- Email: [email protected]
Combined ribavarin treatment and cryotherapy for efficient potato vitus M and potato virus S eradication in potato in vitro plantlets
Abstracts / Cryobiology 71 (2015) 537e573
freezing and re-warming process. For thawing, the heat exchangers of the CryoRACK are directly flowed through by pressurized hot steam triggered by a N2 gas flow. The revitalization system can be used after thawing even in aseptic ambience up to 48 hours without opening the dishes. The system was successfully tested for the cryopreservation of artificial bone and mucosa grafts. It provides the whole process chain of axenic cryopreservation, storage, transport, thawing and revitalization. Funding: This work was supported by EFRE 2000-2006 and the Free State of Saxony (grant numbers 7953/1272; 10957/1700 and 7954/1272; 109871700) and by the Federal Ministry of Economics and Technology of Germany (grant number MF 090193). The work was also granted by the Association of Industrial Research Associations, “Otto von Guericke”, e.V. (AiF) (grant number ZIM-KF 2388001FO9). Conflict of Interests: N/A P43. EFFECT OF AGE ON SEMEN FREEZABILITY OF AKSARAY MALAKLI SHEPHERD DOG (TURKISH MASTIFF) K. Tekin 1, M.E. Inanc 1, K.T. Olgac 1, B. Yilmaz 1, Y. Akkurt 1, C. Temizkan 1, U. Nkaya 2, P.B. Tuncer 3, U. Tasdemir 3, S. Buyukleblebici 3, B. Kul 1, E. Durmaz 3, O. Uysal 1, O. Ertugrul 1. 1 Ankara University, Ankara, Turkey; 2 Gazi University, Ankara, Turkey; 3 Aksaray University, Aksaray, Turkey E-mail address: [email protected]
The objective of this study was to determine the effect of age on semen quality parameters in Malaklı (Turkish Mastiff). Therefore, twenty dogs were used in this research and separated into two age groups, age group 1 (3 years of age) and age group 2 (4-6 years of age). Semen samples were collected using digital pressure and massage. Semen volume, pH and motility were determined immediately after collection. Then, fresh semen was extended with a tris based extender, equilibrated (+5 C/2h), loaded into a 0.25 french straw, frozen in liquid nitrogen vapour (-120 C/15 minutes) and stored in liquid nitrogen (-196 C). Frozen straws were thawed in a water bath (37 C/30 seconds) and percentages of progressive motility, total motility, and sperm kinetic parameters (VCL, VSL, VAP, LIN, STR, WOB, ALH, BCF and hyperactivity) were determined with a computer assisted sperm analyzer (CASA, SCA Microptics, Barcelona). Abnormal spermatozoa rates (head, acrosome, neck, tail and cytoplasmic droplets) were assessed with sperm blue (Microptic, Barcelona). The results showed that the highest progressive, and total motility, were recorded as 4,8 ± 2,00; 63,8 ± 7,95 in age group 1 respectively, after freeze-thawed. Among age groups, there was a statically significance differences between progressive and total motility (p<0,05). The highest ALH, BCF and hyperactivity were 4,9±0,56, 6,0±1,10 and 15,9±6,05 in age group 1 respectively (p<0,05). There was considerable variation among age groups in ALH, BCF and hyperactivity, however there were no significant differences in other sperm kinetic motions. Significant differences were found according to abnormal spermatozoa rate (p<0,05). There was a correlation between ALH, BCF, hyperactivity and total abnormal spermatozoa (p<0,05). It appears that total and progressive motility, yet some kinetic parameters such as hyperactivity, ALH and BCF, starts to decline with age when the frequency of sperm abnormalities has increased, as evidence by specific age related sperm defects. We concluded that freezing and thawing processes increased abnormal sperm that are due to cell alteration for old males, and consequently, decrease the freezability of Malakli dog semen. Funding: This study was supported by the Turkish Scientific and Technical _ Research Council TUBITAK (project number: 114O636). Conflict of Interests: N/A P44. INFLUENCE OF DIFFERENT FREEZING PROTOCOLS ON HEMOGLOBIN ENCAPSULATED IN ALGINATE MICROSPHERES S. Rozanova, O. Nardid. Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkov, Ukraine Nowadays hemoglobin-encapsulated microspheres are considered as an artificial erythrocyte substitute in transfusion medicine. However their
569
