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## Recent Publications

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Head of Institute: Prof. Oren Tirosh

Institute of Biochemistry, Food Science and Nutrition,
Robert H. Smith Faculty of Agriculture, Food and Environment,
The Hebrew University of Jerusalem,
Herzl 229, Rehovot 7610001, ISRAEL

Tel: +972 - (0)8-9489385
Fax: +972 - (0)8-9363208

# Publications

2016
Rosianski, Y. ; Freiman, Z. E. ; Cochavi, S. M. ; Yablovitz, Z. ; Kerem, Z. ; Flaishman, M. A. Advanced analysis of developmental and ripening characteristics of pollinated common-type fig (Ficus carica L.). 2016, 198, 98 - 106. Publisher's VersionAbstract
Development and ripening processes differ in pollinated and parthenocarpic fruit. While the facultative parthenocarpic common-type fig fruit serves as a receptacle for flower development, it becomes fleshy by either pollination or through a parthenocarpic process. Here we studied the effect of pollination on common-type fig fruit development and ripening characteristics compared to the parthenocarpic fruit under otherwise identical conditions. The effects of pollination on fruit development were investigated on the tree and in storage. Pollinated fruit showed altered developmental processes. Ripened pollinated fruit were round, in contrast to the pear-like shape of the parthenocarpic fruit. The pollinated fruit also had a larger diameter and weight and improved firmness compared to the parthenocarpic fruit. At harvest, the pollinated fruit exhibited more commercially desirable physical and taste characteristics, with advanced fertile nutlets compared to the sterile undeveloped non-bearing nutlets of the parthenocarpic fruit. During storage, senescence and spoilage of the pollinated fruit were slower than in parthenocarpic fruit, as manifested by firmness, internal texture, weight, size, shriveling, and decay. Thus, pollination of the common-type fig cultivar Brown Turkey delayed senescence and extended the shelf life of its fruit. The external and internal morphological differences throughout post-pollination development make common-type fig an excellent research tool for studies of physiological and molecular aspects of pollination.
Tugendhaft, Y. ; Eppel, A. ; Kerem, Z. ; Barazani, O. ; Ben-Gal, A. ; Kadereit, J. W. ; Dag, A. Drought tolerance of three olive cultivars alternatively selected for rain fed or intensive cultivation. 2016, 199, 158 - 162. Publisher's VersionAbstract
Physiological drought response was evaluated for two olive cultivars commonly grown under rain fed conditions (‘Souri’ and ‘Picual’) and another selected for intensive, irrigated cultivation (‘Barnea’). ‘Souri’ is a traditional local Israeli cultivar, ‘Picual’ originated in Spain and ‘Barnea’ is a modern Israeli cultivar. Trees in pots were alternatively provided well irrigated conditions (100% FC, field capacity) or allowed to dry, first to 33% FC and then to 10% FC. Under conditions of greatest water availability, the ‘Barnea’ cultivar had the highest stomatal conductance and net photosynthesis, significantly higher than that found in ‘Souri’. Stomatal conductance and leaf water potential of ‘Souri’ and ‘Picual’ at 33% FC were not affected relative to the well irrigated treatment but decreased significantly at 10% FC. Photosynthetic parameters of ‘Souri' and ‘Picual’ were not affected by water stress. Stem growth was also not affected by drought in ‘Souri’ but was reduced at 10% FC in ‘Picual’. In contrast, the ‘Barnea’ showed higher sensitivity to low water availability with stomatal conductance and net photosynthesis reduced at 33% FC and sharp decreases in these and leaf water potential occurring at 10% FC. At 10% FC ‘Barnea’ trees showed stem shrinkage, a phenomenon not observed in the other cultivars. These results suggest a tradeoff between selection for suitability in intensively irrigated orchards and tolerance to drought.
Rosianskey, Y. ; Dahan, Y. ; Yadav, S. ; Freiman, Z. E. ; Milo-Cochavi, S. ; Kerem, Z. ; Eyal, Y. ; Flaishman, M. A. Chlorophyll metabolism in pollinated vs. parthenocarpic fig fruits throughout development and ripening. 2016, 244, 491 - 504. Publisher's VersionAbstract
Expression of 13 genes encoding chlorophyll biosynthesis and degradation was evaluated. Chlorophyll degradation was differentially regulated in pollinated and parthenocarpic fig fruits, leading to earlier chlorophyll degradation in parthenocarpic fruits.
