Due to the complexity of anatomical relationship between superior mesenteric artery (SMA) and left colic artery (LCA), there is no unified anatomical concept of “Riolan’s arch.” There is no consensus as to tie off the inferior mesenteric artery (IMA) at its origin or just below the origin of the left colic artery during radical surgery of sigmoid colon and rectal cancers. The aim of the study is to investigate the anatomy of shortcut anastomotic branches (adjacent branches) of SMA at splenic flexure and to explore how the shortcut pathway (Riolan’s arch) was formed, as the compensation of anastomotic branches between MCA and LCA under pathological conditions and the reconstruction and the mechanism of pathological Riolan’s arch after high ligation of the inferior mesenteric artery.Between January 2018 and May 2020, patients with colorectal cancer who underwent CTA before surgery were enrolled in the study. The anatomy of shortcut anastomotic branch of SMA and LCA was investigated by volume rendering technique (VR) and maximum-intensity projection (MIP). GE’s small vessel extraction technology (selected VR) was used to directly display these shortcut anastomotic branches on a map and to establish their three-dimensional anatomical classification. Then, we used the axonometric drawing to make the model more exact. Next, combining with some cases of pathological Riolan’s arch and basing on hydrodynamic principle, we speculate the mechanism of collateral circulation. Finally, based on the retrospective study of high ligation cases and combined principles of fluid mechanics, we show how these shortcut anastomotic branches evolved into Riolan’s arch.We report the classification of the ascending branch of LCA (which approaches the splenic flexure) and the left branch of MCA, display these shortcut anastomotic branches on a map, and establish their three-dimensional anatomical classification. We found that Riolan’s arch is a shortcut pathway for the compensation of anastomotic branches, between MCA and LCA under pathological conditions, and that the formation mechanism of shortcut path accords with the principle of hydrodynamics.Our results show the mechanism of pathological Riolan’s arch formation and provide new anatomic thinking for the battle between high and low ligation of IMA in colorectal cancer surgery. In recent years, laparoscopic surgery has been widely used in the management of colorectal cancer. However, no clear consensus has been established, as to whether it is most appropriate to tie off the inferior mesenteric artery (IMA) at its origin (high ligation) or just below the origin of the left colic artery (LCA) (low ligation) during radical surgery of sigmoid colon and rectal cancers .Many surgeons prefer to perform high ligation of the inferior mesenteric artery (IMA) during rectal cancer surgery, in order to accomplish a total lymphadenectomy and better mesenteric mobilization . Other sThese arterial patterns were established previously by means of postmortem angiography or digital subtraction angiography (DSA) . HoweverRiolan’s arch’s existence has been debated for years. It is believed to be an important anastomotic artery between the left branch of the middle colic artery (MCA) from the SMA and the ascending LCA branch from the IMA . It is bThrough computed tomography angiography (CTA) of the IMA, we observed some Riolan’s arch under pathological conditions and plan to elucidate the formation of Riolan’s arch under physiological conditions. We reviewed the literature surrounding Griffiths’ research , whereinBecause the anastomosis of the splenic flexure is formed in three-dimensional space, we can only investigate and display this relationship accurately using a three-dimensional model. All previous research methods (such as DSA) consist of two-dimensional modeling, and the branch construct of the SMA and IMA could not be investigated at the same time. In addition, the branches at the splenic flexure are grades 2 and 3 branches of the main artery supply, and only three-dimensional small-vessel imaging technology can track this clearly.Therefore, we sought to investigate the small vessels through adjusting the shaft position angle of maximum-intensity projection (MIP) and selective VR , then chose a three-dimensional perspective angle to demonstrate their relationship. What is more, previous studies did not address the principle of pathological Riolan’s arch formation. Through the study, we found that the formation mechanism of blood flow pathway conforms to the principle of fluid mechanics.In this report, we try to investigate the existence form of the “physiological Riolan’s arch” by using VR and adjusted-angle MIP. We summarize six types of shortcut anastomotic branches. Retrospective study of 57 cases of the actual blood flow path after high ligation of IMA also supports the hypothesis that the shortcut pathway was formed by the compensation of the shortcut anastomotic branches, following the principle of fluid mechanics, and the two-way blood supply to the far side together with the limbic arch. All of them followed the principle of fluid mechanics.Finally, we use small blood vessel extraction technology to display the three-dimensional anatomical classification of the shortcut anastomotic branches and show the whole blood flow path after high ligation of the inferior mesenteric artery. These studies provide an anatomical thinking for part settling dispute between high ligation and low ligation of the IMA in rectal surgery.In this study, we reviewed a total of 300 patients with colorectal cancer who underwent routine abdominal and pelvic enhancement CT before surgery at Jinan Central Hospital from January 2018 to March 2020. The scan data of arterial phase were reconstructed into 1.0 mm thin-layer image data. There were 57 cases of routine reexamination after IMA high ligation from March 2019 to March 2020 that were used for further analysis. The 1.0-mm CT arterial phase thin-slice image data were used for CTA reconstruction and analysis. Because this study was both of basic research in cross discipline and retrospective study, information was provided to the participants in place of informed consent, and they were given the right to opt out. This study was approved by the Ethics Committee of Jinan Central Hospital.® 2). The selected VR image  was evaluated using AW VolumeShare 5, GE Medical Systems.The 64-slice multidetector-row computed tomography (MDCT) scanner generates 0.75 mm image slices that can be reconstructed into 1.0 mm image data. Image processing analysis was performed using a 3D volume rendering (VR) technique with the Extended Brilliance Workspace (EBW) software. The MIP images were evaluated using VITAL Image Management System  was used to directly display these shortcut anastomotic branches on a map and to establish their three-dimensional anatomical classification. We then used the axonometric drawing to make the model more exact. During the imaging process, we found a specific perspective angle that can better display the three-dimensional relationship of each anastomotic branch  and madeFinally, we investigated the blood flow pathways of 57 high ligation cases and found that the collateral circulation (compensatory pathway) evolved from the shortcut anastomotic branches .Most MIP images and almost all GE selective VR images can show the descending colon and the limbic arch. Through adjusting the angle, we can directly show that the ascending branch of LCA and left branch of MCA are anastomosed in the splenic flexure, which also proves that we have been able to image all the three to four branches of each blood vessel. The ascending branch of the LCA travels to the left colic angle in order to constitute the left pillar of the marginal artery.As reviewed by Due to the limitation of techniques and the diverse locations of the transverse colon’s left mesocolon, the main splenic artery of the transverse colon has not been clearly reported.Here we classified the left branching pattern of the MCA and the AMCA  into thrIn order to better display the shortcut anastomotic branches, we used GE’s small-vessel extraction technology to display the classification and distribution of shortcut branches intuitively . We sougWe investigated the blood flow pathways of 57 high ligation cases and found that the collateral circulation (compensatory pathway) evolved from the shortcut anastomotic branches: The left branch of MCA and anastomotic site dilated and blood flow increased, anastomotic branch of ascending branch of LCA dilated, and reverse blood flow occurred. The formation mechanism follows the fluid mechanics. Therefore, the second blood supply to the distal intestine is combined with the marginal arch. Of note, the occurrence rate of the accessory colon middle artery is higher .For gastrointestinal surgeons, an operation’s success hinges on a full preoperative understanding of the blood supply of the splenic flexure. The splenic flexure is the junction of the transverse and descending colon; unfortunately, its blood supply has rarely been investigated. The number and exact location of the colic arterial arches that connect the superior and inferior mesenteric arteries at the splenic flexure is largely unknown . This coPrevious studies yielded contradictory results mainly due to limitations in methodology. Analysis and interpretation of two-dimensional images cause some misunderstandings that can only be corrected with three-dimensional modeling. In recent years, multidetector-row computed tomography (MDTC) and three-dimensional computed tomography angiography (3D-CTA) have become frequently used for the evaluation of vascular anatomy.In this study, we evaluated the vascular anatomy of the splenic flexure in a large number of patients, using 3D-CTA to get a three-dimensional perspective. The selected VR technology enables us to show small and curved blood vessels and provides evidence of shortcut anastomosis between the MCA and the LCA. As shown in Our results show how the left branch of the MCA distributes to the left 1/3 of the mesocolon. This is very important, because in rectal cancer surgery, this left branch serves as a compensatory pathway for upstream blood supply.The AMCA is a branch artery originating directly from the SMA and is more proximal than the first jejunal artery. It has recently been identified preoperatively, because of recent advances in imaging technologies. The role of the AMCA in colon surgery is very important, but there is little known about its branching pattern.In the past, the anatomical structure of the MCA was mainly obtained by DSA, which demonstrates the projection of blood vessels but loses information by simplifying three-dimensional space into a two-dimensional model. The depth of the angiography catheter entering the SMA may affect the appearance of the branch vessels that originate higher than the SMA. Therefore, DSA cannot provide the exact location of blood vessels. Without a precise reference system, it is difficult to determine a blood supply map of the upper SMA branches, and some vessels could not be named accurately.Our method accurately identified the AMCA and demonstrated that in some patient the left branch point of the MCA originated higher and separately from different origins of the SMA (not from a common trunk) , and there was no “potential blood vessel.” It has been previously established that the blood supply of the colon is divided into different sections . When these secondary and tertiary branch vessels extend into the marginal arch, they are also the main arteries of the corresponding colon under physiological conditions.In this experiment, we sought to explain why the collateral circulation, Riolan’s arch, is formed by compensation of shortcut anastomotic branches. This can be explained by the original principle of fluid mechanics . As showWe took advantage of selective VR, which can be used to track the blood flow of axial sections. It is similar to tracking the blood flow of an angiography, so as to find the marginal anastomosis. Selective VR can accomplish simultaneous imaging of the SMA and the IMA and can rotate at any angle during the mapping process. Thus, it is able to accurately confirm the starting point and arrival position of each vessel. We were able to conclude that a high proportion of AMCA and L1 and L2 correlates with classic central Riolan’s arch.The shortcut anastomotic branches all connect at the marginal