application is restricted by the protein hem oxidation during storage. Cryopreservation may be a proper way of encapsulated hemoglobin storage. The research aim was to investigate the effect of different freezing protocols on encapsulated hemoglobin properties. Hemoglobin-loaded alginate microspheres were obtained by ionotropic gelation. Microspheres were frozen down to -20 C or -196 C at 1-2 C/min and 300 C/min cooling rates respectively. Thawing was carried out at 22 C. Hemoglobin functional activity was analyzed by ability to release oxygen in anaerobic conditions using sodium dithionite. The percentage of different hemoglobin forms in the microspheres was detected by alterations in protein absorption spectra. ABTS+ radical decolorization assay was used to investigate protein stability. Freeze-thawing of hemoglobin loaded microspheres has been shown to lead to partial hemoglobin loss and to the lowering of protein ABTS+ scavenging ability, the most expressed in the case of freezing down to -20 C. It has been demonstrated that hemoglobin loaded into alginate microspheres is able to release oxygen in anaerobic condition. Freezing does not affect this protein ability. The results obtained have revealed freezing of encapsulated hemoglobin down to -196 C as more effective. Funding: N/A Conflict of Interests: N/A P45. CRYOPROTECTANT TOXICITY STUDIES ON CRASSOSTREA ANGULATA SPERM lix 1, Sonia Martinez-Pa ramo 1, Domitília Marta F. Riesco 1, Francisca Fe 3, Marc Suquet 4, Elsa Matias 2, Sandra Joaquim 2, Catherine Labbe Cabrita 1. 1 CCMAR, University of Algarve, Faro, Portugal; 2 IPMA, IP, Avda 5 ~o, Portugal; 3 INRA, Fish Physiology and Genomics, Rennes, Outubro, Olha France; 4 IFREMER, Departement PFOM/Laboratoire ARN Station Experimentale d’Argenton, Argenton, France E-mail address: [email protected]
The preservation of genetic material of both improved stocks and of original population is very important for the oyster aquaculture industry to avoid potential impacts from epidemic diseases and natural disasters. Nowadays, purely wild populations of Crassostrea angulata are rare to find due to multiple factors that have affected this industry. Cryopreservation technology could promote alternative techniques to contribute to the resource management efficiency of the Portuguese oyster and associated economic activity. This report describes the first attempt on cryoprotectant sperm toxicity in the Portuguese oyster and discusses a set of interacting variables at different steps from sperm collection to thawing (initial sperm concentration, sperm dilution in cryoprotectants, caffeine addition (10 mM) and type and osmolarity of extender). Sperm motility, sperm viability (using fluorescent dyes), and enzymatic activities were used to evaluate the exposure of sperm at different cryoprotectants (Me2SO, PEG, EG, Methanol) using different concentrations (5%, 10% and 20% v/v). Toxicity assays revealed that PEG and Me2SO are significantly less toxic than other cryoprotectants at the same concentrations. Other parameters such as caffeine supplementation, low dilution ratios (1:2) and extender (isosmotic) significantly improved sperm quality after cryoprotectant exposure. This work summarizes the factors influencing sperm management prior to cryopreservation. Funding: This work was supported by CRIOBIV 31-03-05-FEP-59 PROMAR program and COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE). Conflict of Interests: N/A P46. COMBINED RIBAVARIN TREATMENT AND CRYOTHERAPY FOR EFFICIENT POTATO VITUS M AND POTATO VIRUS S ERADICATION IN POTATO IN VITRO PLANTLETS S.V. Kushnarenko 1, N.V. Romadanova 1, M.O. Bekebayeva 1, A.M. Alexandrova 2, B.M. Reed 3. 1 Institute of Plant Biology and Biotechnology, Almaty, Kazakhstan; 2 M.A. Aithozhin Institute of Molecular Biology and Biochemistry, Almaty, Kazakhstan; 3 USDA-ARS National Clonal Germplasm Repository, Oregon, USA E-mail address: [email protected]
570
Abstracts / Cryobiology 71 (2015) 537e573
