Felis catus and ferrets Mustela furo) and a mesopredator (stoats Mustela erminea), in relation to their primary prey  and habitat use. Further, we tested for mesopredator release by selectively removing cats and ferrets in a pulse perturbation experiment. We found compelling evidence of niche partitioning; spatiotemporal activity of apex predators maximized access to abundant invasive prey, with ferrets targeting lagomorphs and cats targeting rodents. Mesopredators adjusted their behavior to reduce the risk of interference competition, thereby restricting access to abundant prey but increasing predation pressure on diurnal native birds. Stoats were only recorded at the treatment site after both larger predators were removed, becoming the most frequently detected predator at 6 months post‐perturbation. We suggest there is spatial and resource partitioning within the invasive predator guild, but that this is incomplete, and avoidance is achieved by temporal partitioning within overlapping areas. Niche partitioning among invasive predators facilitates coexistence, but simultaneously intensifies predation pressure on vulnerable native species.Predators compete aggressively for resources, establishing trophic hierarchies that influence ecosystem structure. Competitive interactions are particularly important in invaded ecosystems where introduced predators can suppress native prey species. We investigated whether niche partitioning exists within a guild of invasive mammalian predators and determined the consequences for native species. Over 4405 camera‐trap days, we assessed interactions among three invasive predators: two apex predators (feral cats  Predators are an important driving force in ecosystems, regulating food webs and the distribution of resources  exhibit a reciprocal distribution with stoats (M. erminea) in parts of their native range, being rare or absent in optimal prey habitats, but occurring more frequently in less productive areas , ferrets (M. furo), and stoats are the largest introduced predators and are responsible for declines and extinctions of many native species  and bottom‐up  processes in determining niche partitioning and intraguild interactions. In New Zealand, invasive predators depend on other introduced pests as their primary prey, a resource that fluctuates due to natural or management‐driven perturbations Norbury, . In turnOryctolagus cuniculus, rodents and birds) to test if niche partitioning was driven by prey resources and/or competition. We made the following a priori predictions: (1) dominant predator (cat and ferret) activity would correspond to the activity patterns of preferred prey (resource matching); (2) subordinate predators (stoats) would avoid periods of high activity by dominant predators, as opposed to matching the activity of preferred prey (safety matching); (3) site occupancy and abundance of dominant predators would correspond to the occupancy and abundance of preferred prey (resource matching), and; (4) site occupancy and abundance of the subordinate predator would be negatively correlated with those of dominant predators (safety matching). We predicted that: (5) removal of cats and ferrets would lead to increased abundance and/or occupancy of stoats, with no corresponding increase in a non‐treatment site. To test these predictions, we investigated fine‐scale spatial distributions, habitat use, prey availability, and activity patterns of sympatric invasive predators . A pulse perturbation experiment was implemented to test whether the subordinate predator (stoat) responded to dominant predator (cat and ferret) removal. We used occupancy models  and kānuka (Kunzea robusta) at higher elevations (range: 220–635 m), and remnant broadleaf woodland at lower elevations (range: 120–385 m). The study sites were 15 km apart, with no recent history of predator control.This study was undertaken at two farmland sites in Hawke's Bay, New Zealand . Study sites were predominantly grazed pasture with a mosaic of scrub patches dominated by mānuka , lagomorphs (rabbits and hares Lepus europaeus), birds, and invertebrates , to ensure species’ activity could be matched to the diel cycle.In total, 80 (40 per study area) Reconyx PC 900  trail cameras were deployed, one camera at each monitoring station, with ~500‐m spacing between cameras Figure . Home raCameras were fixed to a wooden post, with the base 7 cm from the ground, the approximate shoulder height of mustelids. Cameras were orientated horizontally, the most appropriate set‐up for our focal species  and fresh rabbit meat  site and a non‐treatment site , early winter (May–June) and late spring (November) 2014 – to monitor focal species at both study sites. Each monitoring session was 21 days after the day cameras were activated. Sampling grids, each covering 7 kma priori prediction for the influence of habitat on cats and stoats, which are generalist predators. However, interference competition tends to be stronger in homogeneous habitats  design was implemented to test whether stoats responded to dominant predator (cat and ferret) removal. After 3 weeks of pretreatment monitoring in May 2014, feral cats and ferrets were removed by professional trappers during a pulse perturbation on the treatment site, while the non‐treatment site remained unperturbed.An additional camera‐trap trial was conducted at the non‐treatment site in summer (January–February) 2014 as part of a separate project  independent observations, and (2) site use. To avoid autocorrelation, we considered independent observations to be photographs of the same species on the same camera more than 30 min apart, except when individuals could be reliably distinguished  but used the lowest threshold (∆1) for stoats, based on the number of observations ; nocturnal (between 1 h after sunset and 1 h before sunrise); cathemeral (throughout the 24 h period) and crepuscular (up to1 h before and after sunrise and sunset)  to ensure that activity patterns did not differ between trials at the non‐treatment site. Following the methods of Linkie and Ridout  and could therefore be combined. Prior to combining data, we confirmed, using the coefficient of overlap (Δ1), that there were no significant differences in the activity patterns of focal predators among sites and between seasons  was calculated in other seasons.To assess mesopredator occupancy as a function of dominant predators, we ran a single‐season stoat model. Apex predator (cat/ferret) and prey (rodent/lagomorph/bird) were included as linear covariates in the psi (ψ) conditional model, where psi refers to the probability that a site is occupied by a species of interest  = ψ (Rb + Rd + B + S + P), p(Rb + Rd + B + S + P), where prey covariates relate to rabbits (Rb), rodents (Rd), and birds (B), habitat covariates are scrub (S) or pasture (P), p is detection and ψ is occupancy. Covariates were selected because they have been shown to influence the distribution of our predators or represent landscape habitat attributes that we predicted may influence predator occurrence or detectability. Prey were included as linear covariates based on the conditional occupancy estimates (ψ‐cond) of prey occurrence for each site and habitat covariates were coded as binary variables where a monitoring station was either located on scrub or pasture. The scent lure was not included in our hierarchical models as every site received the same lure treatment and a previous study demonstrated the attractiveness of the scent lure for our target species  was included based on the observed presence (1) or absence (0) of this predator at monitoring stations. Following MacKenzie et al. (c (QAICc) where we identified overdispersion , and Akaike weights (w) were used to determine the best supported models for each species  of the top ranked model were considered as final candidate models. To be consistent with our AIC‐based approach to model assessment, we present beta coefficients with ~85% confidence intervals (CIs) based on unconditional, VIF‐inflated SEs , ferrets 72 (27%), stoats 23 (8%), and a weasel 1 (<1%). There were an additional 35 stoat observations in the preliminary camera‐trap trial. Livestock  accounted for the majority of photographs at both sites. Cameras were deactivated on 11 of 240 sampling occasions due to livestock disturbance or camera malfunctions. The number of active camera traps, trap days, and photographs are summarized in Table A trapping effort of 4405 trap days generated 769,408 photographs, of which 19,120 were of the focal mammalian predators and prey. There were an additional 26,212 photographs of 27 bird species   at night.In contrast, stoats were almost entirely diurnal, with a 0.91 kernel probability of being active during the day, and a third of activity taking place 1 h either side of noon. There were nocturnal observations of stoats on five (9%) occasions during the summer months (January–February). Stoats displayed minimal overlap with the two dominant predators Table ; stoat aCathemeral activity of cats corresponded broadly with all three prey groups Table . FerretsψB/A = 0.50, SE = 0.80) than absent . Ferrets were more than twice as likely to occupy  and be detected  at a site where a cat was recorded. Cats and ferrets co‐occurred at 36% of camera traps within a monitoring period, with sequential sightings less than 5 h apart on three occasions.The composition of the predator guild differed between sites, although cats were the most frequently detected predator at both locations  as the pA single‐season occupancy model revealed the importance of apex predators on stoat site use Table . Cats b. The modPrey resources varied considerably between study sites , with naïve site use of 49%. There were approximately four‐fold more independent observations of birds at the non‐treatment site (430 vs. 118). Introduced bird species, which made up more than 80% of all independent observations, were primarily in pasture (10 of 14 bird species recorded in this habitat were introduced), while native species were more common in scrub/forest patches (9 of 13 species recorded were native) . At the treatment site, prey were associated with cat site use, appearing in every model; the top four models all included the rabbit covariate, which was selected against . Cat probability of detection was positively associated with rodents , the most common prey at the site. Habitat did not influence detection probabilities as neither variable appeared in the top supported models.Predator observations were driven by prey and habitat type, particularly for apex predators Table . For catc <2. Ferret probability of detection was not associated with prey covariates, with the null model having greatest support.The top model for ferrets identified just two important covariates; ferrets were positively associated with rabbits  and pasture . The top model with both these covariates was the only model supported with AICThe stoat model revealed that stoat site use was negatively associated with the lagomorph and bird covariates, which had a combined model weighting of 0.55, while the cat covariate accounting for the remaining model weight.Of the five camera traps where stoats were detected at 6 months post‐control, both cats and ferrets had been previously recorded at one camera trap and cats had been found at two others prior to the pulse perturbation. Rodents were recorded at all five of the camera traps where stoats were detected, birds at four, and rabbits at two.p = 0.0004) and ferrets (p = 0.0021) following predator removal, representing a fall of 82% and 76%, respectively . A third of the sites where stoats were detected had been previously occupied by both apex predators. At the non‐treatment site, stoat observations were relatively constant over the recording periods .Naïve site use was used to assess stoat distribution as low numbers of detections precluded the use of occupancy estimates. No stoats were recorded at the treatment area prior to the removal of cats and ferrets, but stoats were observed at 14% of monitoring stations 6 months post‐perturbation , as stoats approached ferret odor during daylight but never at night. Predators were recorded with prey on two occasions: a cat was photographed with a rodent at the treatment site in May and a stoat dragged a dead rabbit to the lure at the non‐treatment site during November   and selected different habitat types. Surprisingly, given the ubiquity of interference competition among top order predators, there was no