Tg(fli1a:eGFP)y7 and the synergistic interaction of IA and SA in crafting the archetypical angio-architecture of the CVP. Dynamic in vivo observations and quantitative analyses revealed that the primitive CVP during development was initiated through SA. Further vascular growth and remodeling occurred by IA. Intussusception contributed to the expansion of the CVP by formation of new pillars. Those pillars arose in front of the already existing ones; and in a subsequent step the serried pillars elongated and fused together. This resulted in segregation of larger vascular segments and remodelling of the disorganized vascular meshwork into hierarchical tree-like arrangement. Blood flow was the main driving force for IA, particularly shear stress geometry at the site of pillar formation and fusion. Computational simulations based on hemodynamics showed drop in shear stress levels at locations of new pillar formation, pillar elongation and fusion. Correlative 3D serial block face scanning electron microscopy confirmed the morphological substrate of the phenomena of the pillar formation observed in vivo. The data obtained demonstrates that after the sprouting phase and formation of the primitive capillary meshwork, the hemodynamic conditions enhance intussusceptive segregation of hierarchical vascular tree i.e. intussusceptive arborization resulting in complex vascular structures with specific angio-architecture.Intussusceptive angiogenesis (IA) is a complementary method to sprouting angiogenesis (SA). The hallmark of IA is formation of trans-capillary tissue pillars, their fusion and remodeling of the vascular plexus. In this study, we investigate the formation of the zebrafish caudal vein plexus (CVP) in  It comprises two discrete types, sprouting angiogenesis (SA) and intussusceptive angiogenesis (IA). Expansion of capillary networks by a phenomenon called “growth within itself” was termed intussusceptive microvascular growth (IMG). IA is also known as splitting angiogenesis or non-sprouting angiogenesis or inverse sprouting angiogenesis; it is characterized by formation of transluminal tissue pillars and subsequent vascular splitting. These trans-capillary or intussusceptive pillars are formed by protrusion of opposing capillary endothelial cells into the capillary lumen thus creating a zone of contact, leading to perforation of the bilayer of endothelial cells and increase in girth of the newly formed interstitial pillar due to invasion by fibroblasts and pericytes8. In the strict sense, only IMG could be considered as an angiogenic mechanism due to the fact that it creats new vascular entities. IAR and IBR are important in creation of the local organ-specific angio-architecture as they are associated mainly with reduction in the vascular complexity and pruning could be considered for this reason, as pruning is defined as reduction of the vascular complexity. Under physiological conditions, three facets of IA  work hand in hand and are essential processes for the formation and restoration of an organ-specific angio-architecture. IA has been studied in several experimental models and was first described in vessels of the developing rat lung9 and was later demonstrated in other organs including the chicken chorio-allantoic membrane (CAM)10, in the developing chicken choroid vasculature11, metanephric avian kidney12 and chicken embryo lung13. IA also occurs in different pathological situations like tumor growth, murine colitis, and in neurotoxic disease14.The primitive capillary plexus formed by vasculogenesis, sprouting or intussusception is a finally mesh-like structure, i.e. consists of multiple pillars of different sizes. The subsequent growth and remodelling are achieved through different forms of IA, namely: Intussusceptive microvascular growth (IMG), Intussusceptive arborisation (IAR) and Intussusceptive branching remodeling (IBR). Intussusceptive microvascular growth reflects initiation of pillar formation and their subsequent expansion with the result that the capillary surface area is greatly enhanced, but the capillary plexus remains primitive and disorganized. In contrast, intussusceptive arborization entails formation and subsequent fusion of serried pillars delineating straight capillary segments and generations of smaller future feeding and draining microvessels. By this process, fusing pillars allocate hierarchical microvascular segments restructuring the primitive capillary meshwork to a vascular tree. Optimization of local vascular branching geometry occurs through intussusceptive branching remodelling, so that the vasculature is remodelled to meet the local hemodynamic demand. The latter process occurs via transluminal pillars that are formed close to arterial or venous bifurcation sites. Enlargement of such pillars, their subsequent fusion with the connective tissue at the bifurcation narrows the bifurcation angle. Eccentric formation and expansion of pillars at the bifurcation often results in a sub-form of IBR, namely intussusceptive vascular pruning20. These observations clearly point towards a significant role of blood flow and shear stress mediating intussusceptive vessel splitting. However, the molecular link between shear stress and its effects on regulating intussusceptive angiogenesis are not well understood. To this date, there is no single mechanotransducer clearly linked to IA, but several mechanosensing biological components, which could potentially regulate blood flow-driven pillar formation, have been documented21.Although the morphological aspects of IA have been described in our previous works, unlike SA the molecular mechanisms of IA are yet to be investigated. Hemodynamic parameters of blood vessels such as wall shear stress and red blood cell (RBC) velocity are known to be the key modulators in the process of angiogenesis. One of the main differences between sprouting and intussusceptive angiogenesis is that sprouts appear  in regions lacking perfusion, whereas pillars are formed in the perfused vascular areasThe synergistic interaction between IA and SA during development is largely unknown. We utilized zebrafish CVP as a model to better understand the synergistic interaction between IA and SA. CVP is mainly to study venous angiogenesis and how this plexus remodels gradually and simplifies to become a single vascular tube. In this study by employing the developing zebrafish caudal vein plexus (CVP) we have identified intussusceptive remodeling and shown its response to blood flow alterations. On the basis of our findings, we hypothesize that intussusceptive angiogenesis is promoted and governed/regulated by the specific hemodynamic profile. We show that intussusception is responsible for capillary expansion and in a subsequent step to vascular remodeling and intussusceptive arborization forming in this way the hierarchical angio-architecture of the CVP.Tg(fli1a:eGFP)y7;Tg(kdrl:EGFP)s843 transgenic zebrafish lines were used throughout this study. Genetically modified embryos were obtained from naturally spawning transgenic lines and staged according to Kimmel et al.22. Zebrafishes (Danio rerio) were raised in a dedicated zebrafish facility with the system water maintained at 28.5 °C with 14 h light 10 h darkness diurnal rhythm. Adult male and female zebrafishes were separated for a week prior to breeding. Subsequently they were put together at male to female ratio of 2:1. The embryos were maintained in standard embryo medium (1 × E3 medium) throughout the experiments. The transgenic lines were obtained from aquatic resource program . All the animal experiments were performed according to the guidelines of Swiss animal welfare act (license number is BE413). According to the Swiss government guidelines, experiments based on zebrafish embryos aged less than 48 hours of post fertilized embryos are exempted from animal permission.Tg(fli1a:eGFP)y7 transgenic zebrafish embryos were incubated at 28.5 °C until 24 h post fertilization (hpf). Embryos were screened for GFP expression and the positive ones were treated with 0.003% 1-phenyl-2-thiourea (PTU) solution in E3 medium to prevent pigment formation at 24 hpf. The chorions were removed and mounted on low melting point agarose gel to enable imaging of CVP formation. Establishment of the vascular pattern and blood flow were monitored between 24 – 42 hpf by fluorescence stereomicroscopy . Still images were captured and blood flow was recorded as video files using Leica camera (DFC365X) and software (Leica AF600). These videos were processed further for morphometric quantifications and simulations.Dechorionated embryos were mounted on 0.4% of low melting point agarose gel containing 0.01% of tricaine in embryos medium (standard E3 medium). Time-lapse images were recorded in Axiovert 200 M microscope with laser scanning module LSM 5 Duo live  using 20X objective between 24 and 40 hpf, and z-stacks were also performed. Z-stack image projections were processed using Imaris software  for 3-dimensional (3D) visualization of pillars in the CVP.in vivo observation with emphasis on the CVP starting from 24 hpf up to 42 hpf. The images acquired were used to quantify sprouts and pillars. Pillars were identified as dark holes in the green vascular plexus with diameters roughly ≤ 2.5 µm. All holes greater than 2.5 µm were considered to be meshes (large pillars). A borderline between perfused and the non-perfused area in the CVP was drawn with the help of blood flow videos captured along the still pictures. The following parameters were calculated between perfused and non-perfused areas using Cell^D software The prevalent angiogenic mode (sprouting vs. intussusception) was tightly monitored by 2 [Pp(A)]. Thus; VA = Pp(Vs) * Pp(A).Vessel area (VA) was obtained as the ratio of total number of points falling on the vascular (green) surfaces, [Pp(Vs)] and the point-associated area in µm2 of vessel area, NA(Pr). Thus; NP = NA(Pr)/VA.Numerical density of the pillars [NP] was estimated as the total number of pillars counted per µm2 of vessel area. NA(Spr). Thus; NS = NA(Spr)/VA.Numerical density of the sprouts [NS] was calculated as the total number of sprouts counted per µm24 was already successfully applied to model blood flow through blood vessels in chick embryos and to analyze the creation of pillars25. This method is used in this paper to simulate blood flow through the caudal artery and caudal vein of the zebrafish. In numerical simulations the units for physical dimensions of the domain are micrometers, obtained velocity distribution is shown in micrometers/second, obtained pressure and wall shear stress distributions are shown in Pascal (N/m2). Characteristics of blood are defined as follows: density is set to 1.05 g/cm3 and dynamic viscosity is set to 3.675·10−3 Pa·s. For detailed description of the simulation calculation, please