An in vitro collection of 30 potato cultivars and hybrids were evaluated for five viruses: Potato leafroll virus (PLRV), Potato virus M (PVM), Potato virus S (PVS), Potato virus X (PVX) and Potato virus Y (PVY) by two methods: ELISA and RT-PCR. PLRV was not detected in any accessions. The majority of accessions were singly infected by PVM (26.7%) or mix-infected by PVM and PVS (43.3%) or PVM and PVY (13.3%). One accession had PVM, PVS and PVX. No viruses were detected in four cultivars. For cryotherapy of shoot tips, the PVS2-vitrification protocol was used. The effect of culture with 100 mg/L ribavirin on viruses eradication was also investigated both alone and combined with cryotherapy. Cryotherapy eliminated all PVY infections but only 33.3% were free of PVM. No totally virus-free plants were found in mix-infected accessions. Treatment with ribavirin alone was not effective when used for one or two subcultures, but three subcultures on ribavirin or followed by cryotherapy was effective for both PVM and PVS. RT-PCR did not detect PVS and PVM after two subcultures on ribavirin or following cryotherapy, but ELISA tests indicated a low PVM titer in some accessions. Future studies will include cultivars infected with PVX. Conflict of interests: N/A Source of Funding: This research was supported by the grant No. 0047/GF of Ministry of Education and Science, Republic of Kazakhstan P47. MANUFACTURING A BIO-ARTIFICIAL LIVER (BAL) SUPPORT DEVICE Nick Medcalf 1, Andreea Iftimia-Mander 1, Andy Picken 1, Qian Liu 1, Alexandra Stolzing 1, Rob Thomas 1, John Morris 2, Stephen Lamb 2. 1 Centre for Biological Engineering, Loughborough University, UK; 2 Asymptote Ltd., Cambridge, UK E-mail address: [email protected]
An efficient, cost effective and ‘off-the-shelf’ Bio-Artificial liver (BAL) would be a game-changer in the clinical treatment of liver failure and liver disease. Since the liver is one of the few organs that can repair and regenerate, therapies enabling regenerative medicine that are creating living functional tissues to repair or replace organ function lost due to damage, are expected to play a role in several areas of liver disease. A bioartificial liver machine can temporarily replace the functions of the liver, allowing the damaged liver to regenerate whilst protecting the patient’s other organs from the life-threatening damage that ensues during liver failure. Currently, almost 80% of patients with severe acute liver failure will die within a few days unless they receive a liver transplant. Moreover, a
third of patients on the waiting list for a transplant, die before they receive an organ. This project will develop and deliver an ‘off the shelf’, disposable cassette, consumables and associated cryogenic equipment to allow the clinical delivery of a bio-artificial liver (BAL). The technology also has potential in other applications, including decellularization of a range of organs and tissues, and for the delivery of primary human liver cells spheroids and pancreatic cells. The project is an example of the unit operations associated with presentation and delivery of advanced therapies as part of the systems-based approach at Loughborough University. Conflict of Interests: N/A Funding: Innovate UK (101 620) P48. MICROSCOPIC INVESTIGATION OF ANTIFREEZE PROTEINS ACTIVITY AT CRYOGENIC TEMPERATURES Liat Bahari, Victor Yashunsky, Ido Braslavsky. The Hebrew University of Jerusalem, Israel E-mail address: [email protected]
Antifreeze proteins, are one of the natural mechanisms that are found in cold adapted organisms to cope with subzero temperatures. In the laboratory, they have been demonstrated to improve preservation of cells either under chilling conditions or ultra-low temperatures. AFPs inhibit ice growth in slightly suppercooled conditions, and inhibit recrystallization of ice in frozen tissues. These activities were tested in temperatures down to a few degrees celsius lower than melting point. It is not known yet if and how AFPs act at ultra-low temperatures. We investigate the activity of AFPs in vitrification, an “ice-free” method of cryopreservation in which ice crystal growth may occur during the rewarming step, at low temperatures slightly above the glass transition temperature (devitrification). An examination of this process under a microscope equipped with a controlled cold stage showed that hyperactive AFP from the Tenebrio Molitor mealworm (TmAFP), influenced ice growth even at temperatures of -110 C, during the warming step. For example, using SnowMax as nucleators in this process, TmAFP in Me2SO solutions reduced the number of ice crystals observed. These results indicate that AFPs can be active at cryogenic temperature ranges as growth inhibitors of ice crystals and this function may improve cryopreservation procedures. Funding: This research was funded by the European Research Council Conflict of Interests: N/A
