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Head of Institute: Prof. Oren Tirosh

Administrative manager: Ms. Yael Fruchter

Office Address:
Institute of Biochemistry, Food Science and Nutrition,
Robert H. Smith Faculty of Agriculture, Food and Environment,
The Hebrew University of Jerusalem, 
P.O.Box 12, Rehovot 7610001, ISRAEL

Tel: +972 - (0)8-9489385
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Email Address: yaelf@savion.huji.ac.il

Publications

2017
Nimri, L. ; Spivak, O. ; Tal, D. ; Schälling, D. ; Peri, I. ; Graeve, L. ; Salame, T. M. ; Yarden, O. ; Hadar, Y. ; Schwartz, B. A recombinant fungal compound induces anti-proliferative and pro-apoptotic effects on colon cancer cells. Oncotarget 2017, 8 28854-28864.Abstract
Finding intracellular pathways and molecules that can prevent the proliferation of colon cancer cells can provide significant bases for developing treatments for this disease. Ostreolysin (Oly) is a protein found in the mushroom Pleurotus ostreatus, and we have produced a recombinant version of this protein (rOly).We measured the viability of several colon cancer cells treated with rOly. Xenografts and syngeneic colon cancer cells were injected into in vivo mouse models, which were then treated with this recombinant protein.rOly treatment induced a significant reduction in viability of human and mouse colon cancer cells. In contrast, there was no reduction in the viability of normal epithelial cells from the small intestine. In the search for cellular targets of rOly, we showed that it enhances the anti-proliferative activity of drugs targeting cellular tubulin. This was accompanied by a reduction in the weight and volume of tumours in mice injected with rOly as compared to their respective control mice in two in vivo models.Our results advance the functional understanding of rOly as a potential anti-cancer treatment associated with pro-apoptotic activities preferentially targeting colon cancer cells.
Bein, A. ; Zilbershtein, A. ; Golosovsky, M. ; Davidov, D. ; Schwartz, B. LPS Induces Hyper-Permeability of Intestinal Epithelial Cells. Journal of Cellular Physiology 2017, 232, 381-390. Publisher's VersionAbstract
Necrotizing Enterocolitis (NEC) is a severe inflammatory disorder leading to high morbidity and mortality rates. A growing body of evidence demonstrate the key role of the Toll like receptor 4 (TLR4) in NEC. This membranal receptor recognizes lipopolysaccharides (LPS) from the bacterial wall and triggers an inflammatory response. The aim of the present study was to elucidate the effect of LPS on paracellular permeability known to be severely affected in NEC. IEC-18 cells were treated with LPS and the effects on morphology, paracellular permeability and their associated gene and protein expressions were measured. Our results show that LPS down regulated the expression of occludin and ZO-1 mRNAs while up regulating Cdkn1a. In addition LPS caused a significant increase in paracellular permeability and epithelial barrier damage. Finally ZO-1 protein was found to be spatially disarrayed in the intercellular junctions in response to LPS. We conclude that LPS adversely affected the functionality of the intestinal epithelial barrier suggesting a new mechanism by which bacterial infection may contribute to the development of NEC. J. Cell. Physiol. 232: 381–390, 2017. © 2016 Wiley Periodicals, Inc.
Avni, S. ; Ezove, N. ; Hanani, H. ; Yadid, I. ; Karpovsky, M. ; Hayby, H. ; Gover, O. ; Hadar, Y. ; Schwartz, B. ; Danay, O. Olive Mill Waste Enhances α-Glucan Content in the Edible Mushroom Pleurotus eryngii. Int J Mol Sci 2017, 18.Abstract
Mushroom polysaccharides are edible polymers that have numerous reported biological functions; the most common effects are attributed to β-glucans. In recent years, it became apparent that the less abundant α-glucans also possess potent effects in various health conditions. Here we explore several species for their total, β and α-glucan content. was found to have the highest total glucan concentrations and the highest α-glucans proportion. We also found that the stalks (stipe) of the fruit body contained higher glucan content then the caps (pileus). Since mushrooms respond markedly to changes in environmental and growth conditions, we developed cultivation methods aiming to increase the levels of α and β-glucans. Using olive mill solid waste (OMSW) from three-phase olive mills in the cultivation substrate. We were able to enrich the levels mainly of α-glucans. Maximal total glucan concentrations were enhanced up to twice when the growth substrate contained 80% of OMSW compared to no OMSW. Taking together this study demonstrate that can serve as a potential rich source of glucans for nutritional and medicinal applications and that glucan content in mushroom fruiting bodies can be further enriched by applying OMSW into the cultivation substrate.