arch, when the superior branch is occluded or ligated. As long as the exit point of the anastomotic branch is not broken, the anastomotic branch compensates by expanding and the shortcut branch shows retrograde blood flow. The inside path is a shortcut relative to the marginal arch, which is the so-called Riolan’s arch.The efficiency of the shortcut path is stronger than that of the marginal arch. Before the shortcut path reaches the marginal arch, there is no branch and minimal change in blood flow. The marginal arch has many small branches, and the total vessel diameter keeps increasing, resulting in a continuous decrease in blood flow. After a certain distance, the blood flow is very slow and the blood supply efficiency is low.These six shortcut routes can provide an anatomical basis for ending the controversy between high and low ligations. In order to ensure the blood supply of the proximal colon of the anastomotic opening, high or low ligation should each be considered based on the circumstances. When there is only L1 or no right anastomotic branch, the best method is to perform a low ligation.The shortcut branch becomes an important vascular pathway under certain circumstances. Our discovery is helpful to international surgeons. Oriental doctors tend to maintain a longer sigmoid colon in the operation, which means there should be twice as many channels to supply blood to the distal colon, instead of relying on the marginal arch alone.Western doctors tend to keep only the descending colon, but they need to disconnect the inferior mesenteric vein at a high level in order to free the splenic curve and extend the proximal colon of the anastomosis. Therefore, they need to pay attention to the relationship between the inferior mesenteric vein and the shortcut pathway. For example, in the case of the middle accessory colon artery, they should disconnect the lower position of the inferior mesenteric vein.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Human Ethics Committee of Jinan Central Hospital Affiliated to Shandong University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.GL designed the study. YW and WS analyzed the data and drafted the manuscript. AO participated in the design of the study and helped draft the manuscript. LW performed the statistical analysis. YS helped interpret the data. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


New methodologies and technologies developed in the last few decades have highlighted the precise spatial organization of the genome into the cell nucleus, with chromatin architecture playing a central role in controlling several genome functions. Genes are expressed in a well-defined way and at a well-defined time during cell differentiation, and alterations in genome organization can lead to genetic diseases, such as cancers. Here we review how the genome is organized in the cell nucleus and the evidence of genome misorganization leading to cancer diseases.The last decade has seen significant progress in understanding how the genome is organized spatially within interphase nuclei. Recent analyses have confirmed earlier molecular cytogenetic studies on chromosome positioning within interphase nuclei and provided new information about the topologically associated domains (TADs). Examining the nuances of how genomes are organized within interphase nuclei will provide information fundamental to understanding gene regulation and expression in health and disease. Indeed, the radial spatial positioning of individual gene loci within nuclei has been associated with up- and down-regulation of specific genes, and disruption of normal genome organization within nuclei will result in compromised cellular health. In cancer cells, where reorganization of the nuclear architecture may occur in the presence of chromosomal rearrangements such as translocations, inversions, or deletions, gene repositioning can change their expression. To date, very few studies have focused on radial gene positioning and the correlation to gene expression in cancers. Further investigations would improve our understanding of the biological mechanisms at the basis of cancer and, in particular, in leukemia initiation and progression, especially in those cases where the molecular consequences of chromosomal rearrangements are still unclear. In this review, we summarize the main milestones in the field of genome organization in the nucleus and the alterations to this organization that can lead to cancer diseases. In all organisms, including bacteria , the genIn the last two decades, chromosomes have been intensely studied inside the cell nuclei to understand if they occupy specific positions to correctly drive the expression of genes they contain. Radial chromosome positioning is evident in all animal cells, as demonstrated in human ,4,5,6, mHere, we review the recent advances in knowledge about gene and genome organization in the cell nucleus. We focus on the organization of genes in the nuclear chromosome territories and variation in the chromatin structure as a consequence of chromosomal rearrangements leading to repositioning in the nucleus of specific genes. This repositioning can lead to an ectopic alteration in gene expression and replication timing of the involved genomic sequences, two features related to cancer diseases.Using DNA probes to “paint” individual chromosomes with the fluorescence in situ hybridization (FISH) technique, it has been possible to visualize whole chromosome territories in the interphase nuclei allowing precise analyses to understand the organization of each chromosome in the nucleus . Indeed,Chromosomes 18 and 19 occupy different intranuclear positions  because they are small and relatively homogeneous in their nucleotide composition and gene density, as previously shown ,34,35,36Conversely, all the GC-poor bands, along with the centromeric sequences, are located at the nuclear periphery or close to the nucleolar periphery . Indeed,These results demonstrate that chromosome territories are endowed with a GC-level gradient increasing from the periphery to the inner nuclear compartment . This reMore recent techniques were developed to study the organization of the genome in 3D within the cell nucleus. These are derived from the chromosome conformation capture (3C) method , namely The analysis of the human genome’s spatial organization through Hi-C confirmed what was initially found by FISH, namely that gene-rich chromosomal regions are spatially linked, as demonstrated by the high number of contacts observed . InteresThe Hi-C data continued to enhance our knowledge on chromatin organization in the cell nuclei, revealing the presence of Topologically Associated Domains (TADs), a structural organization of the chromatin endowed by a loop structure where the zinc finger CCCTC-binding factor (CTCF) sites play an essential role ,43. CTCFThese biochemical–molecular approaches have disclosed the presence in interphase of two genomic compartments A and B , organizSince genes are part of chromosomes, it also matters where those genes sit in relation to the body of their home interphase chromosome territory, i.e., intrachromosomal organization. Indeed, gene loci can be located deep within chromosome territories, more towards the surface of the chromosome territories, or even at some distance from the main body of the chromosome territory, out on a chromatin loop. There is evidence that even within interphase chromosomes, there is the spatial organization of gene-rich and gene-poor areas, which has been revealed by FISH, with inactive genes more likely to be located in the interior of chromosome territories ,52,53,54MYC and CCND1 in cancer cells located at the edges of their chromosome territories. The same genes, when silenced, were located deep within the interior of the chromosome territories. Experimentally silencing the over-expressed genes leads to alterations in the genes’ locations, which become more internal in the chromosomal territories . Due toDue to a chromosomal rearrangement , genes located close to the breakpoints might alter their position in the nucleus, and this occurs when two chromosomal bands characterized by a very different GC-level, and normally located in different nuclear compartments, find themselves close together in the nucleus, with one of them located in a noncanonical nuclear compartment. Indeed, this is usually the case when two chromosomal bands endowed with opposing features (GC-rich/gene-rich vs. GC-poor/gene-poor) are involved in the rearrangement resulting in close contiguity of sequences normally positioned very far from each other. Depending on the cell type affected by the rearrangement, this may imply that a transcriptionally inactive gene could be ectopically activated by being positioned in a transcriptionally active nuclear compartment. Conversely, a gene could be inactivated if it is repositioned from the internal part of the nucleus to the nuclear periphery. In the case rearrangement happens between two compositionally similar genomic regions, the affected chromosomal bands retain the same radial position in the nucleus . This tyEWSR1/FLI1 and ABL1/BCR were observed in an intermediate nuclear position when compared to the wild type EWSR1 and FLI1 genes and that this location seems to depend to the chimeric chromosomes obtained by the translocation [BCR and ABL and the derivatives resulting after the formation of the Philadelphia chromosome [MLL gene and five of its translocation partners showed that the resulting fusion genes changed their nuclear location according to the gene density of the 2 Mb window of the regions involved [HOX genes has provided evidence that nuclear reorganization does not depend on gene expression but can operate upstream to it [On the Ewing sarcoma cells carrying a cancer-related translocation t, it has been noted that the fusion genes location . Howeverromosome . Some auinvolved . Meaburninvolved  have demam to it .MNX1 gene  was related to its altered radial nuclear position as a consequence of the translocation of the 7q36 band (containing the MNX1 gene) on the telomeric end of the chromosome 12 [MNX1 gene more internally in the nucleus than the wild type allele. The ectopic activation and overexpression of MNX1 were also described in relation to other chromosomal rearrangements observed on cell lines GDM1 [MNX1 gene was related to the type of breakpoints in the deleted chromosome 7, namely when the deletion joined the region with the MNX1 allele to a chromosomal band with high GC-level, thus finding a transcriptionally active nuclear compartment [Another interesting case concerns the t translocation frequently observed on acute myeloid leukemia cells of pediatric patients, where the overexpression of the osome 12 . This trnes GDM1  and K562nes GDM1 , as wellnes GDM1 . In thispartment   translocation, further to the alteration in gene expression related to the altered spatial organization of the genes located close to the der (11) breakpoint, highlighted a different position in the nuclei and an altered gene expression profile also for genes located on other chromosomes not directly involved in the translocation . This inCancer is a genetic disease related to the altered expression of genes generally involved in the cell cycle progression. A growing amount of evidence has shown that gene expression alteration observed in cancer cells could be related to a different position of genes in the cell nuclei and consequently to a chromosomal rearrangement. The nuclear position effect reflects the chromatin organization in the cell nuclei. Many different experimental procedures have demonstrated the presence of two very different nuclear compartments, one endowed with a high GC level, a high gene density, a very early replication during the S-phase of the cell cycle, and a position in the inner part of the nucleus. Another compartment, generally located more peripherally in the nucleus, is endowed with opposite properties. Rearrangements between loci belonging to these two different compartments influence the repositioning of genes in a compartment with different environmental properties, determining the ectopic activation or inactivation of the relocated genes. To date, only a few studies have focused on the nuclear positioning of genes in cancer, and further investigations should be carried out to better understand the real impact of this mechanism in the oncogenic process initiation and progression.