see supplementary data Materials and Methods.Experimental images were used to define the geometry of the domain. Pillar positions and dimensions were manually highlighted in these images. The obtained information about geometry was then used to create the finite element mesh of the model. Finite element mesh was generated using FEMAP software version 10 , and was paired with our in-house developed software tool written in C++. This software tool is used to adapt the finite elements mesh to the format that is appropriate for the numerical simulations. The in-house developed software package PakFTg(fli1a:eGFP)y7 embryos were maintained in E3 medium until 26 hpf and the CVP was imaged using epifluorescent microscope. Control groups and treated groups were separated accordingly. Freshly prepared isoprenaline hydrochloride  and 2,3 BDM  was added to E3 medium of the treated embryos at 26 hpf and 24 hpf respectively. The embryos were incubated at 28.5 °C until 30 hpf. The control embryos in E3 medium were maintained in the same incubator until 30 hpf. The drug solution for the treated group was replaced every 45 min with freshly prepared drug solution and incubated at 28.5 °C until 36 hpf. Images of the CVP from both the control and treated groups were obtained.troponin 2a, (tnnt2a)26 and gridlock (grl)27 MOs were purchased from Gene Tools, LLC, USA. MOs were injected with phenol-red (0.05%) in zebrafish embryos at the 1~2 cell stage at a concentration of 3-5 ng/embryo. The MOs of the following sequences were used:Control, Control MO; 5′-CCTCTTACCTCAGTTACAATTTATA-3′,tnnt2aMO; 5′-CATGTTTGCTCTGATCTGACACGCA-3′,grlMO; 5′-CGCGCAGGTACAGACACCAAAAACT-3′,Embryos were treated with 0.003% PTU solution to prevent pigment formation at 24 hpf and then mounted using a 1% low-melting agarose on the glass bottom dish and laterally oriented. CVP in the tail region (within somite boundary 16 and 21) were imaged and processed using a Nikon confocal microscope  and Velocity program . ImageJ and Fiji programs were used to create maximum intensity projections and depth coding of Z-stack images.Tg(fli1a:eGFP)y7 embryos were imaged and fixed in karnovsky solution  overnight at 4 °C. Further, the embryos were washed 5 times for 3 min each in 0.15 M cacodylate buffer. Embryos were then incubated in 0.15 M cacodylate solution containing 1.5% potassium ferrocyanide and 2% aqueous osmium tetroxide (Electron microscopy sciences) for 1 hour at room temperature. The samples were washed in water and incubated in 1% filtered thiocarbohydrazide  solution for 20 min at room temperature. Subsequently, they were washed gently 5 times for 3 min in milliQ water at room temperature and then incubated in 2% aqueous osmium tetroxide for 30 min at room temperature. Following this step, the embryos were washed again 5 times for 3 mins in milliQ water and then incubated in 1% uranyl acetate and left at 4 °C overnight. The following day the embryos were washed again 5 times for 3 min in milliQ water at room temperature and incubated in Walton’s lead aspartate solution28 at 60 °C for 30 min. Then they were washed in milliQ water 5 times for 3 min and dehydrated by immersion in ascending concentrations of ethanol starting from 20%, then 50%, 70%, 90%, and 100%, 5 min each at room temperature. After the final incubation in ethanol, the embryos were transferred again to pure ethanol for 10 min at room temperature. Finally the embryos were placed in mixtures of Durcurpan (Sigma) and ethanol as follows: 25% Durcupan for 2 hours, then 50% Durcupan for 2 h, and 75% Durcupan for 2 hours. Embryos were then placed in 100% Durcupan overnight and then transferred into fresh 100% Durcupan for polymerization in silicon molds at 60 °C for 48 h.The in situ ultramicrotome (Gatan). The block face images obtained were taken with 3 kV acceleration voltage and at 10 Pa pressure (low vacuum). Each slice was 75 nm thick and imaged with a magnification of 626.0×, pixel size of 21 nm and pixel dwell time of 1 µs (min frame time (s) = 34.169). The images were further processed for 3D reconstruction using Imaris software (Ver.7.7.2).The blocks were trimmed and transverse sections were cut through the trunk of the zebrafish embryos. To perform serial block face scanning electron microscopy (SBF-SEM), ultra-thin sections were imaged with a Quanta 250 FEG (FEI) scanning electron microscope equipped with a 3View2.XP p < 0.05; **p < 0.01; and ***p < 0.005).The data obtained were compared by non-parametric Mann-Whitney statistical test using the GraphPad Prism software . Data were presented as mean ± SD and asterisks were used to indicate significant differences (*Tg(fli1a:eGFP)y7 zebrafish embryos between 24 hpf and 42 hpf is presented in Fig. An overview of the developing CVP of 4 µm2 to 4.8 ± 0.3 × 104 µm2 in the developing CVP. In contrast, the vessel area of the non-perfused region decreased from 0.9 ± 0.1 × 104 µm2 (at 24 hpf) to 0.1 ± 0.01 × 104 µm2 (at 42 hpf) in the distal region of the growing CVP  and a ventral vein (VV) y7 zebrafish embryos, were observed by series of high magnification in vivo fluorescence images and real time videos of blood flow in the CVP. The precise location of pillar appearance in the vascular network cannot be predicted in advance. Tiny pillars with a diameter ≤ 2.5 µm appeared after 32 hpf of CVP development in the well-perfused regions of the capillary plexus. Subsequently, we made an effort to track the pillar formation along with pillar fusion and splitting of vessels during development of the CVP in the same embryo at different time points. At 34 hpf, y7 zebrafish embryos was imaged at 38 hpf . The CVP development of hpf Fig. 1 and B1.hpf Fig.  could behpf Fig. . Collagehpf Fig. . Some ofhpf Fig. . In one hpf Fig. . The 3D hpf Fig. . This evTg(fli1a:eGFP)y7 between 25-42 hpf. The velocity profile revealed that it remained high in the dorsal aorta (DA) corresponding to the color code in the scale bar . The in-house developed PakF software was used to simulate blood flow velocity and shear stress profiles in the dorsal aorta and CVP of bar Fig. . During bar Fig. . Shear sbar Fig.  and S2a.bar Fig. . During bar Fig. 1 and B2 bar Fig. 2 and S2bbar Fig. . In Fig.It should be noted that parameters defining characteristics of the blood in numerical simulations were approximated to be equal to the ones in humans. The main goal of blood flow simulations was to observe the changes in distribution of hemodynamic quantities, rather than just quantify them. These parameters only affect the values of the hemodynamic quantities, which was demonstrated in the sensitivity analysis. Additional simulations were performed to observe the effect of change of viscosity Fig.  and inleThe data collected using numerical simulation raise the possibility that alteration within the blood flow and shear stress are essential for intussusceptive pillar formation and fusion, leading to vascular splitting. It seems that there is a specific shear stress constellation on the site of pillar formation . The coRegulation of hemodynamic parameters, like blood velocity and shear stress seems to affect IA. Pharmacological compounds like adrenergic receptor agonist isoprenaline hydrochloride  and 2,3-butanedione monoxime  were used to increase or decrease the heart rate, respectively. These treatments mimic physiological and pathological blood flow conditions that can occur during formation of the CVP in the zebrafish embryo.4 µm−2 in control to 2.1 ± 0.3 × 104 µm−2 in BDM treated embryos  in isoprenaline treated embryos (186.3 ± 3.5 µm/s) compared to non-treated embryos , which inhibits cardiac output and a gridlock (grl), specifically inhibits blood flow in the trunk and the tail of zebrafish embryo y7 zebrafish embryos. The developing CVP from 24 to 42 hpf was captured using in vivo fluorescence imaging, similar to previous findings32. Numerous outgrowths of angiogenic sprouts, which initiate CVP growth at 24 hpf mostly in the non-perfused regions were observed. These sprouts interconnect each other to form the primordial plexus by 28 hpf. In the chick embryo CAM model, intussusceptive microvascular growth was shown to be mainly responsible for the capillary network expansion following an earlier sprouting phase6. Similarly, during embryonic development of the CVP in the zebrafish embryos, increase in the area of perfusion at 36 hpf leads to the appearance of tiny intussusceptive pillars, which fuse with the neighboring ones and result in remodeling of the CVP.We asked if the intussusceptive angiogenesis mechanism does occur during zebrafish vascular development and started to investigate in 11. The 3D reconstruction of transluminal intussusceptive pillars using in vivo confocal laser scanning microscopy and SBF-SEM further confirmed the existence of IA in the developing zebrafish CVP. The 3D finite element models helps to study intussusceptive angiogenesis on various models depending upon the flow dynamics25. Future studies on the IA should focus on dynamics of pillar formation and the concomitant cellular events including loss of endothelial cell polarity, rearrangement of the cytoskeleton, creation of endothelial-endothelial junctions . Plausibly, such investigations would reveal new anti-angiogenic targets applicable in anti-angiogenesis based therapy.Three-dimensional visualization of intraluminal protrusions of intussusceptive pillars is essential for the characterization of IA6. The described specific flow profile with a drop in shear stress resulted in following events running simultaneously (1) “expansion, reshaping and fusion of already existing serried pillars” ; (2) “formation and subsequent fusion of the new pillars with the serried elongated pillars and slits as described in “1”. Repetitive formation and fusion of such kind of pillars resulted in delineation of straight capillary segments and generation of smaller future feeding and draining microvessels. The concept of pruning was introduced by Ashton investigating development of retinal vessels under hypoxia which refers to the process of “removal of superficial branches”33. It is clear that it is conceptually incorrect to name the formation of the new vascular entities described as “pruning” because it reduces the number of vascular segments. The role of the intussusceptive pillar formation in the real pruning of superficial branches has been described previously in different models35. Due to the fact, that the newly arrived pillars are transient structures (they fuse in a short time with the serried elongated pillars and slits) and on the end the vascular complexity decreases. This does not justify naming the process as “angiogenesis”. Therefore, it will be reasonable to stick to the previously introduced term“intussusceptive arborization”35. The latter described exactly the series of events that we observed in