membrane may be responsible for the dysfunction of mitochondria and the dramatic decrease in the rate of respiration of S. cerevisiae under acid stress. Interestingly, the accumulation of ergosterol was found to be a protective mechanism of yeast exposed to organic acids, and the ERG1 gene in ergosterol biosynthesis played a key in ergosterol-mediated acid tolerance, as perturbing the expression of this gene caused rapid loss of viability. Interestingly, overexpressing OLE1 resulted in the increased levels of oleic acid (18:1n-9) and an increase in the unsaturation index of fatty acids in the plasma membrane, resulting in higher tolerance to acetic, formic and levulinic acid, while this change was found to be detrimental to cells exposed to lipophilic cinnamic acid.ConclusionsComparison of lipid profiles revealed different remodeling of lipids, FAs and the unsaturation index of the FAs in the cell membrane in response of S. cerevisiae to acetic, formic, levulinic and cinnamic acid, depending on the properties of the acid. In future work, it will be necessary to combine lipidome and transcriptome analysis to gain a better understanding of the underlying regulation network and interactions between central carbon metabolism (e.g., glycolysis, TCA cycle) and lipid biosynthesis.

Abstract 4130: TRIM37 expression levels dictate susceptibility to centrosome removal, supporting Plk4 inhibition as a potential new strategy for targeting neuroblastoma

Abstract Centrosomes are the major microtubule-organizing centers in animal cells. Centrosomes catalyze microtubule assembly to accelerate formation of the bipolar mitotic spindle that segregates chromosomes during cell division. To analyze the effect of centrosome removal in normal and cancer cells, we developed centrinone, a potent, specific and cellularly-active inhibitor of the protein kinase polo-like kinase 4 (Plk4), which is essential for centrosome duplication (Wong et al., 2015, Science 348:1155-60). Centrosome-less cells take longer to execute mitosis, and trigger a mitotic stress signaling pathway that results in p53 stabilization and apoptosis and/or senescence of the resulting daughter cells. Failure to form a spindle can also lead to apoptosis during mitosis or exit from mitosis without chromosome segregation (mitotic slippage). In a genome-wide CRISPR/Cas9 loss-of-function screen for resistance to centrinone, we had previously shown that loss of the ubiquitin ligase TRIM37 makes spindle assembly more robust to centrosome removal (Meitinger et al., 2016, J Cell Biol. 214:155-66), raising the possibility that high levels of TRIM37 might increase sensitivity to centrosome removal. Here, we describe the use of single-cell live imaging assays to identify determinants of vulnerability of cancer cells to centrosome removal. Among a panel of 37 tested cell lines, neuroblastoma-derived cell lines were the most sensitive to centrosome removal, exhibiting high levels of apoptosis and rapid loss of viability following treatment with centrinone. We found that sensitivity to centrosome removal correlated with TRIM37 expression levels. Upon centrosome depletion, cells with high TRIM37 levels, particularly neuroblastoma cells, exhibited greatly increased mitotic duration and frequency of mitotic failure. p53 positive neuroblastoma cell lines exhibited apoptosis resulting from mitotic stress-mediated p53 activation and from mitotic failure. Surprisingly, p53 negative neuroblastoma cell lines, which are unable to sense mitotic stress, were also highly sensitive to centrosome removal, with cell death in this case resulting from high rates of mitotic failure. TRIM37 deletion restored the ability of cells to proliferate in centrinone. Thus, centrosome removal via Plk4 inhibition appears to be a promising strategy for therapeutic treatment of neuroblastomas and potentially other cancers with high levels of TRIM37 expression. Citation Format: Franz Meitinger, Robert L. Davis, Ruth Kabeche, John V. Anzola, Yao Liang Wong, Andrew K. Shiau, Arshad Desai, Karen Oegema. TRIM37 expression levels dictate susceptibility to centrosome removal, supporting Plk4 inhibition as a potential new strategy for targeting neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4130.

Bioengineered organoid models of human germinal centers

Abstract B cells undergo affinity maturation to T cell-dependent antigens within specialized microenvironments in secondary lymphoid organs termed germinal centers (GCs) resulting in the selective proliferation and differentiation of high-affinity B cells. Selection is driven by the ability of antigen-specific GC B cells to extract antigen presented on follicular dendritic cells and subsequently recruit stimuli from T follicular helper cells. Successful GC B cells ultimately differentiate into plasma cells that secrete large amounts of antigen-specific antibody, or memory B cells that contribute to long-term immunity. However, the factors that influence selection and differentiation of GC B cells in humans are not yet well understood. One challenge arises from the rapid loss of viability of human primary GC B cells ex vivo under conventional cell culture conditions. To overcome this, we bioengineered organoids using a customizable matrix along with modified feeder cell lines. When encapsulated in these organoids, primary human GC B cells exhibit enhanced viability similar to that of B cells in GCs in vivo. The low auto-fluorescent properties of these organoids allow for imaging of cellular interactions and migration. Furthermore, the customizable chemistry of the organoid matrix allows us to screen for factors that influence the differentiation and fate of human GC B cells. Improving our understanding of these processes may provide important insights in areas such as therapeutics and vaccine development.

Effects of seed storage time and salt stress on the germination of Jatropha curcas L.

Jatropha curcas L. has been proposed to be a potential tropical plant for augmenting renewable energy sources. Due to its several merits, J. curcas deserves to be considered as candidate for biofuel production instead of for food seed sources. Its seeds contain 27–40% oil that can be processed to produce a high quality biodiesel fuel that is usable in a standard diesel engine. While J. curcas grows well under low-rainfall conditions, the salt tolerance capacity of this species has received meager attention. Furthermore, another major problem faced by this species is the lack of a safe system capable of guaranteeing seed germination when stored for medium or long periods of time. Thus, J. curcas seeds show two important problems: (i) rapid loss of viability, resulting from the high respiratory rate of the seeds during storage, and (ii) seed sensitivity when germinated under salt conditions. Therefore, the main objectives were (i) to verify if storage in very low humidity boxes can improve germination without affecting the oil content of the seeds and (ii) to compare different genotypes to prove their salt tolerance capacities. The results indicate that from the use of desiccants, all the germination parameters were kept reasonably constant, and germination was essentially unchanged during the entire storage period. Biochemical activity, supported by the drastic reduction in respiratory activity of the seeds, was also decreased during the storage in order to keep the seed reserves intact; therefore, J. curcas seeds may be resistant to storage when stored in a controlled process. On the other hand, it was shown that J. curcas presents a moderate tolerance to salinity. This species is able to germinate in the presence of up to 150 mM of Sodium chloride (NaCl), although a drastic reduction in the biomass accumulation was observed with the increase in salt concentration in the irrigation water. It was shown that the germination was reduced to values close to 4% at 150 mM NaCl; whereas biometric and biomass characteristics were strongly affected by the increase in salt content. However, it was shown that genotypes 114, 171 and 183 were shown to be potentially tolerant, whereas genotypes 218 and 133 were sensitive.