The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . We use the weekly mutation spectrum of the genomes to depict each country and compare their similarities within each 2-week window from Feb 24, 2020 to Aug 16, 2021. The similarities are calculated based on both the Cosine similarity (with windows flattened into row vectors) and the Frobenius similarity .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The organization of biomolecules and bioassemblies is highly governed by the nature and extent of their interactions with water. These interactions are of high intricacy and a broad range of methods based on various principles have been introduced to characterize them. As these methods view the hydration phenomena differently , a detailed insight in each particular technique is to promote the overall understanding of the stunning “hydration world.” In this prospective mini-review we therefore critically examine time-dependent fluorescence shift (TDFS)—an experimental method with a high potential for studying the hydration in the biological systems. We demonstrate that TDFS is very useful especially for phospholipid bilayers for mapping the interfacial region formed by the hydrated lipid headgroups. TDFS, when properly applied, reports on the degree of hydration and mobility of the hydrated phospholipid segments in the close vicinity of the fluorophore embedded in the bilayer. Here, the interpretation of the recorded TDFS parameters are thoroughly discussed, also in the context of the findings obtained by other experimental techniques addressing the hydration phenomena . The differences in the interpretations of TDFS outputs between phospholipid biomembranes and proteins are also addressed. Additionally, prerequisites for the successful TDFS application are presented . Finally, the effects of ions and oxidized phospholipids on the bilayer organization and headgroup packing viewed from TDFS perspective are presented as application examples. The role of the hydration in maintaining the biological function of biomolecules and biomolecular aggregates is unquestionable . Water mIn the case of proteins, water interacts with a heterogeneous partner . Water mvia hydrophobic effect but also serve as plasticising spacer within the lipid headgroups balancing the free volume in the hydrocarbon chains, which enables the formation of liquid phases. Moreover, hydration enhances the configurational space for additional water and lipid populations which assist for instance the peptide binding (Lipid bilayers (core of biomembranes) represent a biomolecular self-assembly essential for the existence of living organisms. Lipid bilayers also show a complex hydration patterns , even wh binding .The closer look on the water distribution along the bilayer normal reveals that majority of water molecules can be found in the interfacial headgroup region . Three bIn order to track the degree and nature of the hydration of biomolecules and bioassemblies, manifold of the experimental approaches have been introduced. Each of these methods is unique from the perspective of the length-scale and time-scale of the followed parameters . InevitaThe main topic of this contribution is Time-Dependent Fluorescence Shift (TDFS) . TDFS d. Quite aBelow, we start with a short overview of the current knowledge about the hydration of biological systems. Further, we shortly describe the theoretical background of TDFS method. Then, we cover the interpretation of the relaxation probed in lipid membranes. We address the applicability of the method and clearly point out its limitations. Description of the TDFS instrumentation as well as the choice of the fluorescent probes is also given.Before focusing on the principles and applications of the TDFS technique for the investigation of lipid bilayers, it is helpful to discuss the current knowledge on the hydration shell of biomolecules obtained by label free techniques. It is essential to acknowledge that within the last decade advances in molecular dynamics simulations as well as in ultrafast vibrational spectroscopy led to a much clearer picture about how one has to understand the water shell of lipid bilayer. The conclusions drawn were summarized in a seminal review by Despite that relatively low number of interfacial water molecules, it is undoubtedly accepted that the structure and function of the biological membranes are strongly affected by the dynamic properties of the hydration water layer. Indeed, it plays a pivotal role in transport and signalling functions, mediating membrane-membrane interactions, as well as the ones with ions, DNA or proteins . This isThe hydration shell in lipid bilayers is highly oriented, due to the tendency of the water molecules to reach the lowest number of hydrogen bonding configuration as well as electric field created by the oppositely charged choline and phosphate groups . ATR-FTIE. Coli, two distinct regions could be distinguished in the relaxation time profile obtained by 2H-NMR. A minimum of 11–16 molecules was found to be part of the first hydration shell, corresponding to a correlation time of 90 ms. Furthermore, an exchange with water molecules not strictly belonging to the first layer has been observed   . NMR and–10−11 s , but thes  being the core of TDFS technique . The TDF solvent . Moreove solvent . This isThe molecules of water hydrating lipid bilayer are the major source of polarity sensed by TDFS. The mobility of those water molecules is, however, strongly restricted. Compared to bulk aqueous solutions the timescale of relaxation probed by TDFS slows down from hundreds of femtoseconds to nanoseconds on the distance of 1–2 nm . Here weThe polarity sensed by TDFS in lipid bilayer is usually determined by the average number of water molecules within the microenvironments of the fluorescent probes. This is the foundation of TDFS sensitivity to membrane hydration. Not only TDFS provides a quantitative measure of membrane hydration , but it also measures it locally (at certain depth within the membrane). This increases the specificity of the obtained information about membrane hydration and allows assessment of the complete hydration profiles of lipid membranes , do not provide sufficient rearrangement during the lifetime of a probe (d) phase, but also to the so-called liquid ordered membranes (Lo). In Lo phase the mobility of the molecules is limited by the presence of sterol that fills the gaps between the phospholipids. Nonetheless, for some cases with high content of cholesterol or at low temperatures, limitations of the technique can be also reached in Lo membranes  of TRES and the time-zero spectrum estimation allow for identification of the number of problems and for evaluation of the completeness of the recorded relaxation process. The basics and applicability of these tools was described in detail in our previous work .The contribution of lipid molecules to the polarity measured by TDFS is usually limited when compared to that of water molecules. Nevertheless, possible contribution of the system components (including lipids) to the measured polarity, as well as possibility of their specific interactions with fluorescent probe should be evaluated in each case. After the issues related to probe relocation (discussed in section “Choice of the suitable dyes for membrane and protein studies”), the specific influence of the studied molecules on the fluorescent probe are the major source of complicacy in TDFS analysis.The polarity probes utilized in TDFS can be useful even when time-resolved instrumentation is not available. The most common steady-state methods that utilize fluorescent polarity probes and can be applied to characterize lipid membranes include red-edge excitation shift (REES)  and geneAs illustrated above, TDFS possesses a great potential to map hydration and packing effects in monophasic model lipid membranes providing interpretable outputs. The homogeneous membranes present an anisotropic system along the membrane normal, yet staying isotropic in the lateral directions. Although the lipid and probe fluctuations along the z-axis cannot be excluded , the aveMuc probes. These experiments revealed that the specific contact sites between the probe and hydrated amino-acid residues  has to be regarded for the quantitative TDFS application. To identify these dye-specific effects the assistance of MD simulations is of high importance.Even more entangled situation in comparison to the complex lipid membranes arises in proteins, which form an anisotropic entity with high chemical heterogeniety in all three dimensions. This results in an extremely complex TDFS response, which was illustrated for example for the dehalogenase enzymes (HLDs). The tunnel mouth region of the selected HLDs was labeled with a set of various fluorophores, based on Coumarin-120 and Prodan-like probes (denoted as Muc) . In spit1. In order to introduce sufficient perturbation by the dye photoexcitation into the system, the probe must show a significant difference between the excited and ground state dipole moments in magnitude and/or direction. This difference is usually translated into the large Stokes shift observed for polar solvents.2. The ideal TDFS dye is to possess a linear solvatochromic behavior, i.e., no specific interactions should interfere with the polarity dependence.3. The knowledge of the location of the probe is clearly of utmost importance for the correct interpretation of data. A probe should have as defined location in the system of interest as possible, be it a lipid bilayer  or a protein .The proper choice of the fluorescent dye is essential for the successful TDFS implementation. The ideal probe should fulfill three basic requirements:Coumarin-153 is one of the best examples of fluorescent dyes appropriate for TDFS studies, especially for the neat solvents  and micePopular strategy for designing the TDFS probes is the insertion of an electron donating and electron withdrawing groups at a certain distance. This promotes intramolecular charge transfer upon excitation, thus inducing large changes in the dipole moment. The family of amino-substituted naphthalene probes designed in this way have been proven to be highly sensitive to polarity and have been common choices for TDFS studies in proteins and biomembranes. For the latter system, Prodan  and its Aminonaphtalene “Prodan-like” dyes have also been utilized for site-specific labelling of proteins for TDFS studies . For exaThe importance of the choice for the proper TDFS probe can be also strengthened by the inspection of the dyes which are unsuitable for TDFS. For example, NBD dyes display a small change of dipole moment upon electronic excitation of ∽2 D . MoreoveAs mentioned above, TDFS response can cover a huge time-span ranging from hundreds of femtoseconds up to microseconds. In addition to the fluorescence lifetime of the probe, which strictly sets the experimental time window, the choice of the instrumentation has to be also considered.For mapping the ultrafast relaxation