CVP.During zebrafish CVP development, the angiogenic sprouts are initially not perfused by blood whereas the intussusceptive pillars are formed in the perfused vascular areas. Blood flow and shear stress have a major role in segregation of straight capillary segments smaller feeding and draining microvessels; a process termed before intussusceptive arborization38. In the recently published review by Richard and Simons39 the authors evaluated different mechanisms of vascular pruning. Lenard et al. reported cellular mechanisms involved in the process; type I pruning involves apoptosis, including endothelial cell apoptosis and type II pruning was apoptosis independent. The latter type entails retraction and migration of endothelial cells40. The second type of pruning mainly depends upon the presence of blood flow where the endothelial cells rearrange accordingly, collapse and undergo self-fusion to separate the lumen40. In the present study, the existence of intussusceptive pillar formation was documented along with vascular regression and intussusceptive arborization. The intussusceptive arborization observed in the CVP during zebrafish embryonic development is similar to type II cellular pruning described by Lenard et al.40.Several studies have shown in different animals models such as mouse, rat and zebrafish that a link exists between hemodynamics and vascular remodeling, regression and pruning7. In our study, shear stress distribution was also mapped between 25 hpf to 42 hpf using RBC velocities in the perfused regions of the CVP. The computational model of CVP confirmed our previous findings41 that the transluminal intussusceptive pillars are formed in regions with drastic drop in shear stress within a short distance. With the increasing blood velocity during development, the areas with shear stress discrepancy become more pronounced, what induces progressive pillar fusion and splitting thus leading to intussusceptive arborization. This alteration to shear stress in the vessels correlates with the structural changes in the microcirculatory network mainly due to transluminal pillar formation42. Experimental changes to shear stress by accelerating blood flow in chick CAM vessels has proven the association between blood flow dynamics and pillar formation8.The 3D computational flow simulations of the chick CAM have shown a significant role of wall shear stress and specifically, the areas of a dramatic drop in shear stress are known to induce pillar formation and remodeling in the developing chick CAM vessels44 by using isoprenaline hydrochloride and 2,3 BDM, similar rates of increase or decrease in the heart rate respectively were observed. To evaluate the role of circulatory flow during morphogenesis of the CVP, isoprenaline hydrochloride treatment accelerated the growth of the CVP. This was mainly by intussusceptive arborization and early remodeling of the CVP was inferred. In contrast, reducing the heart rate using the 2,3 BDM resulted in decreased growth of the CVP, which was dominated by the sprouting phase throughout the drug treatment period and lacked intussusceptive arborization possibly due to absence of blood flow. Our results were congruent with vessel remodeling studies on zebrafish cranial division of internal carotid artery during eye blood vessel development45. Blocking the heartbeat pharmacologically or by genetic manipulation induced blood vessel pruning whereas re-stimulation prevented vessel pruning36. The blood flow dependency of intussusceptive arborization and vascular regression and intussusceptive pruning has motivated further investigations on mechanical forces39. From our analyses and according to the literature, we understood that intussusceptive arborization and vascular regression are driven by hemodynamics and play an essential role in the formation of hierarchical vascular treeIn agreement with previous reports in zebrafish embryos46. The lack of circulation in tnnt-2 morphants severely impaired growth of both ISVs and CVP. No intussusceptive pillar formation, vascular regression and intussusceptive arborization has been observed but numerous angiogenic sprouts drove the growth of the impaired CVP due to lack of perfusion. Interestingly, sequestering blood flow to the fin vessels by aortic coarctation in grl mutants has shown near normal development of ISVs and CVP. Anastomoses of the numerous sprouts appearing in the non-perfused areas mainly drives growth of the CVP, nevertheless the grl morphants lacked pillar formation and remodeling for the segregation of caudal veins into DV and VV. The one possible explanation for the near normal development of the CVP in the grl morphants is that, in these mutants with functional hearts, there develops collateral vessels and they survive to adulthood48. We infer from our results that hemodynamics regulate IA by pillar formation and are also involved in post angiogenic mechanisms, namely blood vessel regression and intussusceptive pruning39.In addition, to overcome the possible nonspecific phenotype due to side effects of pharmacological modulators, a more precise genetic modification by anti-sense oligonucleotides known to inhibit translation of proteins by injecting morpholino in zebrafish embryos was usedin vivo) with the observation that sprouting initiates the primitive CVP and the expansion of the capillary plexus is driven by the intussusceptive arborization. This process occurs in perfused regions showing decline in shear stress confirming that intussusceptive arborization is mainly regulated by hemodynamics. Thus, the synergistic actions between the sprouting and intussusception can be evaluated simultaneously within few hours during the morphogenesis of CVP model in zebrafish embryos.In conclusion, we have studied the role of intussusceptive arborization in the zebrafish CVP model (Supplementary FiguresSupplementary video S1Supplementary video S2Supplementary video S3Supplementary video S4Supplementary video S5Supplementary video S6


Here, we benefit from a tunnel crossing a large and vertical headwall in the European Alps (Eiger), where we measured concentrations of in-situ cosmogenic 36Cl along five depth profiles linking the tunnel with the headwall surface. Isotopic concentrations of 36Cl are low in surface samples, but high at depth relative to expectance for their position. The results of Monte-Carlo modelling attribute this pattern to inherited nuclides, young minimum exposure ages and to fast average denudation rates during the last exposure. These rates are consistently high across the Eiger and range from 45 ± 9 cm kyr−1 to 356 ± 137 cm kyr−1 (1σ) for the last centuries to millennia. These high rates together with the large inheritance point to a mechanism where denudation has been accomplished by frequent, cm-scale rock fall paired with chemical dissolution of limestone.Quantifications of  Our understanding of the spatial scale, rate and timing of denudation, is coined by a very large set of scale-specific data, quantified with a small number of methods. For mountain belts such as the European Alps, quantitative data on surface denudation is often inferred from concentrations of in-situ cosmogenic nuclides9, records of low temperature thermochronology11 and river sediment loads12. These data have also been used to measure the effects related to the coupling of individual erosional mechanisms15. Most of aforementioned studies share the same concept as they rely on sediment deposits of various type17 and scale as archives18. This inherently includes a possible bias because the original denudation signal may be blurred, or shredded, by transport mechanisms, intermediate storage, and mixing with material from other sources. Related possible pitfalls are already outlined in the pioneering studies where local headwall denudation has been quantified based on sediment budgets20. While low-temperature thermochronology techniques can yield information on local erosion rates of currently exposed bedrock, these methods are only suitable if the focus lies on erosion rates over millions of years. In the opposite cases, where remote sensing technologies were employed to measure surface changes at the local scale23, the timescales over which denudation was measured were too short to allow a meaningful interpretation of the underlying controls and mechanisms. Over the last decades, terrestrial cosmogenic nuclides (TCN)25 have been used to quantify the exposure history of rock surfaces, which built the basis for the temporal quantifications of coastal cliff collapse events26, rock fall activities in the high Alps27, and for the calculations of rock wall recession rates of limestone cliffs28. However, these studies were based on single TCN concentrations in surface samples only, which cannot be used to infer a time-averaged rate of denudation, as either a second TCN in the same sample or information on the TCN concentrations within depth profiles are needed for such an endeavor25.Denudation in its broader sense is a complex process, which is initiated and controlled by a variety of mechanisms and conditions such as chemical and physical erosion36Cl along several depth profiles. We employ this methodology because it yields quantitative and in-situ information on the local denudation of the target headwall averaged over millenia32. We focus on the Eiger in the Central Swiss Alps  at the ETH Zürich. Principally, upon inferring denudation rates from TCN concentrations within depth profiles, one considers the time since the studied surface has been exposed to the cosmically induced particle shower, i.e., the exposure time, which started at an unknown point of time in the past  to the time of sampling  and denudation rates averaged between t0 and the present  through Monte Carlo (MC) simulations of 36Cl concentration patterns within each depth profile32. By utilizing the shape of the TCN profiles within the bedrock, we can conceptually constrain the erosional mechanisms and history during this inferred exposure time  between 7 and 39% (Table 36Cl concentrations in near surface samples (≤ 0.47 × 105 at g−1). The related 36Cl concentration patterns result in seemingly truncated profiles  and the SE sites (EM profiles). This includes the total 36Cl concentrations and the decrease in these values over depth. Samples at greater depth (>100 cm) show 36Cl concentrations between 0.07 × 105 at g−1 and 0.21 × 105 at g−1  only increase the ages up to maximum of ~20%. An upper-end estimation on the exposure age can be constrained by calculating apparent exposure ages for the deepest available samples, thereby considering similar conditions . The resulting apparent ages range from 36 ± 18 to 103 ± 87 ka, overlapping within the 1σ confidence interval. These values represent upper boundaries for further MC modelling since they are based on the