Basheer, L. ; Schultz, K. ; Kerem, Z. Inhibition of cytochrome P450 3A by acetoxylated analogues of resveratrol in in vitro and in silico models. 2016, 6 31557. Publisher's VersionAbstract
Many dietary compounds, including resveratrol, are potent inhibitors of CYP3A4. Here we examined the potential to predict inhibition capacity of dietary polyphenolics using an in silico and in vitro approaches and synthetic model compounds. Mono, di and tri-acetoxy resveratrol were synthesized, a cell line of human intestine origin and microsomes from rat liver served to determine their in vitro inhibition of CYP3A4 and compared to that of resveratrol. Docking simulation served to predict the affinity of the synthetic model compounds to the enzyme. Modelling of the enzyme’s binding site revealed three types of interaction: hydrophobic, electrostatic and H-bonding. The simulation revealed that each of the examined acetylations of resveratrol led to the loss of important interactions of all types. Tri-acetoxy resveratrol was the weakest inhibitor in vitro despite being the more lipophilic and having the highest affinity for the binding site. The simulation demonstrated exclusion of all interactions between tri-acetoxy resveratrol and the heme due to distal binding, highlighting the complexity of the CYP3A4 binding site, which may allow simultaneous accommodation of two molecules. Finally, the use of computational modelling may serve as a quick predictive tool to identify potential harmful interactions between dietary compounds and prescribed drugs.
Rosianski, Y. ; Doron-Faigenboim, A. ; Freiman, Z. E. ; Lama, K. ; Milo-Cochavi, S. ; Dahan, Y. ; Kerem, Z. ; Flaishman, M. A. Tissue-Specific Transcriptome and Hormonal Regulation of Pollinated and Parthenocarpic Fig (Ficus carica L.) Fruit Suggest that Fruit Ripening Is Coordinated by the Reproductive Part of the Syconium. Frontiers in Plant Science 2016, 7 1696. Publisher's VersionAbstract
In the unconventional climacteric fig (Ficus carica) fruit, pollinated and parthenocarpic fruit of the same genotype exhibit different ripening characteristics. Integrative comparative analyses of tissue-specific transcript and of hormone levels during fruit repining from pollinated vs. parthenocarpic fig fruit were employed to unravel the similarities and differences in their regulatory processes during fruit repining. Assembling tissue-specific transcripts into 147,000 transcripts with 53,000 annotated genes provided new insights into the spatial distribution of many classes of regulatory and structural genes, including those related to color, taste and aroma, storage, protein degradation, seeds and embryos, chlorophyll, and hormones. Comparison of the pollinated and parthenocarpic tissues during fruit ripening showed differential gene expression, especially in the fruit inflorescence. The distinct physiological green phase II and ripening phase III differed significantly in their gene-transcript patterns in both pulp and inflorescence tissues. Gas chromatographic analysis of whole fruits enabled the first determination of ripening-related hormone levels from pollinated and non-pollinated figs. Ethylene and auxin both increased during fruit ripening, irrespective of pollination, whereas no production of active gibberellins or cytokinins was found in parthenocarpic or pollinated ripening fruit. Tissue-specific transcriptome revealed apparent different metabolic gene patterns for ethylene, auxin and ABA in pollinated vs. parthenocarpic fruit, mostly in the fruit inflorescence. Our results demonstrate that the production of abscisic acid (ABA), non-active ABA–GE conjugate and non-active indoleacetic acid (IAA)–Asp conjugate in pollinated fruits is much higher than in parthenocarpic fruits. We suggest that fruit ripening is coordinated by the reproductive part of the syconium and the differences in ABA production between pollinated and parthenocarpic fig fruit might be the key to their different ripening characteristics.
Barazani, O. ; Keren-Keiserman, A. ; Westberg, E. ; Hanin, N. ; Dag, A. ; Ben-Ari, G. ; Fragman-Sapir, O. ; Tugendhaft, Y. ; Kerem, Z. ; Kadereit, J. W. Genetic variation of naturally growing olive trees in Israel: from abandoned groves to feral and wild?. 2016, 16, 261. Publisher's VersionAbstract
Naturally growing populations of olive trees are found in the Mediterranean garrigue and maquis in Israel. Here, we used the Simple Sequence Repeat (SSR) genetic marker technique to investigate whether these represent wild var. sylvestris. Leaf samples were collected from a total of 205 trees at six sites of naturally growing olive populations in Israel. The genetic analysis included a multi-locus lineage (MLL) analysis, Rousset’s genetic distances, Fst values, private alleles, other diversity values and a Structure analysis. The analyses also included scions and suckers of old cultivated olive trees, for which the dominance of one clone in scions (MLL1) and a second in suckers (MLL7) had been shown earlier.