processes (i.e. shorter than ∼20 ps), the techniques employing the ultrafast femtosecond lasers combined with the up conversion approach , Streak Since the relaxation in biosystems are often dominated by the slow nanosecond components the ultrafast kinetics can be often neglected. The instrumentation of choice in such case would be time-correlated single photon counting (TCSPC) and multifrequency domain fluorometry . AlthougTDFS is nowadays a well-established method for studying model lipid membranes. As discussed before, it is sensitive to membrane hydration and to mobility of lipids. Since these two parameters are easily affected by number of biologically relevant processes, the method can be successfully used to sense lipid packing, phase state of lipids, adsorption of peptides, specific effects of adsorbed salt ions, and oxidation of membrane components, to name just a few of them. Below we present applications that exemplify the sensitivity of TDFS to 1) model lipid membrane oxidation and 2) interaction with calcium ions.Oxidation of membrane components can considerably change both their mobility and hydration . PhosphoNot only phospholipids, but also cholesterol oxidizes. Many oxysterols are products of oxidative stress, but some of them are created by our organisms on purpose to be used in signaling and/or in cholesterol homeostasis . Additio2+ to the mixed lipid bilayer composed of 80 mol% zwitterionic POPC and 20 mol% anionic POPS is definitely enhanced by the presence of POPS, but the major Ca2+ binding sites are not the negatively charged carboxylic groups of POPS. TDFS results based on Laurdan and Dtmac probes, sensing lipid carbonyls and phosphate groups, respectively, together with sum frequency generation measurements, and molecular dynamics simulations revealed a complex picture of calcium adsorption to lipids  overall dynamic Stokes shift Δν and 2) characteristic relaxation time τ can report directly (i.e. without data modelling) on subtle changes in 1) the degree of hydration and 2) mobility, respectively, of the hydrated phospholipid or protein segment at the close vicinity of the fluorophore embedded in the bilayer. This implies that for a meaningful application of the TDFS technique the precise knowledge on the location of the dye is required. In protein science this pre-requisite is achieved by site-selected labelling, while the location of the chromophore of amphiphilic membrane probes can be determined by quenching experiments and molecular dynamic (MD) simulations. Together with MD simulations the TDFS approach identified how molecular parameters like membrane curvature , lipid c


Nature-Based Solutions (NBS) have been gaining importance in many European cities to reduce floods' impacts. However, evidence of their effectiveness in reducing the impacts of droughts in rural areas are scarce. Besides, ignoring future climate conditions or the specific socio-economic context in which NBS is applied could decrease their long-term effectiveness. This study aims to stress the importance of developing scientifically-based and customised information on climate change impacts as a precondition for designing and implementing NBS. For that, a System Dynamic model was developed to analyse and understand the dynamic behaviour of NBS responding to different scenarios of climate change and socio-economic contexts. This article recognises the proactive involvement at all societal levels as an essential component to enhance and maintain ecosystem resilience and, therefore, NBS  •Nature-Based Solutions are inherently multidimensional.•Stakeholders knowledge should have real impact on the NBS assessment process.•NBS may not be sufficient to adapt to an intense climate change scenario.•Climate information is an essential precondition to design adaptable NBS. BesidesAdditionally, climate change impacts on ecosystem processes are expected to compromise important regulatory ecosystem services such as waste-water treatment or erosion prevention. For instance, higher incidences of extreme precipitation events will increase soil erosion, affecting soil productivity and water retention capacity of soils threatening food security and agricultural incomes. . All theSeveral governmental bodies have highlighted the need to shift to more sustainable food systems. New policy frameworks and climate adaptation actions are encouraged, which jointly address water and food security, preservation of agricultural and rural landscapes, mitigation of and adaptation to climate change, and conservation of ecosystems and biodiversity .Among all the proposed strategies, the concept of Nature-Based Solutions (NBS) has occupied the EU political foreground in the past years . NBS incDespite the growing evidence base for NBS to support climate change mitigation and adaptation, most studies investigating NBS effectiveness are limited to empirical studies, thus ignoring factors that cannot be studied empirically, i.e. long-term climate change projections. For this reason, scenario modelling approaches considering temporal projections of climate are crucial to understand the challenges and limitations of NBS in the context of environmental change.Additionally, adaptation and mitigation strategies, including NBS, are often approached from an expert-driven, single-focus and top-down perspective. However, NBS affect a wide range of stakeholders including NGOs, national and local governments, scientists or civil society members.Moreover, even though many biodiversity-based measures have been implemented in rural areas to support the development of sustainable agriculture, several authors have pointed out that the majority of NBS studies are specific from urban settings . For thiIn response to these gaps, we have adopted a system perspective and a multi-sectoral approach to examine the socio-economic dynamics associated with the implementation of NBS in the Medina del Campo Groundwater body (MGWB). MGWB is located in North-West central Spain, where the rural development and the regional economy depend strongly on agriculture. The uncontrolled overexploitation of the Medina del Campo aquifer has decreased groundwater levels and deteriorated the aquifer-dependent superficial ecosystems. Besides, climate change is likely to increase the frequency and intensity of droughts, threating further agricultural productivity, biodiversity and regional landscape. Therefore, the main aim of this study is to assess the potential of NBS to conserve and enhance water resources management without compromising agricultural productivity. For that, we developed a system dynamics model (SD) to analyse the long-term effectiveness of NBS strategies under different scenarios of climate change.System dynamics (SD) is a mathematical modelling technique to mimic and understand the non-linear behaviour of complex systems . SD has This paper purposes an innovative method to analyse the long-term effectiveness of different NBS strategies by integrating regional downscaled climate ensembles for three RCPs scenarios into a quantitative system dynamics model. Participatory modelling techniques were used to obtain relevant bottom-up information and to facilitate the co-design process of the SD model, focusing on the stakeholder's perception on the main dynamics of the Medina del Campo System.The co-development of a system dynamic model along with key stakeholders had four main objectives for this investigation. Firstly, to expose the causes of different system behaviours by analysing the complex network of interactions between physical, ecological and socio-economic factors affecting NBS effectiveness. Secondly, to observe the implementation of different NBS strategies under different input conditions (i.e. climate projections data). Thirdly, to anticipate possible rebound effects or policy resistance and identify suitable NBS strategies to be implemented. Finally, to engage with stakeholders at the beginning of NBS implementation to increase NBS effectiveness by promoting awareness and motivation of those taking part in the decision-making processes, thus providing a platform for the joint-ownership of results.22.12 bordered by the Duero river to the east and the Sierra de Gredos and the Adaja and Trabancos rivers to the west. The rural development and the regional economy depend strongly on agriculture. Irrigated agriculture represents about 19% of the total agricultural land. Groundwater (GW) is the primary source of irrigation; 95% of total GW extractions are due to irrigation in agriculture. GW overexploitation has resulted in an uncontrolled decrease of the piezometric levels. Measurements show that GW levels have declined up to 20 m in some areas in the last 40 years. Simultaneously, the decrease of groundwater levels has increased their lithologic arsenic concentrations. Besides, excessive agricultural fertilization has spread nitrate pollution in the aquifer and superficial ecosystems.The Medina del Campo Groundwater body (MCGB), located in the Duero River Basin, north west-central Spain, forms the case-study for this region . The MCGThese issues have caused a chain reaction resulting in severe deterioration of the main GW dependent ecosystems such as wetlands, rivers, riversides and streams. Many rivers and streams experience longer dry periods than before. More frequent and intense droughts have altered the region's landscape and biodiversity .Climate change impacts are likely to increase climate variability and extreme weather events such as heatwaves and heavy rainfalls . The con2.2The System Dynamic approach was described for the first time in 1950 by Forrester and Cole J.W. . Since tMathematically, the SD approach is based on linked first-order differential  equations that are represented in the simulation model in the form of stocks and flows. Stocks are accumulations within the system (i.e. amount of groundwater in the aquifer) and are represented by rectangles in the model. They represent the (observed) state of the system that can be changed by flows. Flows may be inflow to a stock or an outflow from stock. They are represented by pipes with valves controlling the rate of flow. In this study, the development of a simulation system dynamic model has provided several advantages. Firstly, it has allowed the integration of quantitative and qualitative data from different sources and spatial-temporal scales. Secondly, although SD is not an optimal tool to exactly predict future system states, it is an effective method to indicate how different policy interventions may alter the tendency to move towards any future state by simulating “what-if” scenarios . It was possible to observe how the implementation of different NBS strategies under different input conditions (i.e. climate change scenario) affected the behaviour of the system as a whole. Finally, the conceptual representation of the model in a graphical structure facilitated the engagement of stakeholders in the co-design of the SD model allowing the integration of knowledge of end-users