assumption that all 36Cl concentrations at deeper levels stem from one exposure, which would require a situation of a slowly eroding rock surface over one exposure period.Apparent exposure ages were calculated for surface samples by considering a simple end-member scenario consisting of one single exposure event, assuming no inherited 31 with Monte Carlo (MC) simulations32. We modelled our 36Cl concentrations in the depth profiles by limiting the solution space as little as possible (see methods). This works well for estimations of denudation rate and inheritance, but not for the determination of mean or maximum exposure ages, which strongly depends on the assigned initial constraints on denudation32. In the absence of geological information, we assigned total maximum denudation cut-offs of 12, 15 and 20 m  for all profiles during independent model runs with the purpose to evaluate the dependency of our model outputs on the initial assumptions.Exposure age, denudation rate and inheritance can be estimated from randomized depth profile modelling5 modeled 36Cl concentration depth profiles for each site for EM-02 and EW-03 are within the 2σ confidence interval of the measured 36Cl concentrations, while EM-01 and EM-03 are within 3σ, and EW-02 is within 4σ only  values show a similar trend, where the lowest values are close to one for EM-02 (1.2) and EW-03 (1.1) and are slightly higher for EM-01 (2.9), EM-03 (2.9) and EW-02 (4.0). We note here, that despite needing a larger solution space for reaching 105 good fits between simulation results and data, the best fitting model profiles for EW-02 fall within the 3σ confidence interval of the measured 36Cl concentrations. The results for exposure age, inheritance and denudation have similar χ2 distributions with clear minima for all model runs . The modelled denudation rate probability distributions are consistent for all three estimates of cumulative denudation  with minima of 5.2 × 103 to 1.3 × 104 at g−1. Maximum modelled inheritance ranges from 1.7 × 104 to 2.1 × 104 at g−1, which would account for a significant proportion of the concentrations that were measured for the corresponding surface sample (e.g. > 60% for EW-03-1). The inheritance distributions and their mean values are independent of the total net denudation, as the corresponding values remain constant with a larger total net denudation cut-off ; and (ii) apparent exposure ages are significantly older than 0.11 ka for all profiles, also verified by the MC profile modelling . Nevertheless, by adapting the shieldingng Table . The onl36Cl concentrations at depths exceeding 3 m  could be obtained for a meaningful parameter estimation within a reasonable sigma range32. For 4 out of 5 sites, the model profiles displaying the lowest χ2 values could reproduce the depth profile data within or close to the 2σ range  of apparent exposure ages from the same surface samples (Table 40) in the south and to 19 ka (LGM deglaciation41) in the north face, at least for the last exposure. This is based on the assumption that during these periods the reconstructed ice cover was thick and erosive enough to reset the TCN clock.MC modelling results of TCN concentrations in depth profiles have been used in recent years to estimate these parameters in question0. Such a scenario would imply that our samples had been located at depths, were production is too low to produce the excess of nuclides we have measured in our deepest samples 7 and for glacial/periglacial environments in the Central Alps where average denudation rates range between 1 and 2 mmyr−1 (100–200 cm kyr−1)43. An exception is site EW-02, situated at a higher elevation within the NW face, where modeling implies a lower mean denudation rate between 45 ± 9 and 46 ± 8 cm kyr−1 for 1 kyr, or possibly even longer. This profile is the only one, where the modelled average denudation rate could correspond to the rate of carbonate dissolution and frost weathering alone43  spallation mainly from Ca, K, and less through Fe, Ti, (2) low energy  neutron absorption by 35Cl, and by (3) fast muon interaction and slow muon capture25. This production is thus dependent on the chemical composition of the individual samples46. In limestone, spallation from Ca is the predominant production reaction48, while muogenic production from Ca becomes important with increasing depth51. The in-situ production is dependent on the sample position, which thus needs a scaling to geographic position, elevation, and correction for shielding from cosmic rays37. The measured concentrations of 36Cl and the robustness of the subsequent interpretations thus hinge on site and sample specific conditions, and it relies on the selection of the corresponding production calculation method.Cosmogenic 52. Each sample was about 5 or 10 cm thick and consisted of 1 to 1.5 kg of bedrock. Depth profile sampling was possible through the occurrence of sub-horizontal, several tens of meter short tunnels that link the railway tunnel with the surface of the bedrock. These tunnels were originally used to dispose rock material during the construction of the railway tunnel and were constructed along the shortest path to the surface. We sampled material along the lateral walls of the several meter-high and -wide tunnels 47. This includes whole rock crushing and sieving to recover the 250–400 mm grain size fraction, two steps of HNO3 leaching and rinsing with ultra-pure water, and addition of a 35Cl carrier. The samples were then dissolved with HNO3, precipitated with AgNO3, and filtered by centrifugation. BaSO4 precipitation was done in order to remove 36S. The complete sample preparation took place at the Institute of Geological Sciences, University of Bern.Sample preparation for 35Cl and 36Cl concentrations were measured at the ETH AMS facility of the Laboratory for Ion Beam Physics (LIP) with the 6 MV TANDEM accelerator, using an isotope dilution method56. 35Cl is assumed to represent total Cl content, which should be determined on unprocessed bulk material. This is justified by the overall low Cl content in the samples and their mineralogical homogeneity. Measured sample ratios were normalized to internal standard K382/4 N with a 36Cl/Cl ratio of (17.36 ± 0.36) × 10−12, and a stable ratio for 37Cl/35Cl of 31.98%57. Full process chemistry 36Cl/35Cl blank ratios of (2.9 ± 1.8) × 10−15 were used for correction, amounting to an adjustment of <15% for most samples, with a maximum value of 23.5% (EM-03-7). Major and trace element concentrations, which are required to calculate 36Cl production, were determined on separated 12 g aliquots of etched sample material at the ACTIVATION LABORATORIES LTD (Canada) using an inductively coupled plasma mass spectrometer (ICP-MS). These measurements were conducted on lithium metaborate/tetraborate fused samples (FUS-ICP-MS) for major oxides and trace elements respectively  instead of lithium metaborate/tetraborate was used as flux. Boron levels were measured by Prompt Gamma Neutron Activation Analysis (PGNAA). Uncertainties on reported concentrations , and a geometrical component arising from the slope of the sampled surface itself. A total shielding factor (ST) was calculated through the scaling of the nuclide production rate to the specific sampling site at the surface35. This was done through the consideration of an open sky visibility (as zenith angle) using the ‘skyline graph’ standard routine of ESRIs ArcGISTM Desktop 10.1 licensed to the Institute for Geological Sciences, University of Bern. The calculation was done for 1° azimuthal increments using a high-resolution DEM (2 m resolution) provided by the Swiss Federal Office of Topography, Swisstopo. We corrected the attenuated production from spallation58 at depth below surface using a site-specific effective apparent attenuation length . Both parameters were calculated with the CRONUS Earth online Topographic Shielding Calculator v2.0 (http://cronus.cosmogenicnuclides.rocks/2.0/html/topo)59 based on previous versions37. Apparent attenuation lengths are calculated therein using the analytical PARMA model60 for the cosmic ray spectra in the atmosphere25. For a discussion and evaluation of our shielding correction approach, see Supplement The geometries of the depth profiles required the consideration of shielding effects59 resulting from the CRONUS earth project61. Results were obtained using production pararameters from previous work47 that have been re-evaluated62. This includes a SLHL spallogenic production rate of 52.2 ± 5.2 at 36Cl (g Ca)−1 yr−1, and 150 ± 15 at 36Cl (g K)−1 yr−1, and a low energy production rate of 696 ± 185 neutrons (g air)−1 yr−1. The production rate scaling followed Stone63, which itself is based on the method of Lal24, and considers a scaling of the 36Cl production according to the sample’s longitude, latitude and atmospheric depth. The production rates and the scaling are in good agreement64 with the recently published nuclide-specific scaling38. Apparent exposure ages  were calculated for each sample considering the samples’ chemical composition  and zero inheritance , in close agreement with production rate schematics reported for the CRONUScalc program57. For consistency, we used the same production rates as for the surface exposure age calculation62 . We also updated the shielding macro36, and we scaled the 36Cl production63 accordingly. We refrained from a global muon attenuation length fitting of the profile data68, instead we calculated the corresponding patterns for each modelled profile, for which we used a muon propagation parametrization based on experimentally determined muon stopping power51. This parametrization originally included empirically fitted parameters that led to significant overestimations of 36Cl production by muons for geological settings, especially at depth69. To avoid this bias, we adapted the approach of the CRONUS-Earth project61. We thus employed the parameters derived from this project, which are based on calibration profiles. For 36Cl, the corresponding values are α = 1 for the energy-dependent coefficient of the muon energy cross section69 and σ0 = 8.3 × 10−30 cm2 for the nuclide production cross-section in 10−24 cm2 at 1 GeV through fast muons62. We used the same value for the target elements Ca and K due to the good agreement within errors for the reported values62. For slow muon production, we again used a rate of 696 ± 185 neutrons (g air)−1 yr−1for the epithermal neutron production rate at the rock/air interface. Finally, effective probability values for particle emission to the nuclide of interest after capture of 62 were used. We additionally used the calculated shielding factor (ST) for corrections on spallogenic and muogenic production and apparent attenuation length for spallogenic production  as input (see Supplement −3). Each profile was parametrized using