evidence of competitive exclusion between the two apex predators; ferrets were more than twice as likely to occupy and be detected at a station where a cat was recorded. Ferrets are tenacious, with a bite force equivalent to the much larger domestic dog , but did so at the home range scale . Cats and ferrets were active concurrently but selected for different habitats. Prior to the perturbation, the apparent absence of stoats suggests they are prone to competitive exclusion in pasture‐dominated ecosystems where spatial avoidance is difficult. Mustelids never co‐occurred at a camera within a recording period, but stoats co‐occurred with cats on six occasions. Nocturnal activity patterns displayed by cats at these camera traps contrasted with the diurnal activity of stoats, reducing the risk of an encounter. On occasions when stoats were nocturnal they did not approach the lure at a camera trap, which contrasted with behaviors recorded in daylight hours when the absence of dominant predators could be confirmed visually.The results generally supported our prediction of resource matching, as dominant predators were positively associated with preferred prey across monitoring stations. Cat detection probabilities were driven by rodents and this conclusion was supported by observations of concurrent nocturnal activity. Unexpectedly, cats avoided pasture with rabbits, particularly at the treatment site, where rabbits appeared as a negative covariate in all models. However, ferret distribution at the treatment site was primarily driven by rabbits and differentiation along the prey resource dimension may facilitate coexistence of ferrets and cats. In support of our prediction, stoat distribution corresponded with safety matching, being absent prior to the pulse perturbation, but subsequently recorded after removal of dominant predators.Stoat population densities are frequently low in New Zealand and conclusive evidence for trophic cascades is therefore elusive. In support of our mesopredator release prediction, stoats were only detected after dominant predator removal and, 6 months later, stoats had changed from being undetected to the most common predator at the treatment site. An alternative explanation is that seasonal variations in behavior, such as changes in activity patterns in response to prey availability , may explain increased detections of stoats at the treatment site in the final recording period. However, there was no evidence that predator site use changed at the non‐treatment site and other authors have reported no change in predator activity over similar time periods  from the entire country by 2050 (Russell et al., The New Zealand government recently adopted an ambitious goal to eradicate stoats, ship rats and possums (Where the entire suite of invasive predators cannot be managed simultaneously, our research supports the proposal to initially eradicate stoats, as the removal of apex predators may result in mesopredator release (Rayner et al., Our study provides strong evidence that niche partitioning facilitates invasive predator coexistence. Collectively, the predator guild was active throughout the diel cycle and across habitat types, maximizing exploitation of the fundamental niche. Alterio and Moller  have sugThis research highlights the difficulties in managing invaded ecosystems, where complex interactions between native and introduced biota occur at landscape scales, and well intentioned perturbations can have detrimental impacts. Understanding the interactions among species in invaded systems will inform the timing, extent, and risks of management intervention. Future research could quantify how cascading effects of trophic interactions will influence the predation risk of native species.The authors declare no conflict of interest.Patrick M. Garvey, Alistair S. Glen, Mick N. Clout and Roger P. Pech wrote the main manuscript text and Patrick M. Garvey prepared figures. Patrick M. Garvey, Alistair S. Glen and Margaret Nichols carried out fieldwork. Patrick M. Garvey and Margaret Nichols reviewed camera trap images. All authors reviewed the manuscript.Appendix S1Click here for additional data file.


To determine the rates and risk factors of pedicle screw placement accuracy and the proximal facet joint violation (FJV) using TINAVI robot-assisted technique.Patients with thoracolumbar fractures or degenerative diseases were retrospectively recruited from June 2018 and June 2020. The pedicle penetration and proximal FJV were compared in different instrumental levels to identify the safe and risk segments during insertion. Moreover, the factors were also assessed using univariate and multivariate analyses.p = 0.007), facet angle ≥ 45° , distance from skin to insertion point ≥ 4.5 cm , and lumbosacral instrumentation  were independently associated with intra-pedicular accuracy; the L5 insertion  and facet angle ≥ 45°  were independently associated with the proximal FJV.A total of 72 patients with 332 pedicle screws were included in the current study. The optimal and clinically acceptable screw positions were 85.8% and 93.4%. Of the 332 screws concerning the intra-pedicular accuracy, 285 screws (85.8%) were evaluated as Grade A according to the Gertzbein and Robbins scale, with the remaining 25 (7.6%), 10 (3.0%), 6 (1.8%), and 6 screws (1.8%) as Grades B, C, D, and E. Moreover, in terms of the proximal FJV, 255 screws (76.8%) screws were assessed as Grade 0 according to the Babu scale, with the remaining 34 (10.3%), 22 (6.6%), and 21 screws (6.3%) as Grades 1, 2, and 3. Furthermore, the univariate analysis showed significantly higher rate of penetration for patients with age < 61 years old, sex of female, thoracolumbar insertion, shorter distance from skin to insertion point, and smaller facet angle. Meanwhile, the patients with the sex of female, BMI < 25.9, grade I spondylolisthesis, lumbosacral insertion, longer distance from skin to insertion point, and larger facet angle had a significantly higher rate of proximal FJV. The outcomes of multivariate analyses showed that sex of male (adjusted OR 0.320, 95% CI 0.140–0.732; TINAVI robot-assisted technique was associated with a high rate of pedicle screw placement and a low rate of proximal FJV. This new technique showed a safe and precise performance for pedicle screw placement in spinal surgery. Facet angle ≥ 45° is independently associated with both the intra-pedicular accuracy and proximal FJV.The online version contains supplementary material available at 10.1186/s13018-022-03271-6. The pedicle screw placement has been widely applied in the treatment of spinal fractures, tumor, infections, deformities, and degenerative diseases for the improvement of stability after the spinal reconstruction. However, due to the spinal cord, nerve root, and blood vessels adjacent to the pedicle, the malposition of screws might contribute to severe complications, the rate of which was reported as 4.2% of patients . BesidesInitially, the pedicle screws were placed in open methods with inevitable need for paravertebral muscle dissection resulting in excessive bleeding and trauma of soft tissues. With the wide acceptance of minimally invasive concepts, various insertion techniques, such as the percutaneous fluoroscopy-guided, patient-specific template-guided, the computer-assisted, and robot-assisted (RA) navigation techniques, have been designed to guide the pedicle screw placement for the enhancement of insertion accuracy, protection of proximal facet joint, and reduction of severe complication including neurological, vascular, and muscular injuries , 6. AmonRecent years, a new robot system named TiRobot  has been introduced in orthopedic surgery, which received China Food and Drug Administration approval in 2016  reportedA consecutive series of patients who underwent TINAVI RA percutaneous pedicle screw placement for the treatment of thoracolumbar fractures or the degenerative diseases between June 2018 and June 2020 were, respectively, recruited, and all the operations were conducted by a surgical team. The study was approved by hospital's institutional review board with all the included patients signing informed consent forms.The inclusion criteria were as follows: (1) patients older than 18 years old; (2) patients diagnosed of thoracolumbar fractures or degenerative diseases including lumbar disk herniation, spinal stenosis, grade I spondylolisthesis, and mild degenerative scoliosis; (3) patients undergoing TINAVI RA percutaneous insertion, regardless of purely instrumental fixation or decompression along with intervertebral fusion; (4) insertion segments ranging from T11 to S1; and (5) patient receiving postoperative three-dimensional (3D) CT examination. The exclusion criteria consisted of the following: (1) patients with a history of previous surgery on the thoracolumbar segments, and revision surgery in the same segments; (2) patients with destruction of the vertebral body caused by tuberculosis infection or tumor, and unclear anatomy of the surgical segments; and (3) patients with active local or systematic infection, spinal tumor, or the tuberculosis.Patients information on age, gender, body mass index (BMI), and diagnosis before surgery was obtained from medical records with the consent from patients.The percutaneous pedicle screw placements were performed with the TiRobot system composed of three basic elements: an optical tracking device, a workstation for preoperative planning and intraoperative controlling, and a robotic arm .After general anesthesia with endotracheal intubation, the patient took the prone position. Then, the position plate was located and the projection of the planned vertical body pedicle on skin was marked. The pedicle screws were inserted in according with the original method under the guidance of TiRobot system .As for the patients suffering from fractures, the pedicle screw was inserted directly along the guide wire, and the incision was closed after the fracture rehabilitation performed with the screw rod. In contrast, regarding the patients suffering from the degenerative diseases, the screw is placed on the non-decompression side after the insertion of guide wire under the robotic guidance, whereas the trajectory of screw is prepared along the guide wire on the decompression side, and then, the screw is placed after transforaminal lumbar interbody fusion (TLIF) .The accuracy of pedicle screw placement was assessed in accordance with the Gertzbein and Robbins scale  with theThe following factors that possibly affected the accuracy of the pedicle screw placement and proximal FJV were also collected during the assessment of the postoperative CT scan: proximal facet joint degeneration, facet angle, axial pedicle angle, sagittal pedicle angle, distance from skin to insertion point. The proximal facet joint degeneration was measured according to the Pathria classification . The radThe statistical analyses were performed with SPSS version 23.0. The continuous variables were described as the mean and standard deviation and the categorical variables as absolute (no.) and relative (%) frequencies. For statistical purpose, the rates and different grades of the accuracy of pedicle insertion and violation of proximal facet joint were analyzed in each instrumented level. Moreover, possible factors associated with intra-pedicular and proximal facet joint accuracy were gender, age, BMI, side of screw, diagnosis of grade I spondylolisthesis and mild degenerative scoliosis, facet degeneration, instrumented levels , depth of surgical field (distance of entry point from skin), facet angle, axial pedicle angle, and sagittal angle. After the univariate analyses of the above factors, the remaining issues with a significant level < 0.05 in univariate analyses were summarized to the final logistics regression modal of multivariate analyses. As for the intra-pedicular accuracy, the variables included in the multivariate analyses were gender, age, instrumented levels, depth of surgical field, and facet angle, while for the proximal FJV, the variables were gender, BMI, diagnosis of grade I spondylolisthesis, instrumented levels, depth of surgical field, and facet angle. The level of significance was defined as alpha = 0.05.A total of 72 patients with 332 pedicle screws were included in the study. The demographic characteristics and screws-related data are summarized in Tables p = 0.013).Of the 332 screws concerning the pedicle screw accuracy, 285 screws (85.8%) were evaluated as Grade