freezing and re-warming process. For thawing, the heat exchangers of the CryoRACK are directly flowed through by pressurized hot steam triggered by a N2 gas flow. The revitalization system can be used after thawing even in aseptic ambience up to 48 hours without opening the dishes. The system was successfully tested for the cryopreservation of artificial bone and mucosa grafts. It provides the whole process chain of axenic cryopreservation, storage, transport, thawing and revitalization. Funding: This work was supported by EFRE 2000-2006 and the Free State of Saxony (grant numbers 7953/1272; 10957/1700 and 7954/1272; 109871700) and by the Federal Ministry of Economics and Technology of Germany (grant number MF 090193). The work was also granted by the Association of Industrial Research Associations, “Otto von Guericke”, e.V. (AiF) (grant number ZIM-KF 2388001FO9). Conflict of Interests: N/A P43. EFFECT OF AGE ON SEMEN FREEZABILITY OF AKSARAY MALAKLI SHEPHERD DOG (TURKISH MASTIFF) K. Tekin 1, M.E. Inanc 1, K.T. Olgac 1, B. Yilmaz 1, Y. Akkurt 1, C. Temizkan 1, U. Nkaya 2, P.B. Tuncer 3, U. Tasdemir 3, S. Buyukleblebici 3, B. Kul 1, E. Durmaz 3, O. Uysal 1, O. Ertugrul 1. 1 Ankara University, Ankara, Turkey; 2 Gazi University, Ankara, Turkey; 3 Aksaray University, Aksaray, Turkey E-mail address: [email protected]
The objective of this study was to determine the effect of age on semen quality parameters in Malaklı (Turkish Mastiff). Therefore, twenty dogs were used in this research and separated into two age groups, age group 1 (3 years of age) and age group 2 (4-6 years of age). Semen samples were collected using digital pressure and massage. Semen volume, pH and motility were determined immediately after collection. Then, fresh semen was extended with a tris based extender, equilibrated (+5 C/2h), loaded into a 0.25 french straw, frozen in liquid nitrogen vapour (-120 C/15 minutes) and stored in liquid nitrogen (-196 C). Frozen straws were thawed in a water bath (37 C/30 seconds) and percentages of progressive motility, total motility, and sperm kinetic parameters (VCL, VSL, VAP, LIN, STR, WOB, ALH, BCF and hyperactivity) were determined with a computer assisted sperm analyzer (CASA, SCA Microptics, Barcelona). Abnormal spermatozoa rates (head, acrosome, neck, tail and cytoplasmic droplets) were assessed with sperm blue (Microptic, Barcelona). The results showed that the highest progressive, and total motility, were recorded as 4,8 ± 2,00; 63,8 ± 7,95 in age group 1 respectively, after freeze-thawed. Among age groups, there was a statically significance differences between progressive and total motility (p<0,05). The highest ALH, BCF and hyperactivity were 4,9±0,56, 6,0±1,10 and 15,9±6,05 in age group 1 respectively (p<0,05). There was considerable variation among age groups in ALH, BCF and hyperactivity, however there were no significant differences in other sperm kinetic motions. Significant differences were found according to abnormal spermatozoa rate (p<0,05). There was a correlation between ALH, BCF, hyperactivity and total abnormal spermatozoa (p<0,05). It appears that total and progressive motility, yet some kinetic parameters such as hyperactivity, ALH and BCF, starts to decline with age when the frequency of sperm abnormalities has increased, as evidence by specific age related sperm defects. We concluded that freezing and thawing processes increased abnormal sperm that are due to cell alteration for old males, and consequently, decrease the freezability of Malakli dog semen. Funding: This study was supported by the Turkish Scientific and Technical _ Research Council TUBITAK (project number: 114O636). Conflict of Interests: N/A P44. INFLUENCE OF DIFFERENT FREEZING PROTOCOLS ON HEMOGLOBIN ENCAPSULATED IN ALGINATE MICROSPHERES S. Rozanova, O. Nardid. Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkov, Ukraine Nowadays hemoglobin-encapsulated microspheres are considered as an artificial erythrocyte substitute in transfusion medicine. However their
569
