In-depth structural characterization of lipids provides a new means to investigate lipid metabolism. In this study, we have conducted deep profiling of total fatty acids (FAs) from RAW 264.7 macrophages by utilizing charge-tagging Paternò-Büchi derivatization of carbon-carbon double bond (C=C) and reversed-phase liquid chromatography-tandem mass spectrometry. A series of FAs exhibiting unusual site(s) of unsaturation was unearthed, with their identities being confirmed by observing anticipated compositional alterations upon desaturase inhibition. The data reveal that FADS2  NotablyFA 16:0) , which iFA 16:0) ; howevern lipids . The stun lipids , 15. In n lipids , 18. Iden lipids , 18, whin lipids . Alternan lipids , ultravin lipids , 22, epon lipids , 24, azin lipids , 26, andn lipids , 30, 31,In this work, we employ charge-tagging PB derivatization in combination with desaturate inhibition to uncover a diverse set of novel fatty acids with an unusual site(s) of unsaturation in RAW 264.7 macrophages. The latter method allows us to achieve high confidence in the identification even in the absence of authentic standards. Furthermore, desaturate inhibition provides evidence for studying FAs derived from SCD1 Δ9-desaturation and FADS2 Δ6-desaturation activities. Our data suggest that both enzymes display broad activity toward both even and odd-chain saturated FAs and give the cells access to an expanded repertoire of even-chain MUFAs with n-5, n-7, n-9, n-10, and n-12 C=Cs. Furthermore, even-chain MUFAs derived from these de novo synthesized families also act as substrates for further desaturation and elongation, yielding a wide array of PUFAs, consisting of the n-7, n-9, n-10, and n-12 families.n-x nomenclature, counting from the methyl terminus. The Z/E stereo-configurations of C=C could not be assigned from PB-MS/MS and thus were not indicated for FAs from biological samples.The shorthand notation recommended by LIPID MAPS consortium was used . In brieHigh-performance LC (HPLC) grade iso-propanol (IPA), acetonitrile (ACN), methanol (MeOH), and water were purchased from Fisher Scientific. Methyl tert-butyl ether (MTBE), formic acid (HCOOH), ammonium formate, and 2-acetylpyridine (2-acpy) were purchased from Sigma Aldrich. Lipid standards were purchased from Avanti Polar Lipids. AMP+ MaxSpec Kit was purchased from Cayman Chemical.RAW 264.7 macrophages (American Type Culture Collection (ATCC); Manassas, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution and collected by centrifugation. The cell pellets were washed, frozen in liquid nitrogen, and stored at −80°C. For direct inhibition of enzyme activity, RAW 264.7 macrophages were treated for 72 h with 60 μM FADS2 inhibitor  and 2.5 μM SCD1 inhibitor , while the control group was treated with DMSO.g for 10 min. The upper phase was collected. The lower phase was re-extracted with 2 ml of the upper phase of the system MTBE/MeOH/H2O . Finally, the combined organic phases were collected and dried under N2 flow for further use. The cell medium was also subjected to lipid extraction and total FA profiling. The extracted lipids were saponified in 500 μl ACN:15% KOH  at 60°C for 60 min. The solution was acidified with 1M HCl (1 ml). The hydrolyzed total FAs were extracted twice with 1.5 ml isooctane each time. The organic layer was collected, dried under nitrogen, and redissolved in an aliquot of 500 μl MeOH for further derivatization.The cell samples were extracted using a modified MTBE protocol . In brieAMPP derivatization followed the procedure provided by the vendor (AMP+ MaxSpec Kit). AMPP derivatized sample was extracted twice by 600 μl MTBE and dried under a nitrogen stream. The derivatized sample was resuspended in 100 μl MeOH for RPLC-MS/MS analysis.The 2-acpy PB derivatization was performed using a home-made flow microreactor . Total F2O:ACN  and mobile phase B contained IPA:ACN . The flow rate was set at 0.5 ml/min. Oven temperature was set at 60°C. The chromatographic gradient was as follows: 30% B at 0–0.4 min, 30%–45% B at 0.4–0.9 min, 45%–52% B at 0.9–1.1 min, 52%–58% B at 1.1–1.8 min, 58%–66% B at 1.8–2.5 min, 66%–70% B at 2.5–3.1 min, 70%–75% B at 3.1–4 min, 75%–97% B at 4–4.5 min, 97% B at 4.5–6 min, 30% B at 6.1–7 min. The MS parameters were optimized as follows: capillary voltage, 4500 V; end plate voltage, 500 V; nebulizer, 3 Bar; dry gas, 10 L/min; dry temperature, 