a profile specific density and porosity value (“soil” in the input), whereas for the chemical composition an unweighted average of all samples of the same profile was employed. This is justified by the homogenous chemical composition of the bedrock at all sites (Supplement 2 and CaO content below EW-02-3 (i.e. the uppermost samples are enriched in quartz compared to limestone).Exposure ages, surface denudation rates and inheritance were modelled from nuclide concentrations at depth using a Monte Carlo (MC) randomization approach−1) and by assuming a maximum thickness of eroded bedrock of 12, 15 and 20 m since the starting time t0; (3) inheritance, by using the 36Cl concentration of the surface sample as a largest possible value and zero as lower bound. The inferred maximum thickness of eroded bedrock of 12, 15 and 20 m since the starting time t0 were derived based on the following three considerations. (i) At all sites, the deepest samples were taken at depths close to or exceeding 7 m and consequently, the production of 36Cl almost exclusively has occurred by muon pathways. (ii) The muogenic production at these depths is in the order of 0.5 to 2% of the total surface production68, assuming a simple exponential muon attenuation length (Λμ). (iii) Considering such an attenuation, that scales exponentially25 with reported muon attenuation lengths (Λμ) between ~4000 and 5300 ± 950 g cm−2 for 2.7 g cm−3 rock density68, this translates to depths of ~15 and ~19 m for 1 Λμ and ~30 to ~38 m for 2 Λμ, and accounts for a reduction of muogenic production by ~63% and ~87%, respectively. This means that independent of the attenuation length, our deepest samples would have to be located at a depth of >27 m at the time of t0 to allow for more than 20 m of total erosion. Any potential nuclides inherited from before t0 would then have accumulated at this depth or even deeper at a rate of <13% of the muogenic surface production.For the modelled unknowns, computational constraints were put on: (1) exposure age, thereby using the apparent exposure age of the deepest sample as a maximum constraint for an exposure age; (2) maximum denudation rate during the exposure time interval, by leaving the denudation rate virtually unconstrained  for each MC run, while the inheritance at depth  was parametrized followinginh scales the inherited concentration at depth, based on a site-specific average length for muon attenuation69  solutions were found within 2 σ. We note here that for EW-03 a cut-off of χ2 = 1.8  was employed to sample only the solution space of the best fitting profiles. We report the resulting χ2 minimum values in Table 5 profiles within the desired confidence . We present the total number of simulated depth profiles along with all model input and results in Supplement The general model acceptance was ideally confined to a 2 Supplementary NotesSupplementary Dataset


Myocardial infarction and strokes, which are caused by arterial thrombosis in heart and brain respectively, are the most frequent causes of mortality and morbidity in the western world. Several clinical trials have demonstrated the efficiency of anti-thrombotic agents targeting platelets  in the treatment and the prevention of arterial thromboembolism [There are several risk factors for arterial thromboembolism such as advanced age, hypertension, obesity, metabolic syndrome and type 2 diabetes which is continuously increasing all over the world and a major risk factor for cardiovascular diseases. Many studies have reported platelet hyperactivation in patients with metabolic syndrome and/or diabetes. Associated with a disturbed vascular endothelium layer, platelet hyperaggregability is a predisposing factor for arterial thrombosis. In addition, diabetes is frequently associated with a lower response to antiplatelet drugs. The molecular mechanisms implicated are not yet fully understood. Several studies also point to a critical role of platelets in the progression of reperfusion injury following ischemic stroke. During this process, platelet adhesion to activated endothelial cells appears to be critically involved, unlike platelet aggregation which inhibition has been shown to increases the risk of intracranial hemorrhage and mortality, reducing the benefit-risk ratio. In contrast, preventing platelet adhesion to the damaged vessel wall by blocking platelet surface adhesion receptors particularly GPVI or GPIbα has been found to offer a significant degree of neuroprotection in experimental stroke models without increasing the frequency of intracranial hemorrhage.Hence it’s becoming clear that a better understanding of the molecular mechanisms driving platelet responses to environment cues is crucial to discover new pharmacological targets and to develop novel therapeutic strategies to face cardiovascular diseases.2) to produce the lipid second messenger PtdInsP3 that is able to recruit and activate a wide range of signaling proteins, including Akt. By interacting with specific protein domains, phosphoinositides play a pivotal role in the spatio-temporal organization of cell signaling, membrane remodeling, intracellular trafficking and cytoskeletal dynamics. Class I PI3Ks have been extensively studied in different models in the last decades. Their critical roles in diseases, particularly in cancers, have stimulated the development of isoform-specific inhibitors. Some of these inhibitors are undergoing clinical trials for the treatment of solid cancer and overgrowth syndrome with gain-of-function mutations of the PI3KCA gene (PI3Kα). Moreover, the selective PI3Kδ inhibitor idelalisib has received FDA approval for the treatment of B-cell malignancies. The use of class I PI3Ks inhibitors has also been proposed for the treatment of immune diseases including arthritis and PI3Kδ-selective inhibitors are currently being explored in the treatment of inflammatory diseases.Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that catalyze the phosphorylation of the 3-position of the inositol ring of phosphatidylinositolbisphosphate PWhat is the impact of class I PI3Ks inhibitors in platelets activation and thrombosis?Although all class I PI3Ks isoforms are expressed in platelets, class I PI3Kβ has a major role and has been proposed as a potential antithrombotic drug target. By generating mice with a selective inactivation of PI3Kβ in the megakaryocyte lineage, we  and othein vivo. These results suggest that PI3Kα inhibitors have a weak antithrombotic effect and, on their own, are unlikely to increase the bleeding risk. This is important information for cancer patients treated with these inhibitors. However, the impact on the bleeding risk of PI3Kα inhibitors in association with anti-platelet drugs or in patients with thrombocytopenia remains to be investigated. From a mechanical point of view, PI3Kα and PI3Kβ are not redundant but complementary in platelets [3 and Akt activator [Regarding the other class I PI3Ks, mice deficient for the PI3Kγ isoform are resistant to experimental thromboembolism induced by ADP exclusively, whereas PI3Kδ has no significant role in platelets. We recently generated a mouse model with class I PI3Kα deficient platelets  and obselatelets –8. PI3Kαctivator .Thus, among class I PI3Ks, PI3Kβ is a potential antithrombotic drug target to prevent the formation of occlusive arterial thrombus; whether it is also a target to improve ischemic-reperfusion injury in stroke remains to be established.


The data of the two synthetic methods (including the new green one), presented in this research article, provides sharp adequate chemical data about the challenging synthesis, separation (purification), and characterization of this compound. A new and very fast one-pot solventless greener microwave-assisted method of synthesis, in addition to the much slower old conventional one, is used in this present research; and followed by full precise purification and characterization  of 2nz.The usefulness of the structure of galloyl hydrazide  of galloyl hydrazide through designing and constructing a new one-pot solventless greener microwave-assisted synthetic method, in addition to redesigning and qualifying the old traditional conventional heating one; both methods are followed by very accurate identification and characterization data  of galloyl hydrazide. Specification Table2nz) is a very important chemical intermediate in organic synthesis, which is, mainly, well-known for its antioxidant and antitumor bioactivities [2nz through designing and finding a new one-pot solventless greener MW-assisted synthetic method, in addition to redesigning and qualifying the old traditional conv. one; followed by very accurate identification and characterization data which can be referenced for the efficient reproducibility in the organic chemistry community. As illustrated in 2nz in 2 min without passing through the very slow esterification reaction; unlike the much slower two-step conv. procedure which comprises first the synthesis of the intermediate ethyl gallate  through esterification of gallic acid, which is followed by another slower step of the hydrazinolysis reaction of 1nz to form 2nz. By using the present new chemical data collection, the organic/medicinal chemists and drug designers/discoverers would have the following apparent advantages in the synthesis of 2nz:1.2nz through providing two fixed accurate methods (conv. and benign) of synthesis (supported with complete extrapurification technique) followed by constant and reliable reproducible data of physicochemical properties and spectroscopic/microanalytical analyses of 2nz.Avoiding the clear differences among most of the previous literature procedures for the synthesis of 2.1nz, which is the most common ester , unlike most other literature methods which use the less common propyl ester of gallic acid [1nz was preferred in the present work over the famous propyl gallate ester, i.e., it is the ester of choice for this hydrazinolysis reaction, due to many reasons, such as providing the reaction with an internal autosolvent through giving EtOH as a byproduct of the reaction, which could be condensed, recovered, and reused as a solvent for the same reaction mixture, so less volume of the primary original EtOH would be used leading to reduced reaction costs, unlike the propyl ester which would give propanol instead; also ethyl group is a much better leaving group than the longer propyl group in this reaction, because it is easier to leave than the propyl one which is heavier than it, so a faster reaction with better yield would be obtained; and, in addition to these advantages of using it over the propyl gallate, the ethyl ester is mostly the first choice for this type of reactions).Using the easily synthesized ester of gallic acid, the ethyl ester lic acid  (the eth3.2nz (MW-assisted procedure), which is much