A according to the Gertzbein and Robbins scale, with the remaining 25 (7.6%), 10 (3.0%), 6 (1.8%), and 6 screws (1.8%) as Grades B, C, D, and E, respectively. Optimal and clinically acceptable screw positions were achieved in 85.8% and 93.4% of all pedicle screws, respectively. The cortical encroachments in pedicles were found for 47 screws. Among the penetrations, the lateral cortex was the most frequently breached (74.5%), followed by the medial (21.2%) and inferior (4.3%) cortexes. Meanwhile, the risk of pedicle penetration at L5 level was significantly lower than that at other segments , whereas L5 insertion was associated with significantly higher risk of 3.153 times than others (p < 0.001). Besides, the screw head was the most frequent region responsible for proximal FJV (80.5%), followed by screw shaft (19.5%). There is no violation from the rod. The rates of intra-pedicular and proximal facet joint accuracy and the related raw ORs in different instrumented levels are shown in Tables In terms of the proximal FJV, 255 screws (76.8%) screws were assessed as Grade 0 according to the Babu scale, with the remaining 34 (10.3%), 22 (6.6%), and 21 screws (6.3%) as Grades 1, 2, and 3. The risk of proximal FJV in T12 insertion was 3.390 times than other levels , facet angle ≥ 45° , distance from skin to insertion point ≥ 4.5 cm , and lumbosacral instrumentation  were independently associated with intra-pedicular accuracy. Meanwhile, the L5 insertion  and facet angle ≥ 45°  were independently associated with proximal FJV.After the control of confounders, the multivariate analyses of associated with the intra-pedicular and proximal facet joint accuracy were performed concerning the issues in univariate analyses with a significant level < 0.05 (Table The accuracy of pedicle screw insertion is especially crucial because of the severe consequences followed by malposition including neurological and vascular injuries, revision surgery, and spinal instability , 16. MeaMany studies have concluded that RA technique showed improvement of intra-pedicular accuracy at the rates of 86.16–98.2% than conventional techniques , 24. HowThe importance of proximal facet joint protection has been increasingly realized in recent years, and the violation might lead to the spinal instability in adjacent segments and reduce the load-bearing ability, eventually resulting in the radiographic ASD , 28. TheThe factors that affected the intra-pedicular accuracy and proximal FJV were also explored with univariate and multivariate analyses. As for the pedicle screw placement accuracy, our multivariate regression analysis demonstrated that lumbosacral instrumentation was 0.227 times likely to breach the cortex because of a larger diameter of pedicle in the lower lumbar segments. Thus, the occurrence of screw deviation causing severe penetration in lumbosacral segments might decrease than that in thoracolumbar segments. Moreover, we also found that the facet angle ≥ 45° and the depth of surgical field ≥ 4.5 cm are the protective factors in pedicle screw placement, which might result from the fact that in the lumbosacral levels, the facet angle was larger and the mean distance from the entry point to the skin was further than those in the thoracolumbar levels. Because of the higher rate of accuracy in lumbosacral instrumentation, the distance from entry point to screw ≥ 4.5 cm was less likely to breach the cortex. Moreover, we also found that the male patients suffered from less screw penetration.In terms of the independent factors that affected the cranial FJV, our multivariate analysis showed that the L5 insertion was 2.02 times more likely to breach the facet. Tian et al.  identifiThere are some potential limitations in the current study. First, no control group was included in the study and only the data of RA technique were reported without comparative results. Although we compared the outcomes with those from previously published studies, the higher biases appeared. Second, the overall intra-pedicular accuracy and proximal FJV consisted of thoracic and lumbar segments. The results may be affected due to the different anatomical characteristics. Third, because this study was focused on the RA system, the results were only applicable to the RA insertion instead of the universal operation protocol. Fourth, we only reported the radiographic results in a short-term follow-up period. Nevertheless, the postoperative short-term outcomes are crucial for a novel technique regarding the accuracy of pedicle screw placement. Continued assessment of safety and accuracy of screw placement in long-term follow-up using TINAVI RA technique is needed in the future work.TINAVI robot-assisted technique was associated with a high rate of pedicle screw placement and a low rate of proximal FJV. This technique showed a safe and precise performance for pedicle screw placement in spinal surgery.Additional file 1: Summary and characteristics of adverse events.Additional file 2: Univariate analyses of factors associated with intra-pedicular and proximal facet joint accuracy, respectively.


Pyridoxal 5′-phosphate (PLP)-dependent enzymes are found ubiquitously in nature and are involved in a variety of biological pathways, from natural product synthesis to amino acid and glucose metabolism. The first structure of a PLP-dependent enzyme was reported over 40 years ago, and since that time, there is a steady wealth of structural and functional information revealed for a wide array of these enzymes. A functional mechanism that is gaining more appreciation due to its relevance in drug design is that of protein allostery, where binding of a protein or ligand at a distal site influences the structure, organization, and function at the active site. Here, we present a review of current structure-based mechanisms of allostery for select members of each PLP-dependent enzyme family. Knowledge of these mechanisms may have a larger potential for identifying key similarities and differences among enzyme families that can eventually be exploited for therapeutic development. Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, is a coenzyme that is required for the activity of many proteins from bacteria to humans. These enzymes carry out a number of processes, including amino acid metabolism and biosynthesis of antibiotic compounds . PLP-depTo date, there are seven PLP-dependent protein families that are classified based on their 3-dimensional folds . AlthougIn addition to structural diversity, PLP-dependent enzymes also display a diverse repertoire of allosteric mechanisms. Protein allostery is key for the modulation of enzyme activity given a particular cellular context or binding partner. Protein allostery, a term first coined 60 years ago , is defiRhodobacter capsulatus were captured with both the internal aldimine (PDB 2BWN) and the glycine-bound external aldimine (PDB 2BWP)  . Subsequvalently . Importatrans interactions with the neighboring subunit via an interaction with a conserved arginine residue located in the ALAS catalytic glycine-rich motif. Importantly, mutation of this arginine in yeast or eukaryotic ALAS leads to a decrease in enzyme activity  encompasses numerous allosteric enzymes , includi complex , threoni complex , threoni complex , and O-a complex . Althoug complex . For exa complex . Cystath complex  where it complex   and can bind to each subunit to cause a 2- to 3- fold increase in activity   to inhibit ODC activity  . Difluoromiasis) ; this saomiasis) . Unfortuomiasis) , so ongonterface . Herbaceactivity . Anotheractivity  It is unactivity S)-selective branched-chain L-amino acid aminotransferases (BCATs) (R)-selective D-amino acid aminotransferases (DAATs) (R)-amine:pyruvate transaminases (R-ATAs) (R)- or (S)-stereospecificity and orient PLP differently in the active site  , (R)-sel (DAATs) , (R)-ami(R-ATAs) , and 4-a(R-ATAs) . Out of ive site .Generally, most Fold Type IV enzymes form homodimers for catalysis , but som315CXXC318 participate in thiol-thiolate interaction and are responsible for redox sensitivity. Cys315 acts as a sensor for redox regulation and helps in substrate orientation , which is the first PLP-dependent enzyme to be structurally determined . Fold TyOne of the main allosteric mechanisms of glycogen phosphorylase is phosphorylation at Ser14 located ∼45 Å from the active site  Krebs a. UnphospAll three isozymes have a well-conserved AMP-binding site that is formed by a bundle of helices located at the interface between dimer subunits  Sprang . HoweverThe function of the AMP site corresponds with intracellular energy demands. As energy needs increase, the intracellular concentration of AMP increases because of ATP hydrolysis. AMP binds in this site to stabilize the active relaxed state of GP (R state) that allows for access to the catalytic site . ATP andThe GP oligomeric state is an important facet in mediating GP activity. Homodimeric GP is the active form of the enzyme, but activation by AMP or Ser14 phosphorylation causes a change in the tertiary and quaternary structures of the enzyme to promote pairs of dimers to form tetramers. Tetramerization partially blocks access to the active site, thus decreasing enzyme activity to 12–33% of the fully active dimers . FurtherTwo additional effector binding sites have been described, the inhibitory site  and the indole site. Both sites inhibit glycogen phosphorylase by stabilizing the T state and working synergistically with other GP inhibitors . The inhHomology searches identified many PLP-dependent enzymes belonging to the above five fold types. However, other PLP-dependent enzymes that do not resemble the archetypal enzymes were also discovered and subsequently categorized into new fold types . Fold Ty2β2 tetramer (a dimer of αβ units)  catalyzes the reversible 1,2 rearrangement of the terminal amino group of DL-lysine and L-β-lysine. Unlike other PLP-dependent enzymes, 5,6-LAM requires radical propagation from another cofactor, adenosylcobalamin , to the external aldimine for its reaction mechanism to occur. This protein forms an αβ units) , and theβ units) . The 5,6on of E1 . The funloenzyme . The PLPloenzyme . 4,5-OAM folding . The α sivate E1 .+ cluster as coenzymes  can help activate 2,3-LAM by facilitating cluster reduction  was the first aminomutase to be discovered . It is aoenzymes . Most is radical . Becauseeduction  (Brazeaueduction .Pyridoxal 5′-phosphate is a coenzyme involved in a number of essential cellular processes within nearly all organisms. The diversity of reactions catalyzed by PLP-dependent enzymes is not fully realized solely by examination of their 3-D structures and active site architectures. Rather, this review provides an overview of the diverse mechanisms of protein allostery utilized by members of PLP-dependent enzyme families. It is through allostery that PLP-dependent activities are tuned to meet a particular cellular need. Although there are some reoccurring themes, such as oxidation of a CXXC motif or regulation via an N-terminal or C-terminal extension, there are also many divergent mechanisms. These mechanisms include binding of diverse protein or small molecule effectors at distal sites which induce conformational changes that alter oligomerization or active site accessibility and architecture. Here, we present allosteric mechanisms from various members of each fold type. However, as structural and biochemical investigations continue, it is likely that more allosteric routes will be discovered.A common approach in drug development is the design of competitive inhibitors that directly disrupt catalysis by active site interference. This concept proves difficult with PLP-dependent enzymes because of the common reaction mechanisms within fold types. How can we target a particular enzyme without affecting a litany of other biological processes? Nature surmounts this problem by the evolution of allosteric regulation, where structural changes distal to the active site induce conformational changes to fine-tune catalysis. Allostery does not center around a certain mechanism but rather encompasses a multitude of means. By exploiting the natural phenomenon of allostery, the issue of active site similarity can be circumvented, and PLP-dependent enzymes that have been previously structurally characterized can be reexamined for novel therapeutic development. Not only will this expand drug discovery opportunities, but it will also aid in exploring novel structure-based means of allosteric regulation.