application is restricted by the protein hem oxidation during storage. Cryopreservation may be a proper way of encapsulated hemoglobin storage. The research aim was to investigate the effect of different freezing protocols on encapsulated hemoglobin properties. Hemoglobin-loaded alginate microspheres were obtained by ionotropic gelation. Microspheres were frozen down to -20 C or -196 C at 1-2 C/min and 300 C/min cooling rates respectively. Thawing was carried out at 22 C. Hemoglobin functional activity was analyzed by ability to release oxygen in anaerobic conditions using sodium dithionite. The percentage of different hemoglobin forms in the microspheres was detected by alterations in protein absorption spectra. ABTS+ radical decolorization assay was used to investigate protein stability. Freeze-thawing of hemoglobin loaded microspheres has been shown to lead to partial hemoglobin loss and to the lowering of protein ABTS+ scavenging ability, the most expressed in the case of freezing down to -20 C. It has been demonstrated that hemoglobin loaded into alginate microspheres is able to release oxygen in anaerobic condition. Freezing does not affect this protein ability. The results obtained have revealed freezing of encapsulated hemoglobin down to -196 C as more effective. Funding: N/A Conflict of Interests: N/A P45. CRYOPROTECTANT TOXICITY STUDIES ON CRASSOSTREA ANGULATA SPERM lix 1, Sonia Martinez-Pa ramo 1, Domitília Marta F. Riesco 1, Francisca Fe 3, Marc Suquet 4, Elsa Matias 2, Sandra Joaquim 2, Catherine Labbe Cabrita 1. 1 CCMAR, University of Algarve, Faro, Portugal; 2 IPMA, IP, Avda 5 ~o, Portugal; 3 INRA, Fish Physiology and Genomics, Rennes, Outubro, Olha France; 4 IFREMER, Departement PFOM/Laboratoire ARN Station Experimentale d’Argenton, Argenton, France E-mail address: [email protected]
The preservation of genetic material of both improved stocks and of original population is very important for the oyster aquaculture industry to avoid potential impacts from epidemic diseases and natural disasters. Nowadays, purely wild populations of Crassostrea angulata are rare to find due to multiple factors that have affected this industry. Cryopreservation technology could promote alternative techniques to contribute to the resource management efficiency of the Portuguese oyster and associated economic activity. This report describes the first attempt on cryoprotectant sperm toxicity in the Portuguese oyster and discusses a set of interacting variables at different steps from sperm collection to thawing (initial sperm concentration, sperm dilution in cryoprotectants, caffeine addition (10 mM) and type and osmolarity of extender). Sperm motility, sperm viability (using fluorescent dyes), and enzymatic activities were used to evaluate the exposure of sperm at different cryoprotectants (Me2SO, PEG, EG, Methanol) using different concentrations (5%, 10% and 20% v/v). Toxicity assays revealed that PEG and Me2SO are significantly less toxic than other cryoprotectants at the same concentrations. Other parameters such as caffeine supplementation, low dilution ratios (1:2) and extender (isosmotic) significantly improved sperm quality after cryoprotectant exposure. This work summarizes the factors influencing sperm management prior to cryopreservation. Funding: This work was supported by CRIOBIV 31-03-05-FEP-59 PROMAR program and COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE). Conflict of Interests: N/A P46. COMBINED RIBAVARIN TREATMENT AND CRYOTHERAPY FOR EFFICIENT POTATO VITUS M AND POTATO VIRUS S ERADICATION IN POTATO IN VITRO PLANTLETS S.V. Kushnarenko 1, N.V. Romadanova 1, M.O. Bekebayeva 1, A.M. Alexandrova 2, B.M. Reed 3. 1 Institute of Plant Biology and Biotechnology, Almaty, Kazakhstan; 2 M.A. Aithozhin Institute of Molecular Biology and Biochemistry, Almaty, Kazakhstan; 3 USDA-ARS National Clonal Germplasm Repository, Oregon, USA E-mail address: [email protected]
570
Abstracts / Cryobiology 71 (2015) 537e573