Charni, M. ; Molchadsky, A. ; Goldstein, I. ; Solomon, H. ; Tal, P. ; Goldfinger, N. ; Yang, P. ; Porat, Z. ; Lozano, G. ; Rotter, V. Novel p53 target genes secreted by the liver are involved in non-cell-autonomous regulation. 2016, 23, 509 - 520. Publisher's VersionAbstract
The tumor-suppressor p53 is a transcription factor that prevents cancer development and is involved in regulation of various physiological processes. This is mediated both by induction of cell cycle arrest and apoptosis and by controlling the expression of a plethora of target genes, including secreted proteins. It has been demonstrated that p53 may exert its effect in non-cell-autonomous manner by modulating the expression of genes that encode for secreted factors. In this study, we utilized our microarray data to identify and characterize novel p53 target genes expressed in human liver cells and associated with steroid hormones processing and transfer. We identified the steroid hormones binding factors, sex hormone-binding globulin (SHBG), corticosteroid-binding globulin (CBG) and cytochrome P450 family 21 subfamily A polypeptide 2, as novel p53 target genes. Their expression and secretion was increased following p53 activation in various hepatic cells. We observed that p53 wild-type mice exhibited higher levels of CBG compared with their p53 null counterparts. We demonstrated that the induction of the steroid hormones binding factors can be mediated by binding to specific p53 responsive elements within their promoters. In addition, utilizing conditioned medium experiments we have shown that p53-dependent induction of SHBG secretion from liver cells enhances apoptosis of breast cancer cells. Moreover, depletion of SHBG abolished the induction of breast cancer cells death. The newly identified p53 target genes suggest a novel non-cell-autonomous tumor-suppressive regulation mediated by p53 that is central for maintaining organism homeostasis.
Swinstead, E.  E. ; Miranda, T.  B. ; Paakinaho, V. ; Baek, S. ; Goldstein, I. ; Hawkins, M. ; Karpova, T.  S. ; Ball, D. ; Mazza, D. ; Lavis, L.  D. ; et al. Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions. 2016, 165, 593 - 605. Publisher's VersionAbstract
SummaryThe estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER-binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Moreover, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1.
Rozenblit-Susan, S. ; Chapnik, N. ; Genzer, Y. ; Froy, O. Serotonin suppresses food anticipatory activity and synchronizes the food-entrainable oscillator during time-restricted feeding. 2016, 297, 150 - 154. Publisher's VersionAbstract
The serotonergic and circadian systems are intertwined as serotonin modulates the response of the central brain suprachiasmatic nuclei (SCN) clock to light. Time-restricted feeding (RF) is characterized by increased food anticipatory activity (FAA) and controlled by the food-entrainable oscillator (FEO) rather than the SCN. Our objective was to test whether serotonin affects the FEO. Mice were treated with the selective serotonin reuptake inhibitor (SSRI) fluvoxamine (FLX) or the tryptophan hydroxylase inhibitor parachlorophenylalanine (PCPA) and locomotor activity under ad libitum feeding, RF and different lighting conditions was monitored. Under AL, FLX administration did not affect 24-h locomotor activity, while mice treated with PCPA exhibited increased activity. RF-FLX-treated mice showed less FAA 2h before food availability (ZT2–ZT4) compared to RF- or RF-PCPA-fed mice. Under DD, RF-PCPA-treated mice displayed increased activity, as was seen under LD conditions. Surprisingly, RF-PCPA-treated mice showed free running in the FAA component. These results emphasize the role of serotonin in SCN-mediated activity inhibition and FEO entrainment and activity.
Rozenblit-Susan, S. ; Chapnik, N. ; Froy, O. Metabolic effect of fluvoxamine in mouse peripheral tissues. 2016, 424, 12 - 22. Publisher's VersionAbstract
Genzer, Y. ; Dadon, M. ; Burg, C. ; Chapnik, N. ; Froy, O. Effect of dietary fat and the circadian clock on the expression of brain-derived neurotrophic factor (BDNF). 2016, 430, 49 - 55. Publisher's VersionAbstract
Brain-derived neurotrophic factor (BDNF) is the most abundant neurotrophin in the brain and its decreased levels are associated with the development of obesity and neurodegeneration. Our aim was to test the effect of dietary fat, its timing and the circadian clock on the expression of BDNF and associated signaling pathways in mouse brain and liver. Bdnf mRNA oscillated robustly in brain and liver, but with a 12-h shift between the tissues. Brain and liver Bdnf mRNA showed a 12-h phase shift when fed ketogenic diet (KD) compared with high-fat diet (HFD) or low-fat diet (LFD). Brain or liver Bdnf mRNA did not show the typical phase advance usually seen under time-restricted feeding (RF). Clock knockdown in HT-4 hippocampal neurons led to 86% up-regulation of Bdnf mRNA, whereas it led to 60% down-regulation in AML-12 hepatocytes. Dietary fat in mice or cultured hepatocytes and hippocampal neurons led to increased Bdnf mRNA expression. At the protein level, HFD increased the ratio of the mature BDNF protein (mBDNF) to its precursor (proBDNF). In the liver, RF under LFD or HFD reduced the mBDNF/proBDNF ratio. In the brain, the two signaling pathways related to BDNF, mTOR and AMPK, showed reduced and increased levels, respectively, under timed HFD. In the liver, the reverse was achieved. In summary, Bdnf expression is mediated by the circadian clock and dietary fat. Although RF does not affect its expression phase, in the brain, when combined with high-fat diet, it leads to a unique metabolic state in which AMPK is activated, mTOR is down-regulated and the levels of mBDNF are high.