and stakeholders within the model.In this study, the development of the computer-simulated model started from a participatory modelling phase. Stakeholders input was used to develop a Qualitative System Dynamics Model (QSDM) representing key variables and relationships within the system. The qualitative model developed with stakeholders was used to develop the dynamic hypotheses before the quantitative stock-flow model was developed. The methodological process adopted is schematized in the following 2.2.1The implementation of participatory modelling techniques allows for the integration of end-users and stakeholder's knowledge with scientific analysis, e.g. with development and application of scientific models. It has proven to be a suitable methodology to engage non-scientists with scientists as it introduces a new perspective for both the modeller and the stakeholder . StakehoDuring the exercise, stakeholders identified essential factors and issues that were relevant for modelling the system. Additionally, modelling components needed to assess the long-term effectiveness of NBS were identified. The identification was carried out after analysing the causal connections and influences involving the multiple dimensions of NBS, ranging from economic, social and environmental factors.The participatory modelling exercise was divided into two phases see . FirstlyThe participatory modelling process resulted in the development of a Qualitative System Dynamics Model (QSDM) . It characterises the state of relevant variables in the system, storing the memory of their state at previous time steps, thus enabling description of their evolution. Flows affect the state of stocks via inflows and outflows, thus supporting interconnections among the variables within a system. For example, the change of the state of aquifer level depends on rain (inflow) or water extractions (outflow). Mathematically, stocks integrate their net flows; the net flows are the derivative of stocks. Identifying and modelling the behaviour of stocks is crucial to analyse the dynamic evolution of the essential variables and potential mutual influences .Given the interdisciplinary and complexity of NBS, a modular structure was adopted to quantify the SD model. Different modules coming from different disciplines were linked together by mutual feedbacks.The validation of the SD model was done in two steps. Firstly, the qualitative SD model was validated in a stakeholder's workshop. The essential premise in SD is that the structure of the system determines the behaviour of the different elements. Therefore, validating the model structure with key stakeholders increases confidence.Secondly, the quantitative model was simulated with historical data of precipitation and evapotranspiration (time-period 1979–2012). The simulation results show a decreasing trend of the Medina del Campo aquifer levels, going in concordance with the results of other studies .2.3The system dynamics model represented in The grey variables in the model describe the principal connections between different sub-models. These variables help to identify relationships and influences that are often difficult to recognize. The polarity of the relationship was represented by a green arrow (positive polarity) or red arrow (negative polarity). The polarity is used to indicate how the dependent variable changes when the independent variable changes.The model is run for 100 years allowing the long-term assessment of NBS effectiveness under different projected climate change scenarios. The time step of the simulation is one month. To facilitate the simulation of groundwater control measures, the model assumes that water extractions are constant over the year. The main dynamic assumptions of each sub-model are summarised in the following:2.3.1Based on the knowledge obtained from the individual interviews, the environmental sub-model  assumes The infiltration coefficient is dependent on the slope, the vegetation cover and the basic soil infiltration (fc). The fc is a characteristic of the soil that corresponds to the permeability of the soil when it is saturated. The model assumes that the lower the slope of the land and the more prominent the vegetation cover, the higher the percentage of rainwater which infiltrates . Soil coThe vegetation leaves retain a portion of the rain, preventing water from reaching the soil. The interception is a function of the foliage coefficient which differs depending on the type of vegetation cover. For example, a forest may intercept 40% of rain while crops such as alfalfa can intercept between a range of 10 and 35% of the total rainfall.The irrigation return is defined by the irrigation efficiency (percentage of irrigation returning) determined by the type of irrigation system used in agriculture—highly efficient irrigation methods such as drip irrigation use virtually 90% of the irrigated water . The modThe monthly rain that is not retained by the vegetation foliage or infiltrated into the soil flows away in the form of surface run-off to the superficial ecosystems .The amount of water stored in the soil level (stock) depends on the amount of water that infiltrates (explained above) and the amount of water that evaporates and percolates into the aquifer. Both processes depend on the field capacity (maximum accumulation of water that an unsaturated soil can have) and the wilting point (minimum humidity to maintain vegetation). The model assumes that when the water stored in the soil equals the wilting point, the evapotranspiration stops, as it assumes that the plants close their stomata .The percolation is described in the model as a function of saturation. The maximum percolation capacity occurs when the soil reaches its field capacity (100% saturated).The total recharge (groundwater inflow) is determined by the amount of water that percolates, the underground run-off coming from other groundwater bodies  and water drainage from superficial ecosystems (Trabanco and Zapardiel river). The model assumes that the groundwater lateral movement is constant over the year. It also implies that rivers drainage into the aquifer is a constant percentage of the monthly river flow.Based on the individual interviews and the discursion generated during the group model building activities, the groundwater extractions and the discharge to superficial ecosystems constitute the outflow of the groundwater level. GW extractions include legal and illegal extractions. Legal extractions are regulated by the irrigation demand and the maximum volume of water allowed for extractions. Illegal extractions are a function of environmental awareness (defined in the socio-economic sub-model). An increase of environmental awareness results in a reduction of the illegal extractions.The decrease of the piezometric level (level to which water is confined in an aquifer) occurs when the total recharge is lower than the GW extractions. If the piezometric level is less than a certain level, the surface ecosystems disconnect from the aquifer. On the contrary, if the piezometric level reaches a certain threshold, water flows to the surface ecosystems.Following stakeholder's perception on the Medina del Campo system, the superficial ecosystems are represented in the model with five stocks, the four most important rivers in the region  and one stock representing the currently-dried wetlands.The model assumes that the only waters that flow into these levels are the surface run-off and the groundwater discharge into superficial ecosystems. GW discharge depends on the piezometric level and the height at which the ecosystem is in the region. The total run-off entering into each of the ecosystems depends on the river/wetland catchment area. The model includes an additional inflow variable for Trabancos and Zapardiel river. This variable can be activated to simulate an artificial recharge of the aquifer through surface techniques. The model assumes that when the artificial recharge variable is activated an additional 0.77Hm3 of water will flow into the Zapardiel river and 0.6 Hm3 will flow into Trabancos.Evapotranspiration and drainage water into the aquifer are the only variables considered to calculate the outflow of water.2.3.1.1The climate component has been highly relevant for this study as the intention is to analyse the performance of NBS strategies under different climate change scenarios. Therefore, climate projection information for three Representative Concentration Pathways scenarios  has been integrated into the SD model. Specifically, monthly precipitation and monthly mean temperature were obtained from the EURO-CORDEX ensemble . RCPs deClimate simulations for past-time periods (hindcasts) from different models were compared with observational data. The comparison was made plotting both time series (for precipitation and temperature) and comparing the mean and the standard deviation. The objective was to select the model that most accurately represented the real-world climate system of MCGWB. Finally, the climate projections data used was generated by the driving model MOHC-HadGEM2-ES and downscaled with the Regional climate model (RCM), KNMI-RACMO22E. See After the appropriate model was selected, time series of precipitation and evapotranspiration (time-period 2019–2100) were integrated into the SD model.0C/month), and Ps corresponds to the percentage of hours of monthly sunlight compared with the annual mean of sunlight.Potential Evapotranspiration was calculated following the equation developed by Blaney & Criddle  and expressed in Eq. Certain parameters of climate models carry considerable uncertainty depending on the climate model used. Thus, it is difficult to determine the actual effect that they have on the final results. It is necessary to use multi-model ensemble simulations to show the uncertainty range produced by climate models.The main aim of the SD model presented in this article is to provide a valid description of the essential system structure and the resulting system dynamics; it does not intend to provide detailed quantitative results. For this reason, multi-model ensemble simulations were not carried out in all simulation scenarios.However, a climate model-ensemble was used for the two climate variables (precipitation and evapotranspiration) in order to show the uncertainty range produced by climate models. This approach was also used for the variable “piezometric level” in the BAU scenario. Piezometric level was considered as the main indicator to describe the state of the aquifer. For this reason, a multi-model ensemble was used in this variable to highlight the need to consider climate uncertainty ranges in NBS decision-making.2.3.2Based on Bossel's Systems Zoo , the socThe model assumes that the higher the environmental quality and advertisement, the higher the gain in tourists. A delay function was included in the tourist gain rate to indicate a postponed effect from the increase in environmental quality to the increase in tourists.The increase in tourists