So, it is becoming attractive for medicinal chemists to design novel inhibitors for this class of enzymes with improved potency and selectivity towards the different isoforms. In the present study, three sets of carboxylic acid derivatives 5a–q, 7a–b and 12a–c were designed, developed and evaluated for the hCA inhibitory effects against hCA I, II, IX and XII. Compounds 5l, 5m, and 5q elicited the highest inhibitory activities against hCA II, IX and XII. In summary, structural rigidification, regioisomerism and structural extension, all played obvious roles in the degree of hCA inhibition. This present work could be a good starting point for the design of more non-classical selective hCA inhibitors as potential targets for several diseases.In searching for new molecular drug targets, Carbonic Anhydrases (CAs) have emerged as valuable targets in diverse diseases. CAs play critical functions in maintaining pH and CO Firstly, they are able to bind directly to the catalytic zinc displacing bound water-hydroxide anion, similarly to sulphonamides. Alternatively, certain carboxylic acid derivatives can anchor to the zinc-bound water-hydroxide ion via a hydrogen bonding, similar to the binding mode noticed for phenol-based molecules. Lastly, carboxylates may bind within an adjacent pocket to the entrance which is located outside the active site of the carbonic anhydrase. This interaction results in a block of the proton shuttle His64 residue in its “out” conformation, leading to the inhibition of CA catalytic activityThe carboxylic acid-bearing small molecules represent an important category of the non-classical carbonic anhydrase inhibitorsm-benzoic acid-bearing derivative , as well as potent hCAXII inhibitor (KI = 2.3 µM). Moreover, we developed another ureides series of piperine-based carboxylic acid derivatives that incorporate benzoic acid moieties linked to the natural product piperine through an ureido spacer2 .In 2020, our research team has reported novel series of benzofuran-based carboxylic acid small molecules that tethered with benzoic or hippuric acid motifs as potential CA inhibitors spacer2  exerted a]benzimidazole-tethered carboxylic acid derivatives as potential CA inhibitorsa]benzimidazole tail3 . In the current work, we devoted our effort to developing new non-classical CA inhibitors through the design of three sets of new carboxylic acid derivatives , Also, in 2020 we designed and prepared a novel methylthiazolo[3,2-le tail3  effectiv5a–q) and the second (7a–b) sets incorporated a benzoic acid moiety that is connected to different aryl tails through an enaminone linker, for the first set, or through the more flexible saturated 3-oxo-propylamine linker, for the second set, 5a–q) should be attributed to the unsaturation , as well as due to the formation of an intramolecular hydrogen bonding that resulted in a pseudo-six-membered ring. Lastly, a structural extension approach was utilised to replace the benzoic acid motif with the hippuric acid to furnish the third series 12a–c  to inhibit hCA I, II, IX and XII isoforms was assessed via the stopped-flow carbon dioxide hydrase assay.The first -1-phenylpropan-1-one 6, which subsequently reacted with m- and p- aminobenzoic acids 4b–c in absolute ethanol to furnish targeted carboxylic acids 7a–b with saturated linker between the ZBG and the aryl tail.In this study, the synthesis of the new carboxylic acids-based carbonic anhydrase inhibitors 9 was acylated with 4-nitrobenzoyl chloride 8 in dry dioxan to get (4-nitrobenzoyl)glycine 10, which was then reduced to the corresponding (4-aminobenzoyl)glycine 11. The third set of the target carboxylic acids with the desired structural extension 12a–c was prepared through the nucleophilic substitution reaction of (4-aminobenzoyl)glycine 11 with the previously prepared enaminones 3a,d,f in a boiling glacial acetic acid  equals 8.0 Hz. The latter Z-form was assumed to be established through the formation of two intramolecular hydrogen bonds that resulted in two pseudo-six-membered rings. 1H NMR spectra of ortho-carboxylic acids bearing enaminones 5a–e exhibited a doublet D2O exchangeable signal that was integrated for one proton, attributable to the NH group of the Z-form, at more down-field region δ 13.2–13.7 ppm when compared with those of 3- and 4-substituted carboxylic acids bearing enaminones 5f–k and 5l–q, respectively, at δ 11.5–12.1 ppm  according to the integrations of their signals. The presence of E-isomer was evidenced via the J constant for olefinic protons Ha and Hb (Ha-HbJ = 12.00 Hz) which was determined by the down-field doublet signal of E-Ha around δ 6.4–6.7 ppm  that came from the up-field doublet signals of Z-Ha around δ 6.13–6.41 ppm. Furthermore, the 1H NMR spectra displayed 2 doublet signal sets, integrated for one proton, each belonging to the (NH) proton for the E and Z isomers around δ 10.28–10.59 and 12.03–12.14 ppm  appeared down-field, due to the formation of intra-molecular hydrogen bonding with the carbonyl oxygen. However, this hydrogen bond between C=O and NH groups was absolutely confirmed, in a previous study, by X-ray single crystal analysis of a sulphonamide analog for compound 5 which exhibited a Z configuration around HaC=CHb with the trans HN-CHb.The downfield signals of NH proton could be attributed to the  E and Z  and 5. M1H NMR of meta-substituted carboxylic enaminones 5g and 5i, as well as 1H NMR for para-substituted carboxylic enaminones 5o, among 5f–k and 5l–q, revealed the appearance of down-field Z-Hb signal around δ 8.00 ppm as dd with J = 12.0 and 8.0 Hz due to E-Hb-NHJ and Z-Hb-HaJ, respectively, whereas the up-field E-Hb signal appeared around δ 8.14 as overlapped dd with two J = 12.0 Hz (E-Hb-NHJ = E-Hb-HaJ = 12.0 Hz). It is worthy to note that after the addition of D2O to the DMSO solution of these compounds  in their NMR tube, the coupling of NH proton for Hb in both Z and E forms disappeared and the dd of Z-Hb was converted to doublet signal due to the coupling of Z-Ha only with J = 8.0 Hz parallel with the conversion of overlapped dd of E-Hb to doublet signal due to E-Ha coupling only with J = 12.0 Hz  using acetazolamide (AAZ) as a reference CA inhibitor. The resulted inhibition constants were presented in In the current study, we have explored the inhibitory activities of the synthesised carboxylic acid derivatives hCA I exhibited the lowest inhibition in this study. Both the ortho and meta carboxylic acids bearing enaminones (5a–e and 5f–k) didn’t exert any noticeable inhibitory activity towards this isoform (KI > 100 µM), whereas the para carboxylic acids bearing enaminones 5l, 5m, 5n, 5p and 5q exhibited weak inhibitory effect , 7a–b with a saturated 3-oxo-propylamine linker showed the same behaviour; the meta carboxylic acid derivative 7a emerged as inactive, whereas the para carboxylic acid derivative 7b emerged as a weak inhibitor for the hCA I isoform (KI = 63.8 µM), The stopped-flow assay outputs disclosed that the off-target cytosolic hCA II isoform was affected by herein reported carboxylic acids bearing enaminones 5a–q and carboxylic acids 7a–b. Both the ortho and meta carboxylic acids bearing enaminones (5a–e and 5f–k) moderately inhibited hCA II isoform with inhibition constants spanning in the ranges 24.3–49.0 µM and 68.2–92.3 µM, respectively, whereas the para carboxylic acids bearing enaminones 5l–q effectively inhibited this isoform (KI range: 7.4–15.3 µM). In addition, carboxylic acids 7a–b with a saturated 3-oxo-propylamine linker displayed KI values equal to 64.3 µM and 16.7 µM, respectively. In particular, the best inhibitory activity in the CO2 hydrase assay was demonstrated by the para carboxylic acids bearing enaminones 5l, 5m and 5q that showed KI values in the single-digit micromolar range equal to 9.8 µM, 8.7 µM and 7.4 µM, respectively. Also, carboxylic acids 5n, 5o, 5p and 7b exhibited good activity against hCA II isoform with KI = 13.5 µM, 15.3 µM, 12.6 µM and 16.7 µM, respectively.Moreover, the stopped-flow assay outputs showed that the physiologically relevant 5a–q, it was found that shifting the carboxylic acid group from the ortho position (compounds 5a–e) to the meta position (compounds 5f–k) resulted in decreasing activity. For example, the phenyl and naphthyl-bearing enaminones 5f and 5k displayed about 2-fold decreased potency , in comparison to their ortho counterparts 5a and 5e , 5a–e) to the para position (enaminones 5l–q) led to about 2–6.5 fold enhancement of the inhibitory activity against hCA II isoform. For example, the phenyl and naphthyl-bearing enaminones 5l and 5q emerged as more potent inhibitors  than their ortho analogues 5a and 5e . Similarly in the second set, the para carboxylic acid derivative 7b displayed better activity (KI = 16.7 µM) than its meta counterpart 7a (KI = 64.3 µM) against hCA II isoform, Regarding the effect of the regioisomerism with the enaminones series hCA IX, the third examined isoform, was effectively inhibited by all carboxylic acids bearing enaminones 5a–q and carboxylic acids 7a–b evaluated here, with inhibition constants spanning in the range 0.76–32.5 µM. The best inhibitory action towards hCA IX isoform has been observed for enaminones 5m and 5q with sub-micromolar inhibition constants , whereas compounds 5g, 5l, 5n, 5o, 5p and 7b exerted good inhibitory activity with KIs in the single-digit micromolar range equal to 9.6 µM, 1.2 µM, 3.4 µM, 5.6 µM, 4.1 µM and 8.3 µM, respectively.The trans-membrane hCA II, grafting the carboxylic acid functionality at the para position (enaminones 5l–q) resulted in a much-enhanced activity (KI range: 0.76–5.6 µM) in comparison to the ortho  and meta  counterparts, 5l and 5q showed about 9- and 25-fold enhanced potency  in comparison to their ortho counterparts 5a and 5e , and showed about 13- and 15-fold increased activity in comparison to their meta counterparts 5f and 5k . In like manner, the para carboxylic acid derivative 7b exerted better hCA IX inhibitory activity (KI = 8.3 µM) than its meta counterpart 7a (KI = 23.7 µM), As discussed above for the inhibitory activity against 5l–q) revealed that only grafting the small fluorine substituent  can enhance the activity of the unsubstituted phenyl-bearing enaminone 5l (KI = 0.92 µM). In turn, substituting the phenyl tail with 4-Cl, 4-Br or 4-NO2 substituents reduced the activity. Moreover, the replacement of the phenyl tail with a naphthyl one resulted in the most potent hCA IX inhibitor in this work , 5f–k.On the other hand, examining the impact of the substitution of the phenyl tail within the most potent enaminones series  than their flexible counterparts 7a–b that bear the saturated 3-oxo-propylamine linker  which hints out that the conformational restriction for herein reported compounds is more favourable for hCA IX inhibitory action.It is worthy to mention that the phenyl-bearing enaminones 5a–q and 7a–b) efficiently inhibited the trans-membrane hCA XII isoform with KI range of 0.85–15.7 µM. Uniquely, the para carboxylic acids bearing enaminones 5l and 5p induced inhibition in the submicromolar range, with KIs of 0.97 µM and 0.85 µM, respectively. Similarly, to the inhibition profile for both hCA II and IX isoforms, the para carboxylic acids bearing enaminones (5l–q) exerted more potent activity (KI range: 0.85–6.2 µM) than their ortho  and meta  counterparts, 2 substitution , neither substitution of the phenyl tail nor its bioisosteric replacement with the naphthyl moiety enhanced the hCA XII inhibitory activity.The newly reported carboxylic acids  were synthesised and evaluated for their CA inhibitory activity. The first (5a–q) and the second (7a–b) sets incorporated a benzoic acid moiety that is connected to different aryl tails through an enaminone linker, for the first set, or through the more flexible saturated 3-oxo-propylamine linker, for the second set. For the third set, a structural extension approach was utilised to replace the benzoic acid motif with the hippuric acid to furnish the carboxylic acid derivatives 12a–c. The inhibitory activity for the prepared carboxylic acids was variable across the four tested CA isoforms, where for example, most compounds showed no or weak inhibition towards hCA I. Furthermore, compounds 5q, 5m, and 5l exhibited a very similar inhibition pattern towards hCA II and IX with KI values equal to 7.4, 8.7, 9.8 and 0.76, 0.92, 1.2 µM respectively. Regarding hCA XII, the para nitro-substituted derivative 5p showed the highest inhibitory activity. Structural extension as done in the third set 12a–c, showed no inhibitory actions towards all tested hCA isoforms, which suggested that this extension might lead to undesired interactions within the CA active site. Generally, ortho/meta carboxylic acid substituted derivatives exhibited weaker inhibitory activities against the tested hCAs. Future studies could reveal the desired pharmacophoric features which could enhance hCA inhibitory actions, as well as selectivity across different isoforms, that belong to this family of enzymes.Three sets of our designed non-classical CA inhibitors . HRMS spectra were recorded by a Bruker MicroTOF spectrometer.Stuart melting point apparatus was used for melting point measurements and were uncorrected. Schimadzu FT-IR 8400S spectrophotometer was used to record Infra-red (IR) spectra as KBr discs. NMR Spectra were recorded on a Bruker NMR spectrometer, at 400 and 100 MHz for 4.1.2.3a–f6113-(Dimethylamino)-1-phenylprop-2-en-1-ones  of o-, m-, p-aminobenzoic acid 4a–c was added to the reaction mixture. Reflux was continued for 3 h, and then the precipitated solid was filtered off while hot, washed with hot 70% ethanol (3 × 2 ml), dried and recrystallized from DMF/methanol mixture (1:4) to yield the target enaminones 5a–q (Supporting Information).An amount of 0.6 mmol from the appropriate enaminone intermediate 4.1.4.6  in absolute ethanol (12 ml), an equivalent amount of m- or p-aminobenzoic acid 4b–c  was added, and the mixture was heated under reflux for 4 h. The formed solid, after cooling, was collected and washed with aq. potassium bicarbonate solution and cold methanol (2 × 3 ml), then recrystallized from isopropanol to get the 3/4-((3-oxo-3-phenylpropyl)amino)benzoic acids 7a–b, respectively (Supporting Information).To a hot solution of 3-(dimethylamino)-1-phenylpropan-1-one 4.1.5.12a–c were prepared via the same method described previously for enaminones 5a–q, utilising (4-aminobenzoyl)glycine 11 instead of aminobenzoic acids 4a–c (Supporting Information).Enaminones 4.2.4.2.1.2 hydration activities utilising an Applied Photophysics stopped-flow instrument as described previouslySupporting Materials).The target carbonic anhydrase inhibitors reported in this work were evaluated for their potential CA-catalysed COClick here for additional data file.