more preferred than the conv. heating method , because it has/is:a.Decreased number of synthetic steps (just one direct step).b.Extremely decreased heating/reaction time (very rapid synthesis of just 120 sec).c.Much more benign and environmentally safe (green MW procedure).d.Extremely more economic and much cheaper .e.Highly efficient .f.Solventless .g.Self-catalyzed as there is no catalyst needed (used) at all.h.Much less energy consumption (extremely minute amount of 0.02667 KWhr is only needed).i.Higher overall yield of the hydrazide (98.2%) and with excellent purity.j.Decreased waste production .k.Simpler manipulation and work-up (automatic MW oven).l.).Higher selectivity of the reaction .n.Increased carbon and mass efficiency (100.0%).Designing a newer and greener method of synthesis of Galloyl hydrazide  operated at 2.45 GHz. TLC was used to monitor the progress of all reactions and to check the purity of products, it was carried out on TLC silica gel 60 F254 plates  as the stationary phase using pet. ether/EtOAc/abs. EtOH  mixture as the mobile phase (the eluting solvent system) for monitoring the esterification reaction for the synthesis of 1nz, while a mixture of 5% abs. MeOH in CH2Cl2 (dichloromethane or methylene chloride) was used as the mobile phase for monitoring the hydrazinolysis reaction for the synthesis of 2nz, and the chromatograms spots were visualized and observed under the used UV light at wavelengths of 254 (mainly) and 366 nm to detect the produced components. Evaporation and concentration was carried out by using rotavap under reduced pressure. A lyophilizer  was used for the lyophilizing purpose. Melting points (°C) of both the synthesized compounds were measured and recorded in open glass capillaries using Fisher-Johns melting point apparatus and were uncorrected. IR spectrum for 2nz was recorded on Nicolet™ iS™ 10 Mid-Infrared (ThermoFisher Scientific) FT-IR spectrometer (υ in cm−1) using KBr (potassium bromide) disk at the Central Laboratory  . 1H-NMR spectrum for 2nz was recorded on Varian Gemini-300 spectrometer (Mercury-300BB "NMR300") at about 300 MHz using TMS as an internal standard at the Microanalytical Center  and its chemical shifts values (δ) were given in ppm downfield from TMS at a temperature of 30 °C using DMSO-d6 as a solvent. 13C-NMR spectrum for compound 2nz was recorded also on Varian Gemini-300 spectrometer (Mercury-300BB "NMR300") at about 75 MHz using TMS as an internal standard at the Microanalytical Center  and its chemical shifts values (δ) were given in ppm downfield from TMS at a temperature of 30 °C using DMSO-d6 as a solvent. MS analysis for compound 2nz was performed on Shimadzu Qp-2010 Plus at 70 eV and results were represented by m/z (Rel. Int. in %) at the Microanalytical Center . Elemental analyses for 2nz were performed at the Microanalytical Center  in order to determine C, H, and N contents in % .All reactions were performed with commercially available reagents. All chemicals (reagents and solvents) were of analytical grade, purchased from commercial suppliers, and were used as received without further purification. MWI for MW reaction was carried out in an unmodified domestic MW oven (2SO4 (5 mL) was added. The resulted reaction mixture was refluxed with stirring at 90–100 °C for 15 hr. After reaction completion , the reaction mixture was cooled to R.T., then xss. EtOH was completely evaporated (distilled off) on a water bath, and the reaction mixture was allowed to cool to R.T. again. The residue remained was taken up in pure distilled H2O in a separatory funnel and the flask was rinsed with a few mL of pure distilled H2O which were also poured into the separatory funnel. The formed aqueous solution was extracted by shaking it vigorously with EtOAc for three times , the combined organic layers (extracts) were returned to a pure dry separatory funnel to be subsequently shaken with a strong solution of NaHCO3 (sodium bicarbonate) until all the free gallic acid was removed , then they were washed with distilled H2O and brine (a highly conc. NaCl (sodium chloride) solution in H2O). The organic layer was taken and dried over anhydrous Na2SO4 (sodium sulfate) during filtration (directly into the pure dry round-bottomed flask of the rotavap), then the filtrate was conc. by evaporation in rotavap under reduced pressure, left in air to be completely dry to give a crude white solid mass of the product 1nz which upon further purification by recrystallization from hexane, it gives the pure product 1nz  in abs. EtOH (500 mL) in a round-bottomed flask, conc. Hreported )) which reported ).1nz  and slight xss. 85% NH2NH2·H2O  was dissolved in abs. EtOH  in a round-bottomed flask, then the resulted reaction mixture was mixed gently and refluxed with stirring for 6 hr. The progress of the reaction was monitored on TLC plates. After reaction completion, the reaction mixture was cooled to R.T., then xss. EtOH was completely distilled off on a water bath, and the reaction mixture was allowed to cool to R.T. again. The reaction mixture was poured onto ice-cold water with stirring and the white mass or precipitate obtained was filtered, washed with distilled H2O several times, and dried under vacuum (or left in air to be completely dry) to give a crude pale white solid mass of the product 2nz which was recryst. twice from DEE and abs. MeOH  and further purified by HPLC analysis  to give about 16.574 g (yield = 90.0%) of the pure 2nz  with M.P. = 294–296 °C.A mixture of 2NH2·H2O  was taken in a 150-mL conical flask, the resulted paste of the reaction mixture was well mixed, then the flask was covered with aluminum foil and subjected to intermittent MWI at intervals of 30 sec for 2 min  at a power level of 800 W. The progress of the reaction was monitored on TLC plates till it was over after the fourth interval. After reaction completion, the reaction mixture was cooled to −20 °C and then it was lyophilized at −50 °C. The white product obtained was washed with distilled H2O several times and dried under vacuum (or left in air to be completely dry) to give a crude pale white solid mass of the product 2nz which was recryst. twice from DEE and abs. MeOH  and further purified by HPLC analysis  to give about 1.808 g (yield = 98.2%) of the pure 2nz  with M.P. = 294–296 °C.•HPLC analysis for separation of2nzand determination of its crude product purity:A mixture of gallic acid  and slight xss. 85% NH•○Column Type: SYMMETRY SHIELD RP C18 .○Column Temperature: Ambient.○Mobile Phase Constituents: A = MeOH; B = 0.1% TFA in H2O.○Diluent Constitution: ACN + TFA.○Injection Volume: 2 μL.○Flow Rate: 1 mL/min.○Run Time: 20 min.○Gradient Solvent System (Time (min)/A (%)): 0/0; 6/0; 10/70; 18/70; 19/0; 20/0.○Wavelengths for UV Detection: 220 & 272 nm.For these two purposes, the following HPLC analytical conditions were used , or directly synthesized from the corresponding gallic acid by MW-assisted method without passing through the esterification step. This scheme provides the best synthetic route for 2nz.The synthetic scheme of the present research  as minor impurities . For detection, separation, and determination  of these closely related impurities from the crude 2nz, preparative HPLC and RP techniques of chromatography were used  is about 99% (the major fraction isolated) and the percentage of related compounds (the four fractions of impurities isolated) in it is only about 1% .Results from 2nz is a pale white to buff fine powder with a M.P. of 294–296 °C. It is mainly freely soluble in DMSO (but its solubility in other organic solvents differs from one to another according to the strength of the solvent).The separated highly pure 2nz was subjected to full spectroscopic and elemental analyses for identification and confirmation of its structure  analyses data obtained for 2nz are shown, respectively, below in The separated highly pure The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The caldera collapse of Deception Island Volcano, Antarctica, was comparable in scale to some of the largest eruptions on Earth over the last several millennia. Despite its magnitude and potential for far-reaching environmental effects, the age of this event has never been established, with estimates ranging from the late Pleistocene to 3370 years before present. Here we analyse nearby lake sediments in which we identify a singular event produced by Deception Island’s caldera collapse that occurred 3980 ± 125 calibrated years before present. The erupted tephra record the distinct geochemical composition of ejecta from the caldera-forming eruption, whilst an extreme seismic episode is recorded by lake sediments immediately overlying the collapse tephra. The newly constrained caldera collapse is now the largest volcanic eruption confirmed in Antarctica during the Holocene. An examination of palaeorecords reveals evidence in marine and lacustrine sediments for contemporaneous seismicity around the Antarctic Peninsula; synchronous glaciochemical volcanic signatures also record the eruption in ice cores spread around Antarctica, reaching >4600 km from source. The widespread footprint suggests that this eruption would have had significant climatic and ecological effects across a vast area of the south polar region. Climate cooling caused by volcanic aerosols has caused crop failure, starvation, disease and civil unrest and has been linked to the rise and fall of civilisations3. Although major climate impacts are often associated with tropical volcanoes, high latitude eruptions may also have wide-ranging environmental consequences and trigger pronounced cooling at hemispheric scales6. Our understanding of the environmental and climatic impacts of many polar volcanic events, however, is limited by a lack of precision regarding the age of past eruptions as well as by the uncertain provenance of numerous tephra layers in ice and sediment cores8.Large volcanic eruptions can have pervasive climatic, ecological and economic effects12. It is located in the South Shetland Islands  of 30–60 km3 of magma, comparable in volume to the catastrophic 1815 Tambora eruption that caused global cooling and resulted in “the year without a summer”12. The age of this major Deception Island eruption has, however, never been firmly established. Estimates have placed the event between the late Pleistocene and 3370 years before present, with 10000 years ago being the most widely repeated age22. During the collapse, rapid volcano-tectonic subsidence along pre-existing, tectonically-influenced faults resulted in a modern 8–10 km-diameter caldera that is of similar dimensions to those of Santorini and Krakatau  in which three major tephra horizons – here referred to as T1, T2, and T3 and whose composition indicated a Deception Island provenance – were correlated based on geochemistry and physical propertiessee