210°C; CID energy for MS/MS, 22 eV.RPLC-MS/MS analyses were conducted on an Acquity UPLC I-Class system (Waters) hyphenated with a hybrid trapped ion mobility-quadrupole time-of-flight mass spectrometer . A CORTECS UPLC C18 column  was used for separation. The mobile phase A contained H1 spectra of AMPP-derivatized fatty acid and corresponding internal standards were imported into the LipidNovelist Extension and subjected to Type-I isotope correction. The most up-to-date version of LipidNovelist and LipidNovelist Extension, along with instructional videos and example data to facilitate its utilization, can be accessed at https://doi.org/10.6084/m9.figshare.22297771.The analysis of RPLC-PB-MS/MS data was carried out using a home-made software, LipidNovelist 1 data which provided relative quantification (I/IIS) at the sum composition level 1 signal to that of all FAs detected in the same RPLC run. Type-I isotope correction was applied to the data prior to relative quanaitation. The associate data are provided in RAW 264.7 cells have been extensively characterized and are well-suited for lipid metabolism studies due to their expression of a wide range of fatty acid desaturation enzymes involved in lipid metabolism , 37, 38.Then, we used 2-acpy derivatization (the PB reaction) followed by RPLC-MS/MS to achieve relative quantitation of unsaturated total FAs at the C=C location level . The PB-A total of 29 groups of unsaturated FA and 9 groups of saturated FA were identified at the sum composition level, covering long-chain (carbon number: 14–21) and very-long-chain FAs (carbon number >21) with up to 6 C=Cs. Our data A showed The retention time (RT) values of AMPP-derivatized FAs were plotted as a function of carbon chain length (14−24) and the number of C=C (0−6) . We clean-xfo and n-xFo, generic structures shown in Since the odd-chain MUFAs and their C=C location isomers have been rarely characterized, we focused our attention to analyze the odd-chain MUFAs. P = 1.6×10−4). These data corroborate that SCD1 is involved in de novo synthesis of these n-8 isomers. However, the %relative composition of FA 21:1 (n-8) showed an increase after SCD1 inhibition . Because FA 21:1(n-8) is the single component found in the culture medium for FA 21:1 , after the inhibition of SCD1 B. It is :1(n-10) . The smaAfter FADS2 inhibition, the relative compositions of the n-11 isomers of the odd-chain MUFAs decreased significantly B, such aAs discussed earlier, FA 17:1 only accounted for 0.43 ± 0.03% of the total FAs in RAW 264.7 cells at the sum composition level, while enzymatic inhibition showed small disturbances to its relative abundance, eg, 0.33 ± 0.03% in the SCD1 inhibition group and 0.47 ± 0.04% in the FADS2 inhibition group A. The anP <0.001, N = 6)  B. The pr2 CID spectrum of FA 18:2. Based on detecting the C=C diagnostic ions (n-xfo and n-xFo), FA 18:2 was identified as a mixture of FA 18:2  (44.2 ± 0.8%), FA 18:2  (29.8 ± 0.5%), FA 18:2 (7.2 ± 0.6%), and FA 18:2 (18.9 ± 0.3%). The presence of FA 18:2  indicates that FADS2 catalyzes Δ8-desaturation of FA 18:1(n-7) to form FA 18:2. Further chain elongation and Δ5-desaturation of FA 18:2 contribute to the n-7 PUFAs identified in FA 20:2, FA 22:2, FA 18:3, and FA 20:3 (P = 3.7×10−11) (P = 9.7×10−12)  B. Simila7×10−12) B. A simi7×10−12) CFig. 6A:P =1.5×10−4) and 3.3 ± 1.1% after FADS2 inhibition (P =1.9×10−5)  indicates that FADS2 catalyzes Δ6-desaturation of FA 18:1(n-9) to form FA 18:2. Furthermore, the n-9 isomer was found to be the most dominant form in FA 20:3 (73.9 ± 0.5%), which is an indicator of essential fatty acid deficiency . Previou2, n-15) . An inhi.9×10−5) B. A simi.9×10−5) .Likewise, the presence of FA 18:2 indicates the possibility for Δ5-desaturation of FA 18:1(n-10) by FADS1, which highlights a point of divergence in enzymatic activity surrounding the FA 18:1(n-10). The subsequent elongation of FA 18:2 results in the n-10 PUFAs, including FA 20:2 and FA 22:2. A similar enzyme branching point can be observed for FA 20:1(n-12), whereby FADS1 mediates Δ5-desaturation towards FA 20:1(n-12) 2, n-15) C.P <0.001, N = 6), except for FA 18:3 and FA 24:5 , FA 20:5 (100 ± 0%), FA 22:5 (100 ± 0%) and FA 22:6 (100 ± 0%), and the n-6 isomers dominated in FA 18:2 (87.5 ± 0.8%), FA 20:3 (75.3 ± 0.8%), FA 20:4 (92.9 ± 0.1%), and 22:4 (64 ± 4%)  and S4. 