Small blood vessel diseases are often associated with impaired regulation of vascular tone. The current understanding of resistance arteries often focuses on how a level of vascular tone is achieved in the acute phase, while less emphasis is placed on mechanisms that maintain vascular tone. In this study, cannulated rat superior cerebellar arteries (SCA) developed spontaneous myogenic tone and showed a marked and sustained constriction in the presence of diluted serum (10%), a stimulus relevant to cerebrovascular disease. Both phosphorylated myosin light chain (MLC-p) and smooth muscle alpha actin (SM-α-actin) aligned with phalloidin-stained actin filaments in the vessel wall, while exhibiting a ‘high to low’ gradient across the layers of vascular smooth muscle cells (VSMC), peaking in the outer layer. The MLC-p distribution profile shifted towards the adventitia in serum treated vessels, while removal of the serum reversed it. Furthermore, a positive correlation between the MLC-p signal and vessel wall tension was also evident. The gradients of phosphorylated MLC and SM-α-actin are consistent with a spatial regulation of the myosin-actin apparatus in the vessel wall during the maintenance of vascular tone. Further, the changing profiles of MLC-p and SM-α-actin are consistent with SCA vasoconstriction being accompanied by VSMC cytoskeletal reorganization. SVD is usually manifest as a narrowing of resistance arteries, ultimately restricting end organ blood flow and promoting ischemia. Importantly, in the aging population, SVD is a major contributor to cerebrovascular pathology, including lacunar stroke and vascular dementia leading to cognitive impairment2. Mechanistically, an exaggerated small artery myogenic response 4 and endothelial cell dysfunction6 are two putative causes of excessive small vessel constriction. To date, our understanding of the myogenic response has predominantly focused on how pressure acutely induces vasoconstriction8, with relatively less emphasis being placed on how a small artery maintains its physiological diameter over the longer timeframe, referred to as myogenic tone. Vascular tone in small arteries is predominantly regulated by Ca2+-induced contraction of the actin-myosin myofilament apparatus. In this regard, Ca2+ mobilization in small artery vascular smooth muscle has been extensively studied in the past few decades12. More recently, mechanisms relating to actin cytoskeleton remodeling have been shown to also play an important role in the regulation of vasomotor tone and are likely to be mediated by mechanisms distinct from acute Ca2+ mobilization16. In addition to these processes, re-elongation of vascular smooth muscle cells (VSMCs) in the vessel wall17, and cross-linking and remodeling of extracellular matrix components18, have also been proposed to play important roles in sustained vasoconstriction or maintaining vascular tone19.Chronic vascular disorders, such as hypertension, are often associated with pathology and dysfunction of distal arteries20. In addition to delayed vasospasm21, several novel mechanisms have been proposed that likely contribute to the extent of brain damage after SAH, including early brain injury (EBI)23, intravascular inflammation24 and impaired regulation of the microcirculation25. Acute spasm of small cerebral vessels has been observed in rodent SAH models in vivo28. Further, leakage of blood and diffusion of serum through the subarachnoid space in SAH can be mimicked in vitro by the addition of serum to isolated and cannulated cerebral arteries. In our own preliminary studies, the presence of diluted serum was observed to induce significant and sustained vasoconstriction of small cerebral arteries ex vivo, which could potentially contribute to the acute cerebral vasospasm in experimental SAH.In addition to enhanced myogenic tone in chronic conditions such as aging and hypertension, common cerebrovascular abnormalities can arise from ischemic stroke and subarachnoid hemorrhage (SAH), with the latter often inducing severe brain damage with debilitating outcomesIn the current study, immunofluorescence microscopy was used to examine myosin light chain (MLC) phosphorylation and smooth muscle alpha actin (SM-α-actin) in the vessel wall of small cerebral arteries that were fixed at a stable level of myogenic or serum-induced tone. This approach provided a level of spatial resolution not possible in classical western blotting assays and allowed us to test the novel hypothesis that sustained serum-induced contraction of cerebral arteries was associated with contractile protein reorganization. Our results demonstrate that in cannulated rat superior cerebellar arteries (SCA), the distributions of both phosphorylated MLC and SM-α-actin actin filaments follow a transmural gradient that peaks at the outer layer of vascular smooth muscle (VSM) and reaches a minimum towards the inner layer of VSMC. Serum treatment modified the transmural gradient of phosphorylated MLC. These results are consistent with small arteries maintaining a stable level of tone through mechanisms involving MLC and actin organization that differ from, and likely support, mechanisms underlying the acute phase of vasoconstriction.Immunofluorescence microscopy Fig. A was emp30. While the distribution of desmin is more discrete and GFAP is more diffuse, labeling for both proteins was apparent across the whole thickness of vessel wall. Dual labeling experiments were also employed to compare the distribution of MLC-p with that of myosin heavy chain A (MHA), and to compare SM-α-actin with that of pan-actin.  Fig. A,B in SCin. Fig. C The conIn SAH, when blood leaks into the subarachnoid space, where blood cells likely coagulate, the soluble blood-borne factors (serum) could potentially diffuse through the cerebral fluid, affecting both neuronal tissues and blood vessels that are bathed in the cerebral fluid. To mimic this condition ex vivo, we tested the effect of 10% serum on the myogenic tone of rat SCA at 70 mmHg. As shown in Fig. The transmural distributions of MLC-p, SM-α-actin and phalloidin labeled actin filaments Fig. A,B were To further investigate the transmural distribution of phosphorylated MLC under control conditions and following serum-induced vasoconstriction, we simultaneously stained total regulatory MLC  and phosphorylated MLC (MLC-p) in the vessel wall of both groups. As shown in Fig. We further examined if the shifted MLC-p profile could be restored by reversing the serum-induced vasoconstriction. Treatment with the endothelium-dependent vasodilator acetylcholine  caused only a moderate (~ 13%) but statistically significant dilation of the serum-induced constriction, while wash-out of serum with PSS buffer restored vessel diameter to ~ 90% level of its original myogenic tone Fig. A. In theBlocking of MLCK activity using the kinase inhibitor, ML-7 (15 μM), abolished the spontaneous myogenic tone of rat SCA at 70 mmHg, and addition of 10% serum induced no further vasoconstriction , while less is understood about how MLC phosphorylation and the actin cytoskeleton are regulated when vessels maintain a level of stable tone. Intriguingly, our results suggest that a graded distribution of MLC phosphorylation and SM-α-actin cytoskeleton exists across the vessel wall of rat SCA, which peaks near the outer layer of VSMCs , and diminishes towards the inner layers of VSMCs . In the presence of rat serum (10%), ex vivo SCAs exhibited robust and sustained vasoconstriction, which could be reversed by wash-out of the serum, and inhibited by pretreatment with ML-7, while only modest reversal was observed in the presence of low does acetylcholine 0.1 (μM). Interestingly, after serum-induced vasoconstriction stabilized, we observed a shift of the MLC-p profile towards the outer edge of the vessel, as well as a decrease in the SM-α-actin and MLC-p fluorescence intensities. The changes in MLC-p intensity and transmural distribution were reversed by removal of the serum, while partial dilation with Ach was accompanied by a left shift of the SM-α-actin transmural distribution. Collectively, these fluorescence-labeling results reveal a spatial regulation of MLC phosphorylation and SM-α-actin in the vessel wall, both in the control condition and following serum-induced vasoconstriction.31. Although highly sensitive, this technique does not allow detection of the spatial distribution of MLC phosphorylation within the vessel wall. The present study demonstrates that immuno-fluorescence microscopy is another sensitive and valuable tool for assessing MLC-p and SM-α-actin in single cannulated small arteries, with the added benefit of spatial resolution. The graded distribution of MLC phosphorylation has been previously reported across the left ventricular wall of the heart in a pattern similar to that observed in our SCAs, with a high MLC-p level at the outer wall that fades towards the inner wall32. In cultured gerbil fibroma cells, Peterson et.al. have shown a concentration of MLC-p at the cell periphery that was significantly greater than in the central region of the cell, while treatment with calyculin A (an inhibitor of myosin phosphatase) further enhanced the concentration of MLC phosphorylation at the cell periphery33. The graded distribution of MLC-p within the vessel wall perhaps challenges the current assumption that sustained vascular tone is achieved through uniform contraction of vascular smooth muscle cells and suggests there is a more refined control of smooth muscle contraction within the vessel wall.The finding of a graded distribution of MLC phosphorylation and SM-α-actin across the vessel wall is novel and somewhat surprising. This pattern was not observed for the immuno-labeling of MHA, GFAP, desmin and pan-actin in the vessel wall, suggesting that antibody diffusion is not a limiting factor in the labeling of target proteins across the vessel wall. Prior analysis of MLC phosphorylation in cannulated resistance arteries has been limited by the small size of these vessels. Previously, a refined western blot technique had been developed by Takeya et. al. to evaluate the MLC phosphorylation in single arterioles34, how this process is regulated in small vessels maintaining a stable level of tone has not been thoroughly investigated. Earlier studies in larger SM tissues have established that an increase of MLC phosphorylation is necessary for the generation of smooth muscle force/stress, although this process does not follow a linear relationship, e.g. the fast contraction phase is usually associated with a higher level of MLC-phosphorylation, while the force-maintenance phase (“latch-state” or steady-state phase) is usually associated with a lower level of MLC-phosphorylation35. After carefully examining the levels of MLC-p and smooth muscle contractile force at steady-state, Ratz et. al. and Rembold et. al. have demonstrated that the smooth muscle contractile force (stress) is positively correlated with the level of MLC phosphorylation37. In myogenically active small arteries maintaining stable vascular tone, the mechanical output of VSMCs is usually measured by the degree of wall tension using LaPlace’s law. In earlier studies, we reported that MLC phosphorylation in small resistance arteries is positively correlated with wall tension38, which is consistent with the relationship found in the smooth muscle layer of larger arteries37. Using a refined western blotting technique31, Johnson et al.39 and Moreno-Dominguez et al.40 have also shown that the degree of MLC phosphorylation in cannulated cerebral and skeletal muscle resistance arteries was increased when intraluminal pressure was raised from 10 to 60 and 80 mmHg, which is likely accompanied by a significant increase of wall tension in these vessels. These studies, together with earlier observations, suggest that smooth muscle MLC phosphorylation is likely positively correlated with the vessel wall tension in small arteries.It should be noted that the graded distribution of MLC-p in constricted SCAs is observed with vessels maintaining stable vascular tone. Whether there is spatial regulation of MLC phosphorylation during