Oren, T. ; Nimri, L. ; Yehuda-Shnaidman, E. ; Staikin, K. ; Hadar, Y. ; Friedler, A. ; Amartely, H. ; Slutzki, M. ; Di Pizio, A. ; Niv, M. Y. ; et al. Recombinant ostreolysin induces brown fat-like phenotype in HIB-1B cells. Molecular Nutrition & Food ResearchMolecular Nutrition & Food ResearchMol. Nutr. Food Res. 2017, 61, 1700057. Publisher's VersionAbstract
Scope Brown adipose tissue (BAT) is the main regulator of thermogenesis by increasing energy expenditure through the uncoupling of oxidative metabolism from ATP synthesis. There is a growing body of evidence for BAT being the key responsible organ in combating obesity and its related disorders. Herein we propose the fungal protein ostreolysin (Oly), which has been previously shown to bind to cholesterol-enriched raft-like membrane domains (lipid rafts) of mammalian cells, as a suitable candidate for interaction with brown preadipocytes. The aim of the present study was therefore to characterize the mechanism by which a recombinant version of ostreolysin (rOly) induces brown adipocyte differentiation. Methods and results Primary isolated brown preadipocytes or HIB-1B brown preadipocyte cells were treated with rOly and the effects on morphology, lipid accumulation, respiration rate, and associated gene and protein expression were measured. rOly upregulated mRNA and protein levels of factors related to brown adipocyte differentiation, induced lipid droplet formation, and increased cellular respiration rate due to expression of uncoupling protein 1. rOly also upregulated ?-tubulin expression, and therefore microtubules might be involved in its mechanism of action. Conclusion rOly promotes brown adipocyte differentiation, suggesting a new mechanism for rOly's contribution to the battle against obesity.
Argov-Argaman, N. ; Mandel, D. ; Lubetzky, R. ; Kedem, M. H. ; Cohen, B. - C. ; Berkovitz, Z. ; Reifen, R. Human milk fatty acids composition is affected by maternal age. J Matern Fetal Neonatal Med 2017, 30, 34-37.Abstract
Human colostrums and transition milk were collected from women under the age of 37 years and women aged 37 years and older. Transition milk of the younger group had lower fat content and 10-fold higher concentrations of omega 6 FA, eicosadecanoic, and arachdonic acids. Gestational age affected the colostrum concentration of total fat and omega 3 and omega 6 FA composition only in the older group. We concluded that age may be a factor in the FA composition of human milk. This should be taken into account when planning diets for pregnant women of different ages.
Bar-El Dadon, S. ; Reifen, R. Vitamin A and the epigenome. Critical Reviews in Food Science and Nutrition 2017, 57, 2404-2411. Publisher's VersionAbstract
ABSTRACTThe epigenetic phenomena refer to heritable changes in gene expression other than those in the DNA sequence, such as DNA methylation and histone modifications. Major research progress in the last few years has provided further proof that environmental factors, including diet and nutrition, can influence physiologic and pathologic processes through epigenetic alterations, which in turn influence gene expression. This influence is termed nutritional epigenetics, and one prominent example is the regulation of gene transcription by vitamin A through interaction to its nuclear receptor.Vitamin A is critical throughout life. Together with its derivatives, it regulates diverse processes including reproduction, embryogenesis, vision, growth, cellular differentiation and proliferation, maintenance of epithelial cellular integrity and immune function.Here we review the epigenetic role of vitamin A in cancer, stem cells differentiation, proliferation, and immunity. The data presented here show that retinoic acid is a potent agent capable of inducing alterations in epigenetic modifications that produce various effects on the phenotype. Medical benefits of vitamin A as an epigenetic modulator, especially with respect to its chronic use as nutritional supplement, should rely on our further understanding of its epigenetic effects during health and disease, as well as through different generations.