5, n-18) C. Intere5, n-18) . We thus5, n-18) . This ph5, n-18) .Fig. 7BiP = 2.3×10−12). In the control group, FA 24:4 only contained the n-6 isomer (100%), likely originating from sequential elongation of the n-6 isomer of FA 20:4 (97.2 ± 1.4%) to FA 22:4 (85 ± 3%). The n-6 isomer of FA 24:5 was observed as the only component (100%) in the control and SCD1-inhibition groups but absent in the FADS2-inhibition group and culture medium  to FA 18:3 and convert FA 24:4  to FA 24:5  by catalyzing the desaturation at the Δ6 position . Thus, ae medium . These dNote that the Δ6-desaturase activity of FADS2 was greatly increased by SCD1 inhibition in the conversion of FA 24:3  to FA 24:4 . %Relative composition of FA 24:4  increased from 0% in control to 100% in after SCD1 inhibition. These results also confirm that SCD1 inhibition consolidates FADS2 activity toward PUFA. The biosynthetic pathways of PUFA observed in RAW cells are proposed in 2 CID  (72.2 ± 0.4%) and FA 24:2  (100%) . The str2 CID B. Similaer cells . Non-meter cells . De novoer cells .Fig. 8MSThe exploration of FA C=C isomers within RAW 264.7 cell lipidome in combination with enzyme inhibition reveals that cellular fatty acid desaturation is far more complex and dynamic than previously considered. The biosynthesis pathways of the odd-chain MUFAs, even-chain MUFAs, and PUFAs are proposed based on the experimental observations. This expanded network of FA desaturation provides plasticity to generate certain FA families arising from extreme metabolic stress. For instance, the FADS2 plasticity allows for multiple substrate acceptances (e.g., FADS2 via OzID . In concAll data concerned with this study are presented within this manuscript.This article contains The authors declare no conflicts of interest.


Bothrops genus. In the French departments of America, B. atrox envenomation in French Guiana may lead to bleeding while B. lanceolatus envenomation in Martinique to thrombosis. Bleeding related to B. atrox envenomation is attributed to vascular damage mediated by venom metalloproteinases and blood uncoagulable state resulting from thrombocytopenia and consumptive coagulopathy. Thrombosis related to B. lanceolatus envenomation are poorly understood. We aimed to compare the effects of B. atrox and B. lanceolatus venoms in the rat to identify the determinants of the hemorrhagic versus thrombotic complications. Viscoelastometry (ROTEM), platelet count, plasma fibrinogen, thrombin generation assay, fibrinography, endothelial , and inflammatory biomarkers  were determined in blood samples obtained at H3, H6, and H24 after the subcutaneous venom versus saline injection. In comparison to the control, initial fibrinogen consumption was observed with the two venoms while thrombocytopenia and reduction in the clot amplitude only with B. atrox venom. Moreover, we showed an increase in thrombin generation at H3 with the two venoms, an increase in fibrin generation accompanied with hyperfibrinogenemia at H24 and an increase in inflammatory biomarkers with B. lanceolatus venom. No endothelial damage was found with the two venoms. To conclude, our data support two-sided hemostasis complications in Bothrops envenoming with an initial risk of hemorrhage related to platelet consumption and hypocoagulability followed by an increased risk of thrombosis promoted by the activated inflammatory response and rapid-onset fibrinogen restoration.Hemostasis impairment represents the most threatening consequence of Viperidae envenoming, notably with  Bothrops venoms contain toxins targeting hemostasis, a physiological process aiming stopping bleeding in case of injury. Envenomation by B. atrox in French Guiana leads to hemorrhage while envenomation by B. lanceolatus in Martinique results in thrombosis. To understand the mechanisms involved in these opposite manifestations related to the two venoms, we designed a comparative rat study of hemostasis based on a comprehensive approach including blood measurement of initiation time and size of the clot, platelet count, plasma fibrinogen, thrombin and fibrin generation, and various inflammatory and endothelial biomarkers. We showed that B. atrox venom induces more marked platelet and fibrinogen consumption, promoting the risk of hemorrhage, while B. lanceolatus venom induces an initial fibrinogen consumption followed by a secondary increase in combination to an enhanced inflammatory reaction resulting in