An in vitro collection of 30 potato cultivars and hybrids were evaluated for five viruses: Potato leafroll virus (PLRV), Potato virus M (PVM), Potato virus S (PVS), Potato virus X (PVX) and Potato virus Y (PVY) by two methods: ELISA and RT-PCR. PLRV was not detected in any accessions. The majority of accessions were singly infected by PVM (26.7%) or mix-infected by PVM and PVS (43.3%) or PVM and PVY (13.3%). One accession had PVM, PVS and PVX. No viruses were detected in four cultivars. For cryotherapy of shoot tips, the PVS2-vitrification protocol was used. The effect of culture with 100 mg/L ribavirin on viruses eradication was also investigated both alone and combined with cryotherapy. Cryotherapy eliminated all PVY infections but only 33.3% were free of PVM. No totally virus-free plants were found in mix-infected accessions. Treatment with ribavirin alone was not effective when used for one or two subcultures, but three subcultures on ribavirin or followed by cryotherapy was effective for both PVM and PVS. RT-PCR did not detect PVS and PVM after two subcultures on ribavirin or following cryotherapy, but ELISA tests indicated a low PVM titer in some accessions. Future studies will include cultivars infected with PVX. Conflict of interests: N/A Source of Funding: This research was supported by the grant No. 0047/GF of Ministry of Education and Science, Republic of Kazakhstan P47. MANUFACTURING A BIO-ARTIFICIAL LIVER (BAL) SUPPORT DEVICE Nick Medcalf 1, Andreea Iftimia-Mander 1, Andy Picken 1, Qian Liu 1, Alexandra Stolzing 1, Rob Thomas 1, John Morris 2, Stephen Lamb 2. 1 Centre for Biological Engineering, Loughborough University, UK; 2 Asymptote Ltd., Cambridge, UK E-mail address: [email protected]
An efficient, cost effective and ‘off-the-shelf’ Bio-Artificial liver (BAL) would be a game-changer in the clinical treatment of liver failure and liver disease. Since the liver is one of the few organs that can repair and regenerate, therapies enabling regenerative medicine that are creating living functional tissues to repair or replace organ function lost due to damage, are expected to play a role in several areas of liver disease. A bioartificial liver machine can temporarily replace the functions of the liver, allowing the damaged liver to regenerate whilst protecting the patient’s other organs from the life-threatening damage that ensues during liver failure. Currently, almost 80% of patients with severe acute liver failure will die within a few days unless they receive a liver transplant. Moreover, a
third of patients on the waiting list for a transplant, die before they receive an organ. This project will develop and deliver an ‘off the shelf’, disposable cassette, consumables and associated cryogenic equipment to allow the clinical delivery of a bio-artificial liver (BAL). The technology also has potential in other applications, including decellularization of a range of organs and tissues, and for the delivery of primary human liver cells spheroids and pancreatic cells. The project is an example of the unit operations associated with presentation and delivery of advanced therapies as part of the systems-based approach at Loughborough University. Conflict of Interests: N/A Funding: Innovate UK (101 620) P48. MICROSCOPIC INVESTIGATION OF ANTIFREEZE PROTEINS ACTIVITY AT CRYOGENIC TEMPERATURES Liat Bahari, Victor Yashunsky, Ido Braslavsky. The Hebrew University of Jerusalem, Israel E-mail address: [email protected]
Antifreeze proteins, are one of the natural mechanisms that are found in cold adapted organisms to cope with subzero temperatures. In the laboratory, they have been demonstrated to improve preservation of cells either under chilling conditions or ultra-low temperatures. AFPs inhibit ice growth in slightly suppercooled conditions, and inhibit recrystallization of ice in frozen tissues. These activities were tested in temperatures down to a few degrees celsius lower than melting point. It is not known yet if and how AFPs act at ultra-low temperatures. We investigate the activity of AFPs in vitrification, an “ice-free” method of cryopreservation in which ice crystal growth may occur during the rewarming step, at low temperatures slightly above the glass transition temperature (devitrification). An examination of this process under a microscope equipped with a controlled cold stage showed that hyperactive AFP from the Tenebrio Molitor mealworm (TmAFP), influenced ice growth even at temperatures of -110 C, during the warming step. For example, using SnowMax as nucleators in this process, TmAFP in Me2SO solutions reduced the number of ice crystals observed. These results indicate that AFPs can be active at cryogenic temperature ranges as growth inhibitors of ice crystals and this function may improve cryopreservation procedures. Funding: This research was funded by the European Research Council Conflict of Interests: N/A