the acute phase of stimulated vasoconstriction is still unknown. Although calcium-mediated MLC phosphorylation has long been recognized as the central mechanism controlling VSMC contractile force44. However, it is often not fully considered that the mechanics of smooth muscle contraction recorded during isometric myography differ substantially from those of cannulated and pressurized vessels, i.e. there is no pressure stimulus and VSMCs are not allowed to shorten during isometric contraction. While the estimation of contractile force is directly measured by the force transducer in a wire myography instrument, in the cannulated and pressurized vessel, the contractile force can be estimated by wall tension45. In the maintained phase (or steady-state) of vasoconstriction for a cannulated artery, the vessel diameter stabilizes, indicating that an equilibrium has been reached between the contractile force generated by VSMCs and the wall tension imposed by transluminal pressure, thus allowing a direct estimate of VSMC generated forces using wall tension.This suggestion could, however, be counter-intuitive in the case of agonist/reagent-induced isobaric vasoconstriction. It is widely assumed that VSMCs will generate greater force during vasoconstriction, which is partly based on the many observations made using isometric tension myography with either arterial rings or vascular smooth muscle strips, where vasoconstrictor agonists induce an increase of contraction forceT = p*r, where “T” is wall tension, “p” is pressure and “r” is vessel radius. In the current study, sustained serum-induced vasoconstriction in SCAs led to a smaller “r” with no change in luminal pressure (70 mmHg). As such, the calculated wall tension (“T”) of the constricted vessel, would be expected to be smaller than that of the control vessels. Thus, the VSMCs in the control vessel need to generate greater force to oppose the resulting wall tension, compared to the serum-constricted vessel, contrary to what might be intuitively assumed. It should be noted that this conclusion only applies to the maintenance phase of vasoconstriction, and does not necessarily describe the active vasoconstricting process. Previous studies of vasoconstriction mostly focused on the active constricting phase, where agonist-induced vasoconstriction is accompanied by either an increase in cytosolic free calcium in VSMCs47 or a decrease of myosin light chain phosphatase (MLCP) activity49, both of which will presumably promote MLC phosphorylation and hence force generation. The discrepancy between these results and the current observation suggested that the regulation of MLC phosphorylation in VSMC could be different during the active constricting phase vs. the maintenance of vasoconstriction, which would suit the changing mechanical demands of each condition. There is currently no clear answer to how this transition is achieved at the molecular level, suggesting this is an overlooked component of our understanding of vasoconstriction. MLC phosphorylation and cytoskeletal organization are key mechanisms linking agonist-induced signaling and VSMC force generation, hence investigating these processes in the wall of cannulated resistance arteries will facilitate answering the questions raised above. Our results indicate that in cannulated rat SCA, and in the conditions described here, the control vessels had a higher level of MLC phosphorylation compared to serum-treated SCAs, and that the MLC phosphorylation is positively correlated with wall tension among the four groups tested  and Rho-ROCK58, which also may contribute to phosphorylation of MLC in the outer layer VSMCs. This result also appears to support the current finding that MLC-phosphorylation positively correlates with vessel wall tension in rat SCA that exhibits a stable level of tone. It is worth noting that how MLC phosphorylation is regulated in other types of small arteries still needs to be elucidated and it is possible that heterogeneity may exist.Serum induced SCA vasoconstriction was blocked by the MLCK inhibitor, ML-7, suggesting that MLCK activity is required for this process. However, the MLC phosphorylation is increased in the group treated with serum plus ML-7 compared to the serum only group, suggesting that the regulation of MLC phosphorylation in steady state dilated rat SCA could be more complex than previously assumed. It is conceivable that, despite ML-7 inhibition of MLCK activity, other MLC phosphorylation regulatory mechanisms, independent of MLCK, are activated in the vessel wall. Candidate signaling mechanisms may include ILK (integrin linked kinase)59. Figure 60, we suggest that enhanced mechano-transduction in the outer layer of VSMCs requires enhanced localization of MLC-p and SM-α-actin. For example, the enhanced activation of mechanosensitive ion channels61 may lead to a higher cytosolic calcium level in the outer layer of VSMCs, which could promote higher levels of MLC phosphorylation. In addition, the activation of integrin-, cadherin- and Angiotensin II type I receptor-mediated mechanotransduction63 could also lead to localized contractile events that are likely to be associated with the activation of MLC phosphorylation. In this regard, the presence of VSMC adherens junctions in the wall of pressurized rat SCA was demonstrated by immunolabeling of β-catenin  approved by the Institutional Animal Care and Use Committee of the University of Missouri, USA and conformed to the Guide For the Care And Use Of Laboratory Animals published by the US National Institutes of Health . The reporting in the manuscript follows the recommendations in the ARRIVE guidelines.2, 1 MgSO4, 1.2 NaH2PO4, 3 MOPS, 5 glucose and 1 pyruvic acid). For each animal, one SCA vessel was used. A chamber heater and a pressure control unit  were used to control the vessel temperature (37 °C) and lumen pressure, respectively. Bath solution was changed every 10 min during an experiment. Vessel intraluminal pressure was initially set to 50 mmHg (in the absence of flow). and passive vessel diameter at 50 mmHg was measured in the absence of Ca2+. After establishing spontaneous myogenic (intrinsic) tone, pressure was raised step-wise to 70 mmHg (and then 90 mmHg in limited experiments). Each pressure step was maintained for 10 min to ensure that the vessel had achieved a stable lumen diameter. For serum exposure experiments, after vessel diameter stabilized at 70 mmHg, the bath solution was replaced with PSS containing 10% Rat serum  or PSS alone (negative control). For ML-7 experiments, after vessel spontaneous tone stabilized at 70 mmHg, the bath solution was replaced with PSS containing 15 μM ML-7. After 10 min of treatment, the bath solution was replaced again with PSS containing 10% rat serum and 15 μM ML-7. Images of the vessel segments were recorded using a video camera and vessel lumen diameter measured offline using an edge-detecting software written in Matlab.Male Sprague–Dawley rats (200 ~ 250 g of weight) were used for all experiments. Following anesthesia by intraperitoneal injection of sodium pentobarbital (10 mg/100 g body weight) and euthanasia by pneumothorax, the brain was removed and placed in cooled (4 °C) dissection buffer. The superior cerebellar artery (SCA) was then isolated and cannulated between two glass micropipettes in a vessel chamber  containing physiological saline solution  at steady-state for 1 h with transmural pressures of 70 mmHg. A relatively high concentration of PFA was used with the goal of achieving rapid fixation across the cell layers in the vessel wall. Vessels were then incubated in glycine buffer (0.1 M glycine in MilliQ water) for 1 h (2×) to quench remaining paraformaldehyde. Vessels were subsequently exposed to a primary antibody buffer (1% BSA (w/v), 0.05% Triton X-100 (v/v) in sodium citrate solution buffer ) plus primary antibody with agitation at 37 °C for 48 h. The vessels were then washed 3 × with buffer (0.05% Triton X-100 in sodium citrate solution buffer) and incubated in secondary antibody solution overnight (37 °C with agitation). After incubation with the secondary antibody, the vessel was washed 3× (0.05% Triton X-100 in sodium citrate solution), mounted on a coverslip, and imaged using confocal microscopy. Details of the antibodies used are provided in Supplemental Table Vessel wall image stacks (with z-slice = 0.3 μm) were analyzed using Image J . An orthogonal cross section was selected at the center bottom of the vessel . For Western blot, the samples were probed using primary antibodies against phosphorylated MLC and SM-α-actin, followed by HRP-conjugated goat anti-rabbit or anti-mouse antibodies. See Supplemental Table P value less than or equal to 0.05.Results are presented as Mean ± SEM. Statistical comparisons were made using ANOVA and Student ‘s T-test. Statistical significance was assumed at a Supplementary Information.


In general, it is known that extreme climatic conditions such as El Niño and positive Indian Ocean Dipole (IOD+) cause prolonged drought in Indonesia's tropical peatlands so that groundwater levels (GWL) drop and peat is prone to fire. However, 27 years of GWL measurements in Central Kalimantan peat forests show the opposite condition, where the lowest GWL occurs several weeks before El Niño and after IOD+ reaches its peaks. We show that the dropped sea surface temperature anomaly induced by anomalously easterly winds along the southern Java-Sumatra occurs several weeks before the GWL drop to the lowest value. Local rainfall decreased, and GWL dropped sharply by 1.0 to 1.5 m, during the super El Niño events in 1997/98 and 2015, as well as remarkable events of IOD+ in 2019. It is suggested that the tropical peatland ecohydrological system (represented by the GWL), El Niño Southern Oscillation (ENSO), and IOD+ are teleconnected. Hence, monitoring GWL variability of peatland over the IMC is a possibility an alert for extreme climate events associated with El Niño and/or moderate IOD+. The active convection over the IMC drives the Walker circulation, and hence sea surface temperature anomaly (SSTA) in this region impacts global weather and climate13. Consequently, the land/islands of Indonesia that straddle along the equatorial band and air–sea interactions over the IMC have a direct link to global ocean circulation and climate via atmospheric and oceanic teleconnections. Intensive solar radiation and abundant water cause IMC to play an essential role in global equatorial circulation. Within the Earth's climate system and the water cycle, latent heat is inputted from the ground to the atmosphere through the cloud-precipitation process, connecting the land to the oceans through the atmosphere14. In the equatorial region with the meridional rainfall peak of approximately 2000 mm/year15, the IMC produces most active convective clouds and rainfall on Earth (approximately 2700 mm/year)17 followed by Central America. Since the most dominant mechanism of convective cloud generation is the diurnal cycle induced by land-sea temperature contrast, any local land modification, as well as SSTA due to El Niño19, changes rainfall of the IMC's tropical peatlands with large water reservoirs, and finally changes the global circulation and climate19.The Indonesian Maritime Continent (IMC), many large and small islands surrounded by the world’s warmest seawater, plays an essential role in the global climate. With its complex geography and narrow passages, IMC provides the only pathway for low-latitude Pacific Ocean water to flow into the Indian Ocean. Transport and water-mass transformation associated with the Indonesian Through Flow (ITF) directly impacts heat and freshwater budgets of the Pacific and Indian Oceans and may have feedback on Asian-Australian monsoons, El Niño Southern Oscillation (ENSO), and Indian Ocean Dipole (IOD) Fig. 1–8. The 1 and the ITF pathways2. Peatlands currently cover 3% of the global land surface and have an essential impact on global climate change through carbon sources in this century23. Land-use change in the last decade has played a significant role in triggering high greenhouse gas (GHG) emissions. It was estimated