increased thrombosis risk. Interestingly, the two venoms exhibited no endothelial toxicity. Its three distinct phases, i.e., primary hemostasis, coagulation, and fibrinolysis, are closely linked . In the culation .Bothrops genus is mostly involved in Latin America [Bothrops venoms targeting hemostasis exhibit highly diverse enzymatic , snake venom serine proteases (SVSPs), phospholipases A2 (PLA2s) and L-amino acid oxidases) and non-enzymatic proteins (disintegrins and C-type lectin proteins) [Hemorrhagic syndrome associated with the absence of blood coagulation are one of the main characteristics of Viperidae envenomation ,8. Other America . Toxins roteins) .B. atrox is the predominant species involved in envenomation in French Guiana [B. lanceolatus is the only venomous snake in Martinique, where it is endemic [B. lanceolatus being included in B. atrox group [In the French departments of America, h Guiana ,13, wher endemic . While tox group ), they aB. atrox bite may result in a typical Viperidae envenomation with the risk of local and systemic bleeding like other Bothrops such as B. asper and B. jararaca [B. atrox- and B. jararaca-envenomated patients admitted to the hospital [B. atrox venom, activates factors V, VIII, and XIII [in vitro clotting of fibrinogen [jararaca –20. Systjararaca ,22. Bleejararaca –26 and tjararaca . Thrombohospital ,29. SVMPhospital –32. Throand XIII ,34. Venoand XIII . Defibribrinogen . Non-coabrinogen .B. lanceolatus envenomation is unusually associated with bleeding and absence of coagulability, but may be complicated by multiple systemic infarctions up to 48 h after the snakebite, even in case of moderate envenomation [B. lanceolatus envenomation is poorly understood. Despite venom-induced thrombocytopenia in mice, B. lanceolatus venom induced no direct effects on platelet aggregation in human platelet-rich plasma [B. lanceolatus venom dose-dependently clotted purified human fibrinogen, indicating the involvement of a thrombin-like enzyme. However, it is devoid of defibrinating activity after intravenous injection in mice [B. lanceolatus envenomation case with diffuse thrombotic microangiopathy (TMA) causing multiple cerebral, myocardial, and mesenteric infarctions on autopsy supported the possible onset of venom-induced endotheliopathy [By contrast, nomation ,39. Thronomation . The exah plasma ,42. B. l in mice ,43,44. I in mice ,43,44, s in mice ,46. Consliopathy .Bothrops species are relatively similar with comparable activities on coagulation in vitro in the whole blood and plasma [in vitro assays have limitations as they only offer a view of the immediate venom-induced effects whereas envenomation is a complex dynamic process. Crude venoms exhibit strong dose-dependent procoagulant effects in vitro [in vivo consumption of clotting factors result in blood hypocoagulability. To observe the time-dependent toxicity of the various enzymes present in Viperidae venoms, it is necessary to investigate the time-course of hemostasis disorders in vivo [Bothrops envenomation. By combining rotational thromboelastometry (ROTEM), a point-of-care viscoelastic test of whole blood hemostasis allowing the global assessment of clot formation and its dissolution in real time [Surprisingly, venom composition of these two d plasma . Howeverin vitro –51, whil in vivo –55. Receeal time , thrombieal time  and fibreal time , it is pB. atrox and B. lanceolatus envenomation aiming to identify the determinants of the hemorrhagic and thrombotic effects of venoms and the contributions of the various key-players of hemostasis, i.e., platelets, coagulation factors, and endothelial cells.We therefore designed a rat model of human Our animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the EU Directive 2010/63/EU for animal experiments and the ethical guidelines established by the National Institutes of Health. The experimental protocols were approved by Paris Cité University Animal Care Ethics Committee and the French Ministry of Research (N° APAFIS #26913-20200-120174-024616-V5).B. atrox venom (batch 211.191) is a pool of samples from wild-caught or born-in-captivity, male and female adult snakes, from French Guiana, Peru and Brazil, whereas B. lanceolatus venom (batch 411.171) is a pool of samples from wild-caught, two male and one female, adult snakes, from Martinique. Venoms were stored at +4°C and dissolved in saline