Wiseman, E. M. ; Bar-El Dadon, S. ; Reifen, R. The vicious cycle of vitamin a deficiency: A review. Critical Reviews in Food Science and Nutrition 2017, 57, 3703-3714. Publisher's VersionAbstract
ABSTRACTVitamin A deficiency (VAD) is a serious and widespread public health problem and the leading cause of preventable blindness in young children. It is also associated with increased rates of death from severe infections, especially in developing countries. Over the past 35 years, researchers have examined the numerous activities of vitamin A in different tissues of the human body. VAD can lead to a series of ocular symptoms, anemia, and weak resistance to infection, which can increase the severity of infectious diseases and the risk of death. Cell development, vision, growth, and normal metabolism are among the vital processes that are insufficiently supported in the presence of VAD. VAD leads to impaired tissue function especially during the developmental periods of infancy, childhood, pregnancy, and lactation. We describe a multidirectional model of VAD that demonstrates how VAD can have progressive, negative effects on vital processes of the human body throughout the life cycle. This model starts with impaired intake and its link to decreased absorption and digestion and includes outcomes such as malnutrition, inflammation, and improper growth processes, including possible mechanisms. Together, these clinical and biochemical manifestations contribute to the vicious cycle of VAD.
Slutzki, M. ; Ben-Shimon, A. ; Niv, M. Y. AnchorDock for Blind Flexible Docking of Peptides to Proteins. In Modeling Peptide-Protein Interactions: Methods and Protocols; Schueler-Furman, O. ; London, N., Ed. Modeling Peptide-Protein Interactions: Methods and Protocols; Springer New York: New York, NY, 2017; pp. 95–108. Publisher's VersionAbstract
Due to increasing interest in peptides as signaling modulators and drug candidates, several methods for peptide docking to their target proteins are under active development. The ``blind'' docking problem, where the peptide-binding site on the protein surface is unknown, presents one of the current challenges in the field. AnchorDock protocol was developed by Ben-Shimon and Niv to address this challenge.
Cheled-Shoval, S. ; Behrens, M. ; Korb, A. ; Di Pizio, A. ; Meyerhof, W. ; Uni, Z. ; Niv, M. Y. From Cell to Beak: In-Vitro and In-Vivo Characterization of Chicken Bitter Taste Thresholds. Molecules 2017, 22.Abstract
Bitter taste elicits an aversive reaction, and is believed to protect against consuming poisons. Bitter molecules are detected by the Tas2r family of G-protein-coupled receptors, with a species-dependent number of subtypes. Chickens demonstrate bitter taste sensitivity despite having only three bitter taste receptors-ggTas2r1, ggTas2r2 and ggTas2r7. This minimalistic bitter taste system in chickens was used to determine relationships between in-vitro (measured in heterologous systems) and in-vivo (behavioral) detection thresholds. ggTas2r-selective ligands, nicotine (ggTas2r1), caffeine (ggTas2r2), erythromycin and (+)-catechin (ggTas2r7), and the Tas2r-promiscuous ligand quinine (all three ggTas2rs) were studied. Ligands of the same receptor had different in-vivo:in-vitro ratios, and the ggTas2r-promiscuous ligand did not exhibit lower in-vivo:in-vitro ratios than ggTas2r-selective ligands. In-vivo thresholds were similar or up to two orders of magnitude higher than the in-vitro ones.
Cheled-Shoval, S. L. ; Reicher, N. ; Niv, M. Y. ; Uni, Z. Detecting thresholds for bitter, umami, and sweet tastants in broiler chicken using a 2-choice test method. Poultry Science 2017, 96, 2206 - 2218. Publisher's VersionAbstract
The sense of taste has a key role in nutrient sensing and food intake in animals. A standardized and simple method for determination of tastant-detection thresholds is required for chemosensory research in poultry. We established a 24-h, 2-alternative, forced-choice solution-consumption method and applied it to measure detection thresholds for 3 G-protein-coupled receptor-mediated taste modalities—bitter, sweet, and umami—in chicken. Four parameters were used to determine a significant response: 1) tastant-solution consumption; 2) water (tasteless) consumption; 3) total consumption (tastant and water together); 4) ratio of tastant consumption to total consumption. Our results showed that assignment of the taste solutions and a water control to 2 bottles on random sides of the pen can be reliably used for broiler chicks, even though 47% of the chicks groups demonstrated a consistently preferred side. The detection thresholds for quinine (bitter), L-monosodium glutamate (MSG) (umami), and sucrose (sweet) were determined to be 0.3 mM, 300 mM, and 1 M, respectively. The threshold results for quinine were similar to those for humans and rodents, but the chicks were found to be less sensitive to sucrose and MSG. The described method is useful for studying detection thresholds for tastants that have the potential to affect feed and water consumption in chickens.