that by 2100 there would be an additional 249 ± 38 Pg (1 Pg = 1015 g) emissions due to changes in peatlands22. On the other hand, the lowering of groundwater level (GWL) driven by a warming climate and anthropogenic forces increase greenhouse gases by 0.86 (0.36–1.36) Pg CO2-equivalent year−1 at the end of the twenty-first century25. However, the role of tropical peat on the global carbon cycle and its possible linkage to climate is still not fully understood due to limited measurements.The three largest islands of the IMC  have peatlands Fig. , an impo19, especially on the IMC's tropical peatlands because their ecosystems have large water reservoirs20. The connection between the IMC and the global circulation means that this circulation impacts the peatland in the IMC. When El Niño occurs, most peatlands experience severe drought, decreasing the GWL22. Lowering the GWL leads to a linear increase in soil respiration, especially in drained tropical peatlands, and increased CO2 emissions27. It was observed that CO2 emissions increased by 79 to 238 gC m−2 per 0.1 m decrease in the GWL30. Based on statistical modeling, it has been shown that the fire strength will increase by considering hydrological conditions, especially during strong El Niño events32. Hot spot and precipitation data with a space-borne Moderate Resolution Imaging Spectroradiometer (MODIS for ten years (2002–2011) shows that peat fires occur in El Niño conditions and reach their peak in the driest conditions (September/ October in those years) in Kalimantan33. While in Central Kalimantan, data on precipitation, hotspots, and GWL (obtained from the model) for the period 1997–2007 show that most hotspots occur when the GWL drops by more than 0.4 m. The data also indicates that precipitation reaches a minimum of about 2 months before GWL reaches its lowest condition34. Recently, Deshmunk et al. (2021) measured carbon dioxide and methane emissions between 2017 to 2020 and showed an increase in emissions from peatlands due to extreme drought caused by the positive IOD phase in conjunction with El Niño28. Those previous studies on the IMC peatland were carried out in a limited period, such as the drought, fires, and carbon emissions from peat decomposition caused by a specific climate34. However, if the temperature and moisture of local land are modified irreversibly by the peatland fire, the cloud generation over IMC, and finally, the global climate should also be modified irreversibly.Climate extremes such as El Niño and positive Indian Ocean Dipole (IOD+) significantly impact the tropical peat forest ecosystem in IMCIn this paper, we compare our long-term measurements of GWL and rainfall in the natural tropical peatlands in Kalimantan with SSTA and winds associated with extreme climate events such as El Niño and positive IOD. Peatland modification indeed triggers irreversible climate changes. We hypothesize that tropical peatland ecosystems (represented by the GWL) and ENSO are teleconnected, although the corresponding teleconnection mechanisms are still not fully resolved. This paper is the first report to show evidence of GWL decreasing before the El Niño and moderate IOD+. We believe this should be confirmed and explained by more observations and theories. In this paper, we describe that this is possible, based on many recent studies showing that local hydro-meteorological processes govern the convective activity on the land of IMC and affect essentially broader climates concerned with ocean–atmosphere interaction, including ENSO and IOD+.To answer the hypothesis described in the previous section, we installed a special station to carry out long-term monitoring of GWL and rainfall in the peatland of Indonesia. We compared those data with regional weather/climate , SSTA, ITF, and wind-induced upwelling variability, which should influence the GWL variability.Taman Nasional Sebangau  near Palangkaraya, Central Kalimantan Province   in the national park were logged during the early 1990s, and shallow trenches were prepared to carry the logged trees. The area was protected after the late 1990s, and the shallow ditches were quickly buried naturally.The special station of GWL and rainfall data are located at the peatland of o S–5o N, 170–120o W), and available from NOAA (https://www.cpc.ncep.noaa.gov/data/indices/). The strength of ENSO is divided into four: normal, weak, strong, and extreme when Niño3.4 is less than ± 0.5 °C, between ± [0.5–1] oC, ± [1.0–1.5] oC, larger than ± 1.5 °C (https://ggweather.com/enso/oni.htm). Super El Niño is defined when Niño3.4 above 2.5 °C, occurred in 1972, 1982, 1997, and 201540. The Dipole Mode Index (DMI) is the mean SSTA difference between the western  and eastern  Indian Ocean5, available at https://stateoftheocean.osmc.noaa.gov/sur/ind/dmi.php. Most of the time , El Niño events coincide with positive IOD, while La Niña events coincide with negative IOD. The impacts of ENSO on precipitation vary over the IMC region14. El Niño/IOD + causes much less rainfall and increases the likelihood of forest fire over the IMC. On the other hand, the La Niña/IOD– causes much more rain and raises the possibility of flooding and overflow on the GWL measurements. Hence, we concentrate on the El Niño/IOD+ events in the following sections.The ENSO phase time series is based on the Niño3.4 index, an average SSTA over the central/eastern equatorial Pacific (537). During the southeast monsoon , easterly wind- induced upwelling. Meanwhile, during the boreal winter, winds-induced downwelling. IOD and ENSO modulate this upwelling. Anomalously stronger early wind during IOD positive and El Niño enhance the upwelling strength and expand the upwelling up to western Sumatra . To quantify the wind- induced upwelling . These results support our hypothesis that tropical peatland ecosystems (represented by the GWL) and ENSO are teleconnected. Therefore, monitoring GWL is essential for understanding the onset of ENSO and IOD.The correlations between GWL and the climate parameters  are depicted in Fig. Detailed relations between GWL and EMT Anomaly (EMTA), EPV Anomaly (EPVA), SST Anomaly (SSTA), rainfall, Niño 3.4, and DMI, during El Nino and/or moderate IOD+ events are presented in Fig. On the other hand, DMI peaked in October, and EPVA did not show significant variation. This year the dry season occurs from mid-May to mid-November. In the 2002 El Niño event see Fig. c,d, GWL oC40. The EMTA parameter also rose several months earlier, and the SSTA in southern Java fell several months before the GWL reached its minimum. The SSTA pattern follows the GWL pattern with little variability from all events, less than 1 °C, while DMI and Niño3.4 were the opposite. In the 1997 El Niño event, the DMI peak rose four weeks before GWL dropped to the lowest depth of –0.95 m, while a peak of Niño3.4 occurred three weeks later. In the 2002 El Niño event, the DMI peak occurred almost simultaneously with the lowest GWL of –1.16 m, and the Niño3.4 peak occurred four weeks after the lowest GWL. In the 2006 El Niño event, the DMI peaked three weeks before the lowest GWL reached –0.9 m depth, and two weeks later, Niño3.4 peaked. Different conditions occurred in El Niño in 2009, where DMI peaked four weeks after GWL reached its lowest depth of –0.98 m. Meanwhile, the peak of Niño3.4 occurred more than two months from the lowest GWL. However, during the 2015 super El Niño, the DMI peak reappeared four weeks before the lowest GWL with a depth of –1.32 m, and the peak of Niño3.4 appeared five weeks later. An interesting thing happened in 2019 when the GWL reached a depth of –1.24 m which was not an El Niño year, but the DMI had a high amplitude of 2.2 °C (extreme IOD), which occurred three weeks before the lowest GWL. If GWL changes before a climate event, we should consider that the peatland modification indeed triggers irreversible climate change. In general, GWL will drop several weeks before Nino34 reaches its maximum in El Niño events. The above conditions enormously occur during super El Niño, namely Nino34 is greater than or equal to 2.5. (2021)20 show that peatland carbon loss has a positive climate feedback loop based on 13 years of continuous eddy covariance flux measurements from Mer Blue Bog, Canada. Using a radiative forcing model and areal data from the Global Peatlands Database shows that forcing CH4 emission due to rewetting activities does not damage climate change. On the other hand, delaying wetting will increase the long-term global warming effect through sustainable CO2 emissions22.Using in-situ measurement data over 27 years, we find that the GWL falls before El Niño reaches its peak. Studies of the interconnection of peat with global warming have begun to be carried out by many researchers because peatlands store a third of terrestrial organic carbon. Rafat et al38. The reduced water vapor will cause the central part of Indonesia  to experience a decrease in rainfall which causes GWL to drop. On the other hand, the presence of EPV, with the combination of La Niña and the IOD negative events, tends to attenuate coastal EMT and EPV, but EMT and EPV tend to be strong during El Niño and positive IOD. In addition to CO2 emissions, the role of peatlands in the world's climate is through climate parameters such as evapotranspiration. A study using the observation of 95 eddy covariance towers showed that peatlands' evapotranspiration (ET) increased more than the ET of forests with increasing vapor pressure deficit (VPD). At high VPD over 2 kPa, the ET of peatlands exceeds the ET of the forest by up to 30%. Therefore, the peatland ET should be included in Earth system models to avoid biases in water cycle projections23. In this paper, we propose the teleconnection between tropical peatland and El Niño, of which the key processes are as follows:The study area is strongly influenced by the monsoon system, where the wind changes twice a year, namely, easterly wind (June–July–August (JJA)) and westerly wind (November–December–January). Anomalous easterly winds become prolonged and intensify during the austral winter  until November or December.36, low rainfall, and a drastic decrease in the GWL. Therefore, negative SST anomalies occur primarily before the GWL troughs.Anomalous easterly winds carry drier (low-humidity) air induced upwelling along the southern coast of Java and extent to west Sumatra and cool the SST region. Hence, it enhances positive feedback of SST (cooler SST causes low humidity)Atmospheric convection moves eastward toward the middle-eastern tropical Pacific, and westerly wind bursts intensify. Simultaneously, oceanic Kelvin waves generated in the western Pacific propagate and carry warm water from the western Pacific warm pool eastward.In this paper, we use time series data to explain the teleconnection mechanism. The emergence of EMT during the southeast monsoon causes a mass of cold water to the surface so that SST will be cold, which causes reduced evaporation43. Research shows a strong indication that extreme ENSO events may become more frequent in the future when external forcing increases 45.Consequently, the dry IMC area, including where the GWL decreased in the studied peatland, would affect El Niño with a time delay of a few weeks, and the IOD peak occurs a few weeks before the lowest GWL. A model of the IMC interconnections with El Niño and IOD events is proposed Fig. . In the 17), which changes the total convective activity on land of IMC. When peat forests exist with high GWL, evaporation is intensive so that many convection clouds are formed, strengthening the tropical updraft, which will be involved in global circulation. The condition is the opposite if GWL decreases where the intensity of evaporation decreases, resulting in a weakening of the tropical updraft, which will impact global circulation. By studying this behavior intensively, we can finally determine a tipping point of the planetary boundary proposed by Steffen et al.46.Finally, we propose a hypothesis that GWL in tropical peat may be used as a parameter to determine the tipping point of anthropogenic effects on climate change in the IMC see Fig. . The mod