Fig. . Our int12 , we conclude that the magma ascent of T3 was rapid and without prolonged shallow storage and associated late-stage crystallisation prior to eruption. T3 is also the only tephra in Byers Peninsula lakes that contains juvenile clasts coarser than 2 mm in diameter, whose deposition here requires an eruption column tall enough to transport the coarse particles over 40 km against the prevailing wind direction. Their presence is thus indicative of a major eruptive event; the finer grain-size distributions 15 characteristic of all other lacustrine tephra layers are consistent with much smaller eruptions.Magmatic temperatures and pressures calculated by applying silica-activity geothermobarometry to volcanic glasses  closely reflect the basaltic-andesite source at 18O) isotopes in ejecta record the modification of the original magmatic signal by interaction with meteoric fluids and seawater. The measured isotopic values from Byers Peninsula provide evidence for the pre-collapse characteristics of the T3 magma as well as the evolution of Deception Island magmas during subsequent eruptions  is consistent with limited fractionation typical of such high-T magmas and the relatively small δ18O differences between magmatic and non-magmatic waters.Hydrogen (δD) and oxygen  and indicate shifts between long-term organic lacustrine deposition and values that more closely reflect the soils of nearby terrestrial environments25, providing parallel evidence of the catchment origin of the sediments in seismic event beds. Above the event deposits, geochemical characteristics returned to ‘typical’ lacustrine values.Whereas geochemical analyses record information about the eruptive process, the stratigraphic setting of lacustrine tephra provides insights into associated palaeoseismicity. Deposits up to 112 cm thick are immediately superposed upon T3 in each of the four studied Byers Peninsula lakes , during (Event) and following (Post-Event) the rapid sedimentation event. Diatom assemblages in Pre- and Post-Event sediments were dominated by taxa that are typical of lakes on the Byers Peninsula plateau, as expected with normal lacustrine deposition26. Interestingly, Post-Event assemblages 23 and, given its much greater size, it is reasonable to infer that seismic events of similar or superior magnitude were associated with the collapse of the Deception Island caldera. Seismic seiches, which have been observed in lakes located hundreds to thousands of kilometres from the epicentres of high magnitude earthquakes, are known to cause rapid post-seismic remobilisation of large volumes of terrestrial material from catchments to lake sediments, and are the likely mechanism for the deposition of the Byers Peninsula mass-wasting beds29.Biological, geochemical and radiochronological data thus showed significant differences between the mass-wasted beds and adjacent sediments, indicating that event beds contained rapidly deposited terrestrial material. We propose that this deposition was triggered by an extreme seismic episode associated with the Deception Island caldera collapse. Distinctive mass-wasting deposits have been generated in lake sediments due to the extreme seismic activity that has accompanied other caldera collapse eruptions; analogous, meter-thick event beds are found in lakes hundreds of kilometres from the 1960 magnitude 9.5 Chilean earthquake, the largest ever recorded14C dates based on aquatic moss samples and three thermoluminescence ages  were used to determine the age of the caldera collapse event based on calibrated ages immediately above the rapid sedimentation event and immediately below T3 , 3940 ± 30 cal y BP  and 3960 ± 50 cal y BP  .In Lake Cerro Negro, the radiocarbon age nearest to event sediments was aged 3130 ± 50 cal y BP, however this age was 8.6 cm above the event’s termination. No material suitable for radiocarbon dating was found below this horizon, either above or below T3. Bayesian age-depth modelling indicated a minimum age for the caldera collapse event of 3635 ± 440 cal y BP, however the Lake Cerro Negro model was based on five moss ages (vs. 17 in the Escondido model) and before 3130 cal y BP was based entirely on a single thermoluminescence age3 and volcanic explosivity index (VEI) of 612 make the Deception Island caldera-forming eruption the largest eruption documented in Antarctica during the Holocene, exceeding the Hudson Mountains Subglacial Volcano  by several orders of magnitude. Although an event of this magnitude would be expected to have widespread environmental consequences, until now, the lack of firm chronological constraint for the Deception Island caldera collapse had precluded an evaluation of the scope of its impacts. We therefore examined Antarctic ice cores, and lacustrine and marine sediments throughout the SSI and the Antarctic Peninsula, in search of contemporaneous volcanogenic layers and comparable event beds, revealing at least 18 such sites around Antarctica that preserve a record of the caldera collapse eruption ; Dome C (4004 ± 200 y BP); Dronning Maud Land (3995 ± 200 y BP); and Vostok (3942 ± 600 y BP)40, as well as Plateau Remote (3969 ± 100 y BP)41, whilst a tephra tentatively aged 3910 ± 200 y BP was deposited in a James Ross Island ice core18, although the correlations remain tenuous until they can be confirmed by geochemical composition data for these tephra. Tephra layers that we correlated to the Deception Island caldera collapse by a combination of chronology and/or geochemistry were also found in sediment cores and terrestrial sites situated across the SSI, James Ross Island and the Antarctic Peninsula, as well as in palaeoceanographic records from Bransfield Strait and the Scotia Sea . The sedimentary record of another two King George Island lakes record tephra and periods of accelerated sedimentation, combined with the deposition of terrestrial diatoms and reductions in organic matter content due to inputs of allochthonous material, that are consistent with our event chronology44. These singular, largely unexplained stratigraphic features mirror those on Livingston Island and underline the magnitude of both the Deception Island eruption and the seismic episode.Now firmly established as a Holocene event, the erupted volume of 30–60 km7. However, our study establishes a precise chronology for the Deception Island caldera collapse, a volcano-climatic event with a likely hemispheric impact. Future, high-resolution re-examination of ice and sediment records should help to clarify the influence of such volcanic forcing on Antarctic climate.Taken together, our study reveals conclusive, multidisciplinary evidence for a major volcanic episode with far-reaching effects at 3980 cal y BP. Ejecta from the caldera-forming eruption were deposited widely across the Antarctic, whilst a major coeval earthquake affected the Antarctic Peninsula region. In addition to resolving the long-lasting controversy regarding the timing of this major volcanic episode, these findings provide a valuable tie point for the calibration of ice and sediment core chronologies and will therefore contribute to an improved understanding of past Antarctic environmental change. Much remains to be elucidated about the effects of large eruptions on climate, and the data available at present are insufficient to provide a detailed characterisation of the climatic and ecological response to this and other profound but short-lived Antarctic eventsDuring Austral summer 2008  and 2012  lakes were cored until the underlying glacial till was reached using a 90 mm diameter UWITEC piston corer mounted on a tripod as well as a 60 mm diameter UWITEC gravity corer to recover the undisturbed sediment-water interface, using the lake ice covers as platforms. Limnopolar Lake sediments were extruded, cut in 60 cm sections, and sealed for shipping, whilst the sediments of the other four lakes were sealed for shipping directly in the plastic core liners. All cores were stored in a cold room at 4 °C until they were opened in the laboratory, when they were split longitudinally and subsamples were taken from the centres of cores.14C data31. All radiocarbon ages were calibrated using the SHCal13 curve for the Southern Hemisphere45 and are listed in calibrated years before present , with the present indicating the year 1950 AD. A full list of 14C samples, accession numbers and data as well as other details about the chronology can be found in the Supplementary Information.Stratigraphic ages were determined throughout cores in order to understand overall trends in sedimentation. In order to constrain event chronologies, particular attention was paid to samples immediately above and below tephra and the rapid, deposition event that was clearly distinguishable from the otherwise regular alternation of organic and mineral sedimentation. Fifty-four AMS radiocarbon measurements were performed, almost exclusively on large moss fragments, with samples from Limnopolar dated at the Poznań Radiocarbon Lab  and those from the other four lakes prepared at the Université Laval Laboratoire de Radiochronologie  and measured at the Keck Carbon Cycle Accelerator Mass Spectrometry Laboratory , respectively, along with three thermoluminescence ages measured at the base of cores in order to corroborate 15N) and C (δ13C). Samples were dried at 60 °C for 48 h and ground by hand using an agate mill. TOC, TN, δ13C and δ15N were determined using a Finnigan DELTAplus elemental analyser-continuous flow-isotope ratio mass spectrometer (EA-CF-IRMS) at the Centres Científics i Tecnològics of the Universitat de Barcelona (CCiTUB). The relative standard deviation (RSD) for TOC and TN measurements was 5% whilst the analytical precision for δ13C and δ15N was 0.2‰. Stable isotope ratios are reported in δ notation using parts per thousand units (‰) and are defined as δ = [(Rsample/Rstandard) − 1] × 1000.Samples were taken throughout two cores for sediment organic matter  and stable isotope analysis of N  geothermobarometer of Albarède46, which precisely matches with the formation conditions of basalt-andesite shield-volcanoes. Water variations gave negligible P-T fluctuations.Magmatic temperatures and pressures were calculated by applying silica-activity geothermobarometry to volcanic glasses. Because the water content was not constrained, we assigned an H2d, where d is the particle diameter in mm). Matrix glass major element compositions (as oxides) were measured on carbon-coated polished grain mounts of the 0–1φ, 1–2φ, and 2–3φ size fractions using the Cameca SX100 electron microprobe at the University of Bristol. Analyses were performed under operating conditions of 15 kV accelerating voltage and 4 nA beam current using a defocused beam (10 μm) to minimise sodium mobility. A combination of mineral and glass standards were used for calibration. Typical 