Oren, T. ; Nimri, L. ; Yehuda-Shnaidman, E. ; Staikin, K. ; Hadar, Y. ; Friedler, A. ; Amartely, H. ; Slutzki, M. ; Di Pizio, A. ; Niv, M. Y. ; et al. Recombinant ostreolysin induces brown fat-like phenotype in HIB-1B cells. Molecular Nutrition & Food Research 2017, 61, 1700057. Publisher's VersionAbstract
Scope Brown adipose tissue (BAT) is the main regulator of thermogenesis by increasing energy expenditure through the uncoupling of oxidative metabolism from ATP synthesis. There is a growing body of evidence for BAT being the key responsible organ in combating obesity and its related disorders. Herein we propose the fungal protein ostreolysin (Oly), which has been previously shown to bind to cholesterol-enriched raft-like membrane domains (lipid rafts) of mammalian cells, as a suitable candidate for interaction with brown preadipocytes. The aim of the present study was therefore to characterize the mechanism by which a recombinant version of ostreolysin (rOly) induces brown adipocyte differentiation. Methods and results Primary isolated brown preadipocytes or HIB-1B brown preadipocyte cells were treated with rOly and the effects on morphology, lipid accumulation, respiration rate, and associated gene and protein expression were measured. rOly upregulated mRNA and protein levels of factors related to brown adipocyte differentiation, induced lipid droplet formation, and increased cellular respiration rate due to expression of uncoupling protein 1. rOly also upregulated β-tubulin expression, and therefore microtubules might be involved in its mechanism of action. Conclusion rOly promotes brown adipocyte differentiation, suggesting a new mechanism for rOly's contribution to the battle against obesity.
Dubovski, N. ; Ert, E. ; Niv, M. Y. Bitter mouth-rinse affects emotions. Food Quality and Preference 2017, 60, 154 - 164. Publisher's VersionAbstract
The sense of taste enables evaluation of food and is an important regulator of food consumption. In general, sweet is an attractive taste modality that leads to ingestion of nutritive food, while sour and bitter are aversive taste modalities that lead to avoidance of spoiled and toxic food. Recent studies suggest inter-connections between taste, emotion and cognition. Here we test the potential effects of two prototypical taste modalities, bitter and sweet, on emotions and on generalized avoidance behaviors, such as risk aversion and mistrust. Three experiments included over 250 participants who tasted, without swallowing, one of the following stimuli: water control, quinine solution, sucrose solution, quinine-sucrose mixture solution, or propylthiouracil (PROP) solution. The participants had to identify the taste, rank its intensity, perform seemingly unrelated behavioral tasks, and fill a PANAS mood questionnaire. Our results indicate that oral exposure to bitter compounds negatively correlates with mood scores; that the effect depends on perceiving the solution as bitter; that bitter mouth rinse can lower PANAS mood score and that there is a potential asymmetry in the effects of bitter and sweet taste modalities on mood.
Dagan-Wiener, A. ; Nissim, I. ; Ben Abu, N. ; Borgonovo, G. ; Bassoli, A. ; Niv, M. Y. Bitter or not? BitterPredict, a tool for predicting taste from chemical structure. 2017, 7 12074. Publisher's VersionAbstract
Bitter taste is an innately aversive taste modality that is considered to protect animals from consuming toxic compounds. Yet, bitterness is not always noxious and some bitter compounds have beneficial effects on health. Hundreds of bitter compounds were reported (and are accessible via the BitterDB http://bitterdb.agri.huji.ac.il/dbbitter.php), but numerous additional bitter molecules are still unknown. The dramatic chemical diversity of bitterants makes bitterness prediction a difficult task. Here we present a machine learning classifier, BitterPredict, which predicts whether a compound is bitter or not, based on its chemical structure. BitterDB was used as the positive set, and non-bitter molecules were gathered from literature to create the negative set. Adaptive Boosting (AdaBoost), based on decision trees machine-learning algorithm was applied to molecules that were represented using physicochemical and ADME/Tox descriptors. BitterPredict correctly classifies over 80% of the compounds in the hold-out test set, and 70–90% of the compounds in three independent external sets and in sensory test validation, providing a quick and reliable tool for classifying large sets of compounds into bitter and non-bitter groups. BitterPredict suggests that about 40% of random molecules, and a large portion (66%) of clinical and experimental drugs, and of natural products (77%) are bitter.