has a positive effect on the creation of new jobs and economic opportunities which, simultaneously, has a positive effect on income . The income is also produced with the use of the water extracted from the aquifer. Therefore, the more water is withdrawn from the aquifer, the higher the income. Income has a positive effect on profit which is a function of income, cost of investing in new infrastructures and cost of groundwater extractions—the cost of extractions increases as the depth of the wells increases (when the piezometric level declines).It is considered that the economy of the area is a critical aspect to maintain the rural population. For this reason, the model assumes that an increase in profit leads to an increase in the rural population. The increase in services and infrastructure also positively affects the increase in the rural population. Environmental awareness can regulate the environmental stress caused by tourists and the local population. However, it is assumed that this regulation requires a specific time to produce effects on environmental stress. The model simulates this phenomenon using a delay function. The level of environmental awareness is defined by the level of information and citizens engagement as well as by the level of the economic growth.2.4Five scenarios have been simulated to ‘test’ the impact of soil conservation practices (NBS) and groundwater extractions control measures on aquifer levels  has been simulated for three RCPs  to assess their long-term performance. The initial dynamic hypothesis is that implementing NBS in the MCGW may benefit both levels, environmental and socio-economic. Climate change may impact ecosystems function; thus, compromising NBS capability to produce benefits (co-benefits). Therefore, NBS multifunctionality may be lower in scenarios where climate change is more intense (i.e. RCP4.5 and RCP8.5). Consequently, the long-term effectiveness of NBS may be compromised in a climate change context.3The following section presents the main results and research findings. The analysis of the dynamic behaviour of four state variables  has been used to analyse the long-term performance of NBS in the economic, social and environmental sustainability dimensions.The most notable increase of the aquifer level is shown when applying NBS and promoting environmental awareness. The improvement of the aquifer levels is produced due to the reduction of illegal extractions. An improvement of the piezometric level is also observed when NBS are applied with GW extractions control. On the one hand, NBS measures focused on improving soils quality, increase the total recharge of the aquifer. On the other hand, measures such as limiting the maximum volume of GW extractions or changing the crop-type to other less-demanding crops, reduce total GW extractions. Although an increase of the piezometric level is observed for RCP2.6, the increase is not enough to reach the basin of the superficial ecosystems preventing the aquifer-superficial ecosystems reconnection.This aquifer level improvement is maintained for the RCP4.5 scenario. However, the results show that in the long-term, these measures would not be sufficient in RCP 8.5, where more investments in adaptation measures will be required.Combining NBS with extractions reduction produces an increase in environmental quality. However, this increase is less pronounced than in the scenario mentioned above (NBS and citizens awareness). Additionally, the positive effect of NBS in EQ requires more time to become visible. The initial increase in EQ produced by these measures (NBS + GW reduction) follows an EQ decrease. Reducing GW extractions may negatively affect the area's economic profit as it is highly dependent on irrigated agriculture. A decrease in profit leads to a decrease in environmental awareness and, thus, on EQ.The same pattern of behaviour is followed by BAU and NBS-NCA (NBS without citizens awareness) scenarios.BAU and GW extractions reductions scenarios do not show any significant improvement in NJEO levels.The model simulation results show a decrease of rural population on all management and climate change scenarios C. ScenarThe general results show that, although the implementation of NBS improves the state of the aquifer, it is not enough to achieve a full recovery. Likely, NBS implementation is not enough to address the main environmental, economic and social challenges.4Adaptation strategies, such as Nature-Based Solutions, are inherently multidimensional. However, NBS complexity is not often captured in research studies and frameworks. NBS are frequently framed in a reductionist way, focusing only on a subset of impacts and ignoring potential trade-offs among NBS and other system elements. For example, climate change impacts are frequently overlooked in NBS assessments. However, it is known that changes in mean temperature, species distribution or precipitation patterns are highly likely to alter ecosystem functions and, thus, NBS functionality. Besides, the long-term capability of NBS to deal with extreme weather events such as droughts may not be sufficient in a climate change context.Additionally, a significant part of the NBS research studies has been contextualised in cities. Usually, the urban-centred evidence on NBS effectiveness cannot be transferred to rural contexts. Rural areas are affected by different pressures and socio-economic circumstances. In this research, we seek to assess the socio-economic dynamics associated with the implementation of NBS in rural areas by analysing the Medina del Campo Ground Water Body system. Special attention has been given to the long-term effects of these measures under different climate change scenarios.We developed a system dynamics model (SD), starting from a participatory modelling phase to analyse the long-term effectiveness of NBS strategies under different scenarios of climate change. The intention was to a) analyse the main dynamics of the system; b) assess the effect of different climate change scenarios; c) see the pathways of development of the system depending on different policy measures to analyse NBS suitability; and d) engage stakeholders in the model development process by contributing to model assumptions and parameters.We believe that stakeholder knowledge and priorities should have a real  impact on the model. Engaging stakeholders in the co-design of the SDM has provided multiple advantages. Firstly, it has supported constructive and targeted discussions relevant to identifying barriers and limitations of NBS implementation. Secondly, it has allowed for the integration of local knowledge in a graphical structure used to set the basis of the quantitative SD model supporting the integration of qualitative and quantitative data. Thirdly, complex interconnections among system elements were revealed, helping to anticipate possible policy resistances or rebound effects and suitable NBS to act on the system. Finally, the process itself has promoted awareness and collective learning of those taking part in the participatory modelling process.The main advantage of SD over other approaches such as statistical modelling is gaining insights and understanding from data-poor problems. The basis of the SD approach is the recognition that the relationships among different system elements are often more important in determining systems behaviour as the individual components themselves. Therefore, the results are highly sensitive to assumptions, increasing uncertainty in results. The simulation of the quantitative SD model does not intend to provide detailed predictions on water level (piezometric level) changes due to NBS implementation. The model was used to study the different patterns of behaviour produced when implementing NBS or other management strategies . It was possible to observe the potential of NBS to improve aquifer levels while delivering additional benefits  in the Medina del Campo system.The results show that the long-term effectiveness of NBS is highly dependent on the socio-ecological context in which NBS are implemented. For example, scenarios with a low level of environmental awareness limit the potential of NBS to deliver certain co-benefits (i.e. creation of new jobs and economic opportunities). Besides, this study also demonstrates that the implementation of NBS may not be sufficient to adapt to a worst-case climate change scenario (RCP 8.5). Therefore, additional measures such as controlling groundwater extractions or increasing environmental awareness by engaging stakeholders before implementing NBS are essential to maintain the delivery of NBS co-benefits. Additionally, we stress the importance of accounting for climate model uncertainty using multi-model ensemble simulations in NBS assessments. Ignoring uncertainty ranges, for example, by only using one climate model, does not allow informed decision-making. Although regional downscaled climate projections were used to analyse the long-term effectiveness of different NBS strategies, evaporation and changes in rain patterns have been the only climate change impacts considered. Soil degradation, plant pathogens, pests, or increase of ground-level ozone are just a few of many climate change impacts that could ultimately compromise the effectiveness of NBS in rural areas. Therefore, new methods and tools supporting the integration of climate change data with other socio-economic elements may contribute to the decision-making process of NBS design and implementation. The uncertainty associated with climate projections has several implications on NBS assessments that, if not correctly understood, may complicate and jeopardise the correct assessment of NBS performance. Climate uncertainty is difficult to quantify and calculate. However, the proper communication and management of this uncertainty represent an opportunity for NBS decision-making. Hence, new research strategies focusing on transforming climate data (and its associated uncertainty) into customised and tailored information to be used in NBS assessments and evaluations are needed.5Developing scientifically based and customised information on the impacts of climate change by assessing uncertainty ranges is an essential precondition to design and implement long-term effective and flexible NBS.The results of this study show that the socio-economic context in which NBS are applied significantly influence the performance of NBS. The benefits delivered by NBS may be compromised if a balance between nature, economic growth and society is not found. NBS should not be considered as a single action to protect or restore nature but as a process that engages with society to merge natural and human systems into a wholly unique system.Fig. A1 Qualitative System Dynamics Model co-developed with stakeholders. Highlighted in green the environmental variables and relationships, in blue the socio-economic elements are represented and in red the risks. The polarity of the relationship is represented by a positive (+) or negative (-) symbol. The processes or decisions that require some time to occur are indicated with a delay mark (//). Relevant feedback loops are numbered and their polarity and direction have been indicated: Reinforcing or positive loop (R), Balancing or negative loop (B).Appendix ATable B.1 List of equations and parameter values.Appendix BAppendix C. Table 1C. List of climate model used in the evaluation and multi-model simulations .Appendix CThe following are the supplementary data related to this article.Eulalia Gómez Martín: Conceptualization, Methodology, Writing – original draft. María Máñez Costa: Conceptualization, Writing – review & editing. Sabine Egerer: Methodology, Writing – review & editing. Uwe Schneider: Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Vagus Nerve Stimulation (VNS) is an adjunctive treatment for patients suffering from inoperable drug-resistant epilepsy. Although a complete understanding of the mediators involved in the antiepileptic effects of VNS and their complex interactions is lacking, VNS is known to trigger the release of neurotransmitters that have seizure-suppressing effects. In particular, norepinephrine (NE) is a neurotransmitter that has been associated with the clinical effects of VNS by preventing seizure development and by inducing long-term plastic changes that could restore a normal function of the brain circuitry. However, the biological requisites to become responder to VNS are still unknown. In this review, we report evidence of the critical involvement of NE in the antiepileptic effects of VNS in rodents and humans. Moreover, we emphasize the hypothesis that the functional integrity of the noradrenergic system could be a determining factor to obtain clinical benefits from the therapy. Finally, encouraging avenues of research involving NE in VNS treatment are discussed. These could lead to the personalization of the stimulation parameters to maximize the antiepileptic effects and potentially improve the response rate to the therapy. Epilepsy is a common neurological condition that affects approximately 50 million people around the world . This diVagus Nerve Stimulation (VNS) has been proposed for over 30 years as an adjunctive treatment for patients with inoperable drug-resistant epilepsy. This therapy is approved in the United States for treatment-resistant depression, and also showed positive effects in multiple other medical conditions, including chronic pain, Parkinson’s disease, essential tremors, gastroparesis, chronic tinnitus, stroke, post-traumatic stress disorder, eating disorders , multiple sclerosis, migraine, and Alzheimer’s disease . VNS conIn this review, after a brief overview of the functional anatomy of vagal stimulation, evidence of the critical involvement of NE in the antiepileptic effects of VNS in animal models and humans are presented and discussed. Moreover, hypotheses are formulated on how inter-individual differences in the noradrenergic system could explain differential responses to VNS treatment. Finally, future perspectives involving NE in VNS are discussed and potential avenues for research on optimization of the treatment with respect to personalized stimulation strategies are proposed.Anatomically, the vagus nerve is the tenth of the cranial nerves and has a mixed composition. It comprises approximately 3/4 of afferent fibers carrying taste, visceral and somatic information to the central nervous system, and 1/4 of efferent fibers, which mainly conduct parasympathetic information to the heart, lungs, gastrointestinal tract and other intra-abdominal organs, but also provide motor innervation to laryngeal muscles . The comThree types of vagal fibers can be distinguished based on their functional properties: A-fibers , B-fibers , and C-fibers , which The nucleus of the solitary tract (NTS), situated in the dorsomedial medulla, is the major sensory nucleus receiving afferences from the vagus nerve . The NTSAlthough the LC is a small, lateralized nucleus of ∼3 mm diameter with only 15.000–20.000 neurons per side in humans, the LC is, by far, the main source of NE in the brain . It is cOriginating from the cerebral cortex, the main feedback influences are shown from the orbitofrontal and anterior cingulate cortex . DespiteIt is worth mentioning that other stimulation techniques have been investigated and may activate pathways that are similar to VNS. For example, trigeminal nerve stimulation has shown to reduce pentylenetetrazol-induced seizures in rats and seizure frequency in humans . The antBased on the widespread connections of the noradrenergic system and on the neurochemical properties of NE, VNS was hypothesized to reduce seizure development and trigger plastic changes within the brain, with the LC-NE system standing as the cornerstone of these effects. We will first review the body of evidence from animal studies, pointing at a critical involvement of NE in both short and long-term antiepileptic effects of VNS. A summary of the results of previous animal studies presented in this section can be found in ex vivo with histological examination of regional brain-dissected samples. This study used the maximal electroshock seizure (MES) model, which is categorized as a model of generalized tonic-clonic seizures that elicits hindlimb extension (HLE) and often used to test anticonvulsant compounds  protein, a marker of neural activity, as a result of increased gene transcription in those nuclei following VNS  . In addiNeurochemical techniques that used tracers highlighted reciprocal connections between the LC and the Dorsal Raphe Nucleus (DRN), a major serotonin brainstem nucleus . Seroton1-adrenoreceptors. Indeed, by increasing the norepinephrine tone on α1-adrenoreceptors located on the cell body of serotonergic neurons, the LC can tonically activate serotonergic neurons in the DRN  in healthy rats . Althoug the DRN . This wa the DRN .Finally, while In addition to the DRN, other studies showed that VNS increased the extracellular concentration of NE in several cortical areas  and in tvia a microdialysis probe  (2-adrenoreceptors located in a subfield of the Cornu Ammonis (CA3) of the hippocampus following VNS administration, while the degree of activation of α1-adrenoreceptors remained unaltered.A study characterizing the role of NE under VNS was further conducted in rats with limbic seizures evoked by intrahippocampal perfusion of the muscarinic agonist pilocarpine is probe . This st effects . Anothermin OFF) . An incr2-adrenoreceptors  the difficulty in obtaining direct neuroimaging, electrophysiological or histopathological findings on LC status; (ii) the complexity – involving a lumbar puncture – and non-established reliability of NE assessment from the cerebrospinal fluid ; (iii) tThe effect of VNS on NE signaling was evaluated with the P300 component of event-related potentials during an oddball task paradigm . The P30Another study further evaluated the potential of P300 features recorded during stimulation to correctly classify responders and non-responders . In contAs in animals, pupil diameter is tightly associated with both phasic and tonic activity of the LC . A functIn addition to the P300 event-related potential and the pupil diameter, the effects of VNS in response inhibition were also studied. Although the neurobiological processes involved in response inhibition are not totally elucidated yet, it was suggested that the noradrenergic system has a critical role. Indeed, the phasic activation of the LC-NE system facilitates responses to task-relevant processes  and is tOne study determined how VNS may influence response inhibition in a stop signal task, with the time needed for the epileptic patient to inhibit a response measured in both VNS ON and OFF conditions, in order to compute the inhibition gain . VNS decConcerning inhibitory capacities, another study used the Eriksen Flanker test in epileptic patients with stimulation ON and OFF to measure their ability to suppress irrelevant information . In the When investigating biomarkers of the effects of VNS on the LC-NE system, research has also focused on sleep characteristics. As part of the ascending activating system, the LC promotes wakefulness, and it needs to be partly silenced for sleep initiation. Further silencing is required to go from slow wave sleep to Rapid-Eye Movement (REM) during which LC neurons are fully silent . IncreasIn order to find biomarkers that could refine the selection of responders to VNS, a prospective study also measured the heart rate variability (HRV) at baseline and after 1 year of VNS treatment . The higA study used machine learning to build prediction models for evaluating responsiveness to VNS therapy preoperatively based on HRV indices in wake and sleep states . 52 HRV While immunochemistry studies evaluating the effects of VNS on the vagal afferent network in humans are scarce due to the limited number of samples available, a first study was recently conducted with aim to extract morphological characteristics of the nuclei linked to this network after chronic VNS . Neuronan = 39, 25 Hz, 0.5 ± 0.47 mA, 250 μs pulse width, 30 s ON, 30 s OFF) after 20 weeks of tVNS therapy, that was not observed in the control group , while the responder rates were similar between the two groups. One recent study reported a robust tVNS-evoked pupillary dilation in healthy volunteers that was not observed under sham stimulation on the earlobe . This non-invasive method uses a surface-electrode to stimulate the cymba concha   and is b earlobe . In cont earlobe . Discrep earlobe  or 0.5 m earlobe  that wasAnother study measured the level of NE released with tVNS by the means of hormonal indices such as the salivary alpha amylase and the salivary cortisol . SalivarFurther research is needed to establish a classification model that intends to predict responsiveness to cervical VNS, based on non-invasive markers of the activity of the noradrenergic system when tVNS is delivered. If such a model reaches a high accuracy, tVNS could be an exciting avenue for assessing responsiveness to invasive VNS before the implantation of a neurostimulator and therefore identify more selectively VNS responders to avoid unnecessary surgery and possibly related complications.The results presented in this review indicate the important implication of NE in the antiepileptic effects of VNS. Based on the results presented, one could hypothesize that responders to VNS show a lower NE reactivity prior to the implantation of the device compared to non-responders , allowinAlthough clear evidence of VNS-induced dose-dependent LC activation is present in animals, more solid findings should still be sought in humans. Particularly, a promising opportunity might reside in the use of LC-NE system markers (such as pupil diameter or P300 responses) as parametric read-outs of the afferent effects of VNS, and studies aiming at a clearer definition of their dose-dependent characteristics are thus advocated. Potential differences in the phasic or tonic LC modulation, depending