Chapnik, N. ; Rozenblit-Susan, S. ; Genzer, Y. ; Froy, O. Differential effect of fructose on fat metabolism and clock gene expression in hepatocytes vs. myotubes. 2016, 77, 35 - 40. Publisher's VersionAbstract
In the liver, fructose bypasses the main rate-limiting step of glycolysis at the level of phosphofructokinase, allowing it to act as an unregulated substrate for de novo lipogenesis. It has been reported that consumption of large amounts of fructose increases de novo lipogenesis in the liver. However, the effect of fructose on ectopic deposition of muscle fat has been under dispute. Our aim was to study the effect of fructose on levels of genes and proteins involved in fatty acid oxidation and synthesis in hepatocytes vs. muscle cells. In addition, as fat accumulation leads to disruption of daily rhythms, we tested the effect of fructose treatment on clock gene expression. AML-12 hepatocytes and C2C12 myotubes were treated with fructose or glucose for 2 consecutive 24-h cycles and harvested every 6h. In contrast to glucose, fructose disrupted clock gene rhythms in hepatocytes, but in myotubes, it led to more robust rhythms. Fructose led to low levels of phosphorylated AMP-activated protein kinase (pAMPK) and high levels of LIPIN1 in hepatocytes compared with glucose. In contrast, fructose led to high pAMPK and low LIPIN1 and microsomal triacylglycerol transfer protein (MTTP) levels in myotubes compared with glucose. Analysis of fat content revealed that fructose led to less fat accumulation in myotubes compared to hepatocytes. In summary, fructose shifts metabolism towards fatty acid synthesis and clock disruption in hepatocytes, but not in myotubes.
Lewis, J. K. ; Bischof, J. C. ; Braslavsky, I. ; Brockbank, K. G. M. ; Fahy, G. M. ; Fuller, B. J. ; Rabin, Y. ; Tocchio, A. ; Woods, E. J. ; Wowk, B. G. ; et al. The Grand Challenges of Organ Banking: Proceedings from the first global summit on complex tissue cryopreservation. 2016, 72, 169 - 182. Publisher's VersionAbstract
The first Organ Banking Summit was convened from Feb. 27 - March 1, 2015 in Palo Alto, CA, with events at Stanford University, NASA Research Park, and Lawrence Berkeley National Labs. Experts at the summit outlined the potential public health impact of organ banking, discussed the major remaining scientific challenges that need to be overcome in order to bank organs, and identified key opportunities to accelerate progress toward this goal. Many areas of public health could be revolutionized by the banking of organs and other complex tissues, including transplantation, oncofertility, tissue engineering, trauma medicine and emergency preparedness, basic biomedical research and drug discovery – and even space travel. Key remaining scientific sub-challenges were discussed including ice nucleation and growth, cryoprotectant and osmotic toxicities, chilling injury, thermo-mechanical stress, the need for rapid and uniform rewarming, and ischemia/reperfusion injury. A variety of opportunities to overcome these challenge areas were discussed, i.e. preconditioning for enhanced stress tolerance, nanoparticle rewarming, cyroprotectant screening strategies, and the use of cryoprotectant cocktails including ice binding agents.
Bar Dolev, M. ; Braslavsky, I. ; Davies, P. L. Ice-Binding Proteins and Their Function. Annual Review of BiochemistryAnnual Review of Biochemistry 2016, 85, 515 - 542. Publisher's VersionAbstract
Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities.Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities.