Di Pizio, A. ; Kruetzfeldt, L. - M. ; Cheled-Shoval, S. ; Meyerhof, W. ; Behrens, M. ; Niv, M. Y. Ligand binding modes from low resolution GPCR models and mutagenesis: chicken bitter taste receptor as a test-case. 2017, 7 8223. Publisher's VersionAbstract
Bitter taste is one of the basic taste modalities, warning against consuming potential poisons. Bitter compounds activate members of the bitter taste receptor (Tas2r) subfamily of G protein-coupled receptors (GPCRs). The number of functional Tas2rs is species-dependent. Chickens represent an intriguing minimalistic model, because they detect the bitter taste of structurally different molecules with merely three bitter taste receptor subtypes. We investigated the binding modes of several known agonists of a representative chicken bitter taste receptor, ggTas2r1. Because of low sequence similarity between ggTas2r1 and crystallized GPCRs (~10% identity, ~30% similarity at most), the combination of computational approaches with site-directed mutagenesis was used to characterize the agonist-bound conformation of ggTas2r1 binding site between TMs 3, 5, 6 and 7. We found that the ligand interactions with N93 in TM3 and/or N247 in TM5, combined with hydrophobic contacts, are typically involved in agonist recognition. Next, the ggTas2r1 structural model was successfully used to identify three quinine analogues (epiquinidine, ethylhydrocupreine, quinidine) as new ggTas2r1 agonists. The integrated approach validated here may be applicable to additional cases where the sequence identity of the GPCR of interest and the existing experimental structures is low.
Nissim, I. ; Dagan-Wiener, A. ; Niv, M. Y. The taste of toxicity: A quantitative analysis of bitter and toxic molecules. IUBMB Life 2017, 69, 938-946. Publisher's VersionAbstract
Abstract The role of bitter taste—one of the few basic taste modalities—is commonly assumed to signal toxicity and alert animals against consuming harmful compounds. However, it is known that some toxic compounds are not bitter and that many bitter compounds have negligible toxicity while having important health benefits. Here we apply a quantitative analysis of the chemical space to shed light on the bitterness-toxicity relationship. Using the BitterDB dataset of bitter molecules, The BitterPredict prediction tool, and datasets of toxic compounds, we quantify the identity and similarity between bitter and toxic compounds. About 60% of the bitter compounds have documented toxicity and only 56% of the toxic compounds are known or predicted to be bitter. The LD50 value distributions suggest that most of the bitter compounds are not very toxic, but there is a somewhat higher chance of toxicity for known bitter compounds compared to known nonbitter ones. Flavonoids and alpha acids are more common in the bitter dataset compared with the toxic dataset. In contrast, alkaloids are more common in the toxic datasets compared to the bitter dataset. Interestingly, no trend linking LD50 values with the number of activated bitter taste receptors (TAS2Rs) subtypes is apparent in the currently available data. This is in accord with the newly discovered expression of TAS2Rs in several extra-oral tissues, in which they might be activated by yet unknown endogenous ligands and play non-gustatory physiological roles. These results suggest that bitter taste is not a very reliable marker for toxicity, and is likely to have other physiological roles. © 2017 IUBMB Life, 69(12):938–946, 2017
Wu, C. ; Du, Y. - W. ; Huang, L. ; Ben-Shoshan Galeczki, Y. ; Dagan-Wiener, A. ; Naim, M. ; Niv, M. Y. ; Wang, P. Biomimetic Sensors for the Senses: Towards Better Understanding of Taste and Odor Sensation. Sensors (Basel) 2017, 17.Abstract
Taste and smell are very important chemical senses that provide indispensable information on food quality, potential mates and potential danger. In recent decades, much progress has been achieved regarding the underlying molecular and cellular mechanisms of taste and odor senses. Recently, biosensors have been developed for detecting odorants and tastants as well as for studying ligand-receptor interactions. This review summarizes the currently available biosensing approaches, which can be classified into two main categories: in vitro and in vivo approaches. The former is based on utilizing biological components such as taste and olfactory tissues, cells and receptors, as sensitive elements. The latter is dependent on signals recorded from animals' signaling pathways using implanted microelectrodes into living animals. Advantages and disadvantages of these two approaches, as well as differences in terms of sensing principles and applications are highlighted. The main current challenges, future trends and prospects of research in biomimetic taste and odor sensors are discussed.