on the cervical or transcutaneous nature of the vagal stimulation, should also be explored.in vivo , could help to refine the selection of patients before the implantation of the device and improve the current understanding of the mechanisms of action of VNS.In addition to the aforementioned biomarkers, which indirectly reflect the activity of the LC-NE system, further studies could evaluate the structural and functional properties of the LC itself with imaging techniques and determine whether differences exist between responders and non-responders to VNS. Magnetic resonance imaging studies might be useful to extract structural and functional properties of structures of the vagal afferent network, which could be critically involved in the antiepileptic effects of VNS. Recently, techniques have been developed to evaluate the integrity of the LC in vivo , using nin vivo . Neuromein vivo . These iin vivo , Parkinsin vivo , schizopin vivo  and latein vivo , but nevvia the nucleus paragigantocellularis, as well as minimize the activation of the inhibitory pathway that relays the information through the nucleus prepositus hypoglossi. Functional magnetic resonance imaging could again be a useful tool to develop a stimulation paradigm based on individualized titration. Indeed, it could be used to assess the activation of the LC during stimulation under different stimulation parameters. However, research about the acute effects of VNS using functional magnetic resonance imaging has been limited due to technical considerations and the requirements specified by the constructor of the VNS device (Further research leading to the personalization of the stimulation strategy to maximize the antiepileptic effects of VNS and potentially increase the response rate to the therapy is warranted. One option could be to investigate how to preferentially activate the excitatory pathway that links the NTS to the LC S device . The devAB, SV, and RE contributed to the analysis and interpretation of the relevant literature and drafting of the manuscript. All authors with their complementary backgrounds have critically revised the manuscript, making a substantial, direct, and intellectual contribution to the work. All authors approved it for publication.AB and PD were employed by the company Synergia Medical SA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Synergia Medical and the Walloon Region. The funder was not involved in the analysis and the interpretation of the literature, the writing of this article or the decision to submit it for publication.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


K, damage variable correction factor δ, and thermal damage variable TD are introduced in a new thermomechanical (TM) constitutive model for the entire process. The compaction stage of rock in uniaxial compression test and the strain softening of rock caused by thermal attack are considered in this article. The damage evolution of rocks is described by a damage variable and a constitutive equation, which are in agreement with the actual thermal experimental breakage. The uniaxial compressive strength of granite subjected to a TM coupling effect can be predicted properly by this new unified constitutive model. The new TM unified constitutive model considering the compaction stage and post-failure stage is in good agreement with the test curves throughout the entire process. The coupling effect of heat and load in the total damage of rock has obvious nonlinear properties, but the coupling effect significantly weakens the specimens. By using the new TM unified constitutive model, the whole process of changes in rock damage with strain after high temperature can be calculated. Meanwhile, the model well represents the stress–strain curve at the post-failure stage. It is expected that this model can provide references for studying the mechanical response of the rock damage propagation characteristics in the future.For thermal and loaded rock in engineering structures for some projects, triple-shear Drucker–Prager yield criteria, compaction coefficient  With the increasing number of projects involving mining, cracked reservoir extraction, and underground nuclear waste disposal, there have been concerns about the stability of rocks under complex conditions, such as in a thermomechanical environment. There are certain situations of rock breakage under the coupling effect of heat and load exemplified all over the world. Research on the thermo-load breakage of some selected rocks from countries around the world was conducted by Sygala et al. . The mecThe Brazilian disc tests and the three-point bending tests were carried out, and the composition and the structure of minerals were regarded as the greatest influences on the mechanical properties of rocks  . In addiTo describe the deterioration and damage in the rock after heat treatment accurately, the damage evolution was presented and applied to the rock under the coupling effect of heat and load  . Based oK and damage variable correction factor δ  [Weibull distribution has been used to establish several thermo-mechanical damage models of rock on the basis of their predecessors, and a new damage model has also been presented  ,20. Howe., 2016) . It is eAs a sort of non-independent physical property, the damage must be incorporated into the elastic, plastic, and viscoelastic materials as one type of degradation factor. Therefore, when the damage variable is defined, it should be combined with an independent physical property. Macroscopic damage in materials includes various kinds of defects , which can be treated as a kind of continuous medium with a microdamage field. Furthermore, the formation, development, propagation, and accumulation of microdamage are regarded as the process of damage evolution. The damage can be regarded as a part of the microstructure, so it should be introduced into the continuous medium model. Since 1980, many researchers have defined several different damage variables for describing the damage state of a material’s structure  ,23; mostAs a continuous evolution and non-independent physical property, the damage variable increases when the rock is impacted by an external force, such as uniaxial pressure. Therefore, the Weibull distribution with two parameters is selected to calculate the damage evolution. Since even infinitesimal quantities of rock material obey the Weibull distribution, the Weibull distribution function is shown as:The cumulative distribution function is:F is the unit with triple-shear Drucker–Prager yield criteria; the formation is shown as:The damage variable under triaxial compression can be shown as:Following the Lemaitre theory of strain equality:δ is added in this formation for controlling and reflecting the post-failure stage of the stress–strain curve under triaxial compression conditions. Therefore, the effective principle equation is:The damage variable correction factor According to (7), the effective principle is obtained as:Following the generalized Hooke’s law:By substituting (7) into (9), the following relationship is derived:Combining (8), (9), and (11):As a result of Following the effective stress, K is:In addition, the compaction coefficient By substituting (4) into the stress–strain formula, and combining (13) and (16), the following relationship is derived:According to the boundary condition (i), when K when the strain arrives at Therefore:The relationship can be derived by (18):F is:According to the boundary condition (ii), when Taking the derivative of the implicit function of Boundary conditions (i) and (ii) can be substituted into (22) for the following result:Combining (22), (23), and (24), the following relationship can be obtained:Solving the system of equations:The parameters T, and According to the experimental program, the elastic modulus is selected to define the thermal damage of rock subjected to thermal treatment. After thermal treatment at a certain temperature, the thermal damage of the granite specimen is a constant value. The thermal damage variable The average values of damage variables of granite specimens after thermal treatment at different temperatures are shown in The total TM damage effect of granite can be expressed by the total damage variable The TM unified constitutive model can be described as:The generalized Hooke’s law is selected to build the TM unified constitutive model. Therefore, the damage of rock caused by high temperature depends entirely on the thermal damage variable Therefore, the scale parameter Based on (31), the picture of the TM total damage variable nd dense a. Comparnd dense b.The stress–strain curve of the uniaxial compression test of granite was selected to verify the theoretical constitutive model. Due to the drastic decrease in the stress after failure under the uniaxial compression test, the machine began fracture protection, and failed to record the full stress–strain curve data. Therefore, in the case of the uniaxial compression test, there was no gentle curve after failure. For this reason, the damage correction coefficient To verify the damage evolution equation further, the experimental results of different temperatures were compared. Although the stress–strain curves of granite specimens become gentler with increasing temperature, the stress of specimens still decreases drastically when the axial stress reaches the uniaxial compressive strength. For this reason, the damage shape parameters at different temperatures were all taken as K and the damage variable correction factor The results show that the TM unified constitutive model based on Weibull distribution and triple-shear Drucker–Prager yield criterion, which introduces the compaction coefficient The relationship between the axial strain and the total damage evolution of granite subjected to different temperatures is shown in (1)After heat treatment, rock is given a thermal load first, and thermal damage appears inside the rock specimen. Based on this condition, the stress load is the second load coupled with the thermal load. The process is not the mechanical superposition of thermal damage and stress damage but, rather, the coupling effect. Meanwhile, the coupling effect of stress damage and thermal damage is lower compared with the mechanical superposition of them;(2)The TM unified constitutive model can describe the whole process, including the compaction stage and post-failure stage. The experimental results of uniaxial compression testing are used for verification of the theoretical model. The results show that the theoretical curves match the experimental curves accurately;(3)The relationship between the total damage evolution ratio and the axial strain of granite subjected to heat treatment at different temperatures can be calculated by the total damage evolution equation. The results show that the peak of the total damage evolution ratio occurs later with increasing temperature. In addition, the peak value of the total damage evolution ratio also decreases with increasing temperature.