Bar Dolev, M. ; Bernheim, R. ; Guo, S. ; Davies, P. L. ; Braslavsky, I. Putting life on ice: bacteria that bind to frozen water. Journal of The Royal Society InterfaceJournal of The Royal Society Interface 2016, 13, 20160210. Publisher's Version
Haleva, L. ; Celik, Y. ; Bar-Dolev, M. ; Pertaya-Braun, N. ; Kaner, A. ; Davies, P.  L. ; Braslavsky, I. Microfluidic Cold-Finger Device for the Investigation of Ice-Binding Proteins. 2016, 111, 1143 - 1150. Publisher's VersionAbstract
Ice-binding proteins (IBPs) bind to ice crystals and control their structure, enlargement, and melting, thereby helping their host organisms to avoid injuries associated with ice growth. IBPs are useful in applications where ice growth control is necessary, such as cryopreservation, food storage, and anti-icing. The study of an IBP’s mechanism of action is limited by the technological difficulties of in situ observations of molecules at the dynamic interface between ice and water. We describe herein a new, to our knowledge, apparatus designed to generate a controlled temperature gradient in a microfluidic chip, called a microfluidic cold finger (MCF). This device allows growth of a stable ice crystal that can be easily manipulated with or without IBPs in solution. Using the MCF, we show that the fluorescence signal of IBPs conjugated to green fluorescent protein is reduced upon freezing and recovers at melting. This finding strengthens the evidence for irreversible binding of IBPs to their ligand, ice. We also used the MCF to demonstrate the basal-plane affinity of several IBPs, including a recently described IBP from Rhagium inquisitor. Use of the MCF device, along with a temperature-controlled setup, provides a relatively simple and robust technique that can be widely used for further analysis of materials at the ice/water interface.
Erratum. AutophagyAutophagy 2016, 12, 443 - 443. Publisher's Version
Abeliovich, H. ; Dengjel, J. Mitophagy as a stress response in mammalian cells and in respiring S. cerevisiae. Biochemical Society Transactionsbiochemsoctrans 2016, 44, 541 - 545. Publisher's VersionAbstract
The degradation of malfunctioning or superfluous mitochondria in the lysosome/vacuole is an important housekeeping function in respiring eukaryotic cells. This clearance is thought to occur by a specific form of autophagic degradation called mitophagy, and plays a role in physiological homoeostasis as well as in the progression of late-onset diseases. Although the mechanism of bulk degradation by macroautophagy is relatively well established, the selective autophagic degradation of mitochondria has only recently begun to receive significant attention. In this mini-review, we introduce mitophagy as a form of mitochondrial quality control and proceed to provide specific examples from yeast and mammalian systems. We then discuss the relationship of mitophagy to mitochondrial stress, and provide a broad mechanistic overview of the process with an emphasis on evolutionarily conserved pathways.
Abeliovich, H. On Hill coefficients and subunit interaction energies. 2016, 73, 1399 - 1411. Publisher's VersionAbstract
The study of cooperative ligand binding to multimeric proteins aims to explain complex cooperative binding phenomena using concepts derived from ideal binding isotherms. The purpose of such efforts is the dissection of the cooperative binding isotherm into its interacting components, a result with a clear mechanistic value. Historically, cooperative binding is usually quantified using the Hill coefficient, $$\hbox {n}_\mathrm{H}$$nH, defined as the slope of the Hill plot at 50 % saturation. It was previously shown that the slope of the Hill plot throughout the titration is equal to the ratio of the binding variance in the system under study, to the binding variance of a reference non-interacting system. In the present contribution, this leads to a broader approach towards quantifying cooperativity, which empirically links cooperativity to the ensemble average of the subunit interaction energy. The resulting equations can be used to derive average differential subunit interaction energies directly from experimental binding isotherms. Combined with recent experimental advances in assessing binding distributions in multimeric proteins, these equations can also be used to calculate individual subunit interaction energies for specific n-ligated protein species.
Tal, Y. ; Anavi, S. ; Reisman, M. ; Samach, A. ; Tirosh, O. ; Troen, A. M. The neuroprotective properties of a novel variety of passion fruit. Journal of Functional Foods 2016, 23, 359 - 369. Publisher's VersionAbstract
Passion fruit is a commercially important crop. The neuroprotective activity of fruit extracts from two hybrid lines of antioxidant ester thiol-rich Passiflora edulis Sims, the commercial “Passion Dream” and novel cultivar 428 (“Dena”) line were studied. Crude extracts from line 428 displayed the strongest dose-dependent neuroprotective activity, preventing glutamate induced cell-death, mitochondrial depolarization and glutathione depletion, when added to the medium of cultured HT4 neurons (p < 0.05). Supplementing diet of mice with the 428 fruit-extract improved survival of dopaminergic neurons by 60% in mice injected with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MTPT) compared to control-fed MPTP-injected mice (p < 0.05). The neuroprotection conferred by passion fruit extracts in vivo and in vitro shows promise for further research into their bioactive potential for medical exploitation.