Kalish-Achrai, N. ; Monsonego-Ornan, E. ; Shahar, R. Structure, composition, mechanics and growth of spines of the dorsal fin of blue tilapia Oreochromis aureus and common carp Cyprinus carpio. Journal of Fish Biology 2017, 90, 2073-2096. Publisher's VersionAbstract
The structural, compositional and mechanical properties of the spines of the dorsal fin in mature anosteocytic blue tilapia Oreochromis aureus and osteocytic common carp Cyprinus carpio are described, as well as their temporal growth pattern and regenerative capacities. The three-dimensional architecture of both spines, from macro to sub-micron levels, is shown to be axially oriented and therefore highly anisotropic and the spines of both species are able to regenerate after partial amputation.
Barazani, O. ; Waitz, Y. ; Tugendhaft, Y. ; Dorman, M. ; Dag, A. ; Hamidat, M. ; Hijawi, T. ; Kerem, Z. ; Westberg, E. ; Kadereit, J. W. Testing the potential significance of different scion/rootstock genotype combinations on the ecology of old cultivated olive trees in the southeast Mediterranean area. 2017, 17, 3. Publisher's VersionAbstract
A previous multi-locus lineage (MLL) analysis of SSR-microsatellite data of old olive trees in the southeast Mediterranean area had shown the predominance of the Souri cultivar (MLL1) among grafted trees. The MLL analysis had also identified an MLL (MLL7) that was more common among rootstocks than other MLLs. We here present a comparison of the MLL combinations MLL1 (scion)/MLL7 (rootstock) and MLL1/MLL1 in order to investigate the possible influence of rootstock on scion phenotype.
Elyasiyan, U. ; Nudel, A. ; Skalka, N. ; Rozenberg, K. ; Drori, E. ; Oppenheimer, R. ; Kerem, Z. ; Rosenzweig, T. Anti-diabetic activity of aerial parts of Sarcopoterium spinosum. 2017, 17, 356. Publisher's VersionAbstract
Sarcopoterium spinosum (S. spinosum) is used by Bedouin medicinal practitioners for the treatment of diabetes. While the anti-diabetic activity of S. spinosum root extract was validated in previous studies, the activity of aerial parts of the same plants has not been elucidated yet. The aim of this study was to clarify the glucose lowering properties of the aerial parts of the shrub.
Ehrlich, Y. ; Regev, L. ; Kerem, Z. ; Boaretto, E. Radiocarbon Dating of an Olive Tree Cross-Section: New Insights on Growth Patterns and Implications for Age Estimation of Olive Trees. Frontiers in Plant Science 2017, 8 1918. Publisher's VersionAbstract
The age of living massive olive trees is often assumed to be between hundreds and even thousands of years. These estimations are usually based on the girth of the trunk and an extrapolation based on a theoretical annual growth rate. It is difficult to objectively verify these claims, as a monumental tree may not be cut down for analysis of its cross-section. In addition, the inner and oldest part of the trunk in olive trees usually rots, precluding the possibility of carting out radiocarbon analysis of material from the first years of life of the tree. In this work we present a cross-section of an olive tree, previously estimated to be hundreds of years old, which was cut down post-mortem in 2013. The cross-section was radiocarbon dated at numerous points following the natural growth pattern, which was made possible to observe by viewing the entire cross-section. Annual growth rate values were calculated and compared between different radii. The cross-section also revealed a nearly independent segment of growth, which would clearly offset any estimations based solely on girth calculations. Multiple piths were identified, indicating the beginning of branching within the trunk. Different radii were found to have comparable growth rates, resulting in similar estimates dating the piths to the 19th century. The estimated age of the piths represent a terminus ante quem for the age of the tree, as these are piths of separate branches. However, the tree is likely not many years older than the dated piths, and certainly not centuries older. The oldest radiocarbon-datable material in this cross-section was less than 200 years old, which is in agreement with most other radiocarbon dates of internal wood from living olive trees, rarely older than 300 years.