1σ standard deviation of counts was between 0.5 and 1%. Analyses of secondary standards  were repeated each day to identify any instrumental drift, for which no corrections were necessary. Counting times were 10 s for Na, Si, Al, K and Ca, and 30 s for Mg, S, P, Ti, Fe and Mn, over a total analysis time of 60 s. A total of 40–50 glassy crystal-poor grains within the 125–250 µm (2–3φ) size fraction was analysed for each tephra layer, with each grain analysed once. Analyses that were contaminated by crystal phases were excluded. All compositions are shown normalised to anhydrous composition.The composition of tephra samples from T1, T2, and T3 was determined by electron microprobe analysis at the Centres Científics i Tecnològics Universitat de Barcelona and the University of Bristol. Byers Peninsula tephra samples (0.5–2 g) were cleaned in an ultrasonic bath for five minutes and dried overnight at 80 °C. Dried samples were then sieved into individual size fractions at phi (φ) intervals from −1φ to <3φ . Isotope ratios were measured on a VG-Isotech SIRA-II dual-inlet mass spectrometer at the Servicio de Isótopos Estables . The preparation systems included both a conventional and a laser fluorination line (δ18O determination in silicates), a D/H high-vacuum line for hydrogen extraction from hydrated minerals with a uranium-depleted reduction system, and a step-heating device for the fractional extraction and purification of fluids  from potential presence of glass inclusions. Both internal and international reference standards  were run to check accuracy and precision. Results are reported in δ18O notation relative to the Vienna Standard Mean Ocean Water (V-SMOW) standard, using a δ18O value of 9.6‰ for NBS-28 (quartz) for the mass spectrometer calibration. Long-term reproducibility for repeated determination of reference samples was better than ± 0.2‰ (1σ).Oxygen isotope analyses were performed on whole-rock powders by laser fluorination2 gas obtained by reduction over hot depleted-U of the water released by induction heating of samples, using a vacuum line, following the procedures described in Godfrey50 with modifications51. Samples were loaded into degassed platinum crucibles that were placed in quartz reaction tubes and heated under vacuum to 125 °C overnight to remove any adsorbed H2O. The yield of evolved gas was used to determine the amount of structural water contained in the sample. Results are reported in δD notation relative to the V-SMOW standard, using a δD = −66.7‰ for NBS-30 (biotite) for the mass spectrometer calibration. Long-term reproducibility for repeated determination of reference samples was better than ± 2‰ (1σ).D/H ratios were determined on a second SIRA-II mass spectrometer on H2O2) and samples were mounted in Naphrax following methods described in ref.27. Three hundred diatom valves from each sample were identified and enumerated on random transects at 1000x magnification under oil immersion using a Zeiss Axio Imager A1 microscope equipped with differential interference contrast optics. See Supplementary information for further details about diatom identification and statistical analyses.Samples for diatom analysis were taken at regular intervals from Lake Limnopolar (core LIM08). Organic matter was oxidised by treatment with hydrogen peroxide (33% HSupplementary InformationSupplementary Dataset 1


Beetroot juice (BJ) is classified as a high-level supplement for improving sports performance. There is some controversy over the benefits of BJ supplementation for endurance exercise performance, especially when referring to well-trained athletes. This study examines the effects of acute BJ supplementation on cardioventilatory responses, exercise economy/efficiency, slow component of oxygen uptake, time trial performance, blood lactate, energy consumption, and carbohydrate and fat oxidation.3−) or placebo (PL). Three hours after taking the supplement, participants completed an endurance test on a cycle ergometer at a constant work rate (W) corresponding to first ventilatory threshold (VT1) (30 min) and second ventilatory threshold (VT2) time trial (~ 15 min).Twelve well-trained, male triathletes (aged 21–47 yr) were assigned in a randomized, double-blind, crossover design to receive 70 ml of BJ  was observed on any of the cardioventilatory variables, efficiency/economy, VT2 time trial, energy expenditure, carbohydrate oxidation and fat oxidation (p > 0.05).Maximal oxygen uptake was 54.78 ± 3.13 mL·minOur findings do not support an improvement in the variables examined in response to acute BJ supplementation. Probably, higher doses are needed for improving time trial performance in male triathletes during a cycle ergometer test conducted at a load intensity equivalent to the first and second ventilatory threshold. Here NO3− is reduced to nitrite (NO2−) by bacteria in the mouth. In turn, NO2− may be further reduced in the stomach and muscle to nitric oxide (NO)  r. (2013)  also detke of BJ  (0.5 L f3−) caused a greater reduction in systolic blood pressure and mean arterial pressure at moderate exercise intensity than lower doses (~ 4.2 mmol of NO3−) in healthy adults [2 steady-state of moderate exercise intensity was reduced significantly after ingestion of 16.8 mmol of NO3−, tended to be lower after intake of 8.4 mmol NO3−, and was unaffected by 4.2 mmol of NO3− [3− per 70 mL) before and during prolonged moderate-intensity exercise might be necessary to attenuate the progressive rise in VO2 and reduce muscle glycogen depletion [3− supplementation could increase sport performance in highly-trained athletes [Previous studies have demonstrated that higher BJ supplementation dose . Subjects with a lower fitness level may be more susceptible to BJ effects regardless of whether the BJ supplementation is acute or chronic. In effect, the literature suggests some interaction between training state and the ergogenic effects of NO3− supplements [− 1 indicating a predominantly aerobic state. During this metabolic stage, such intensity of exercise may be maintained over a long period of time without marked changes in blood lactate concentrations [2 uptake due to a decrease in the aerobic energy required or in the muscular energy used in moderate exercise efforts.The type and/or mode of supplementation or the test used do not seem to play as important a role as cardiovascular fitness level when assessing VOplements , though plements . It is kplements . Thus, aplements . Lactatetrations , 58. Hentrations . We susp2, attributed to reduced ATP resynthesis through oxidative phosphorylation, was not offset by elevated glycolytic ATP provision [3− is produced reflected by increased muscle oxygenation indices and total hemoglobin levels during moderate exercise. The increased blood volume observed in the vastus lateralis muscle after BJ intake is presumably a consequence of improved muscular vasodilation resulting from the increased production of NO from NO2−.The reduction in VOrovision , as indirovision , in lessCompared to VT1, physiological and efficiency (intensity effect) changes were observed here in VT2. Studies have shown that BJ enhances high-intensity endurance exercise performance in moderately-trained subjects , 59 whil3−) improves the time-to-task failure when is compared with a 4.2 mmol dose in young healthy adults [3− doses  [3−) raises the plasma NO2− levels to a greater extent than ~ 8.4 mmol of NO3−, however, no added performance gains are produced [Currently, the scientific literature lacks data on the effects of nitrates on high-intensity exercise  and the y adults . Effectiof NO3−) . A normaproduced . Prolongproduced , 25 and 3− from other sources could lead to adverse effects on health [3− consumed in the form of vegetables have been shown to improve running performance in healthy adults [Furthermore, whole vegetables have been demonstrated to provide important health benefits whereas NOn health . Becausey adults , it is t2, leading to the use of different substrates. Carbohydrates are more efficient as energy substrates than fatty acids. In other words, if more carbohydrates are used as substrate this gives rise to lower oxygen absorption at a given work velocity [2 kinetics (slow component). This change is similar to that observed after an initial bout of high-intensity exercise, giving rise to increased muscle O2 release, increased oxidative metabolic enzyme activity, carbon substrate availability, and abnormal motor unit recruitment patterns [2 over time before and during prolonged moderate-intensity exercise although did not enhance subsequent time trial performance. Interestingly, it was observed that this decrease in VO2 had no impact on time trial performance, which could indicate that supplementation with BJ does not sufficiently reduce muscle glycogen depletion at moderate intensity for decreasing fatigue during cycling time-trial. More research is needed to analyze the BJ supplementation effect on VO2 kinetics during endurance tests over two hours.As a final remark, changes in exercise intensity from VT1 to VT2 involve variations in VOvelocity . Calculavelocity . With thpatterns , 66. It patterns , 67. In patterns  demonstrThere are some limitations in this study which should be considered. Previous research indicates that the plasma nitrite of the participants should increase to show an ergogenic effect, however, nitrite and nitrate concentrations in plasma were not measured in our study. It seems that doses close to or greater than 8.4 mmol are more adequate to determine the positive effects of BJ supplementation on endurance performance in well-trained triathletes .The small sample size in this study should be taken into account when drawing conclusions from the data. Minimal changes in endurance performance are usually observed in well-trained triathletes, therefore, large sample sizes should be required to detect significant changes produced by BJ supplementation on cardioventilatory performance.Although there are several studies that have carried out a similar washout period, it is possible that the washout period established in our study was not sufficient, which could influence the final results.Our findings do not support an improvement in the variables examined here produced in response to acute BJ supplementation. We have yet to elucidate the possible ergogenic effects of BJ in highly trained athletes. However, the slight (not significant) modifications observed in performance variables such as test duration or maintaining work intensity at a given load in several studies prompts numerous questions as the mechanical and physiological mechanisms analyzed so far do not support these improvements and remain poorly understood.Our outcomes suggest a need to analyze individual positive responses to this form of supplementation in well-trained athletes.