before injection. The lethal dose-50% (LD50) of B. lanceolatus venom was previously determined at 6 mg/kg in mice by intraperitoneal route [in vitro experiments conducted in our laboratory using ROTEM in the rat whole blood showed that the procoagulant effect of B. atrox venom was 2-fold that of B. lanceolatus venom, we used 220% of the estimated intraperitoneal LD50 in mice for B. lanceolatus venom , and half of this value for B. atrox venom  to target equipotent doses.Freeze-dried venoms were obtained from Latoxan . al route . Since pB. lanceolatus, B. atrox, B. alternatus, B. asper, B. jararaca, B. jararacussu, B. diporus, B. schlegeii, Lachesis muta, L. melanocephala, L. stenophrys, and Crotalus simus. This antivenom consists of soluble IgG F(ab’)2 fragments. Based on the potency of the supplied batch , 1 mL neutralizes 9.9 mg of B. lanceolatus venom and 2.7 mg of B. atrox venom, as stipulated by the manufacturer.Inoserp South America  is an experimental polyvalent antivenom against ad libitum.We used male Sprague-Dawley rats , weighing 250–400 g, housed for 7 days prior to experiment, maintained under constant temperature conditions (19–21°C), and submitted to a 12h/12h light/dark cycle, with food and water provided The experiment consisted of testing the rat whole blood by ROTEM after adding different concentrations of venom or r ex-tem  or saline (negative control). The triggering reagent was therefore either venom or r ex-tem while the addition of saline evaluates spontaneous coagulation.Whole blood was sampled from the catheterized abdominal aorta in a non-treated rat anesthetized using 10mg/kg xylazine and 70 mg/kg ketamine injected intraperitoneally. Syringe pre-filled with 0.4 mL of buffered sodium citrate  was used to collect 3.6 mL of blood, then gently inverted five times to ensure adequate anticoagulation before transferring blood samples into tubes.2 , 20 μL venom sample, and 300 μL whole blood. Viscoelasticity data were then recorded at 37°C for 60 min. For the other venom concentrations, the volume of venom solution was adjusted to 20 μL using PBS. The positive control consisted on the same procedure using 20 μL of r ex-tem instead of venom solution, whereas the venom solution was replaced with 20 μL of PBS for the negative control (thus corresponding to the spontaneous coagulation activation in whole blood). Parameters assessed using ROTEM included clotting time (CT) and maximum clot firmness (MCF). CT is the time (s) from start of the measurement until initiation of clotting  and depends on the concentrations of coagulation factors. MCF is the maximum strength of the clot (mm) reached during the run, and depends on the platelet count and function and fibrin formation.Rotational thromboelastometry was performed on ROTEM Delta analyzer  to assess the venom effect on coagulation system. For each venom, ROTEM was performed at five different venom concentrations . Venom stock was diluted in saline phosphate buffer (PBS) to obtain a 3.5 mg/mL solution. The non-treated rat citrated whole blood tube was placed in the sample pre-heating station (temperature at 37°C) of the ROTEM analyzer. For the first venom concentration (100 μg/mL), all reagents were pipetted into the cup: 20 μL CaClB. atrox venom, B. lanceolatus venom, and saline) and at each sampling time, rats were randomized (n = 6/group). Saline (control rat group) or venom (2 envenomated rat groups) was injected by the dorsal subcutaneous route, with an average 2 mL/kg volume. Blood was sampled at 3 h (H3), 6 h (H6), or 24 h (H24) after the saline/venom injection, from the catheterized abdominal aorta in a rat anesthetized using 10 mg/kg xylazine and 70 mg/kg ketamine injected intraperitoneally.Secondly, we developed an animal model of envenomation. For each condition  or 20 μL of EDTA were used to collect 3.6 mL and 2 mL of blood, respectively. They were gently inverted five times to ensure adequate anticoagulation then blood was transferred into dry tubes. Antivenom was added into the tubes (10 μL/mL blood) for venom antagonization before analysis.ROTEM experiments were carried out immediately using citrated whole blood and platelet count was performed in EDTA whole blood up to 4 h after blood sampling. Citrate tubes were double-centrifuged at 2500g for 10 min, at room temperature without brake to obtain platelet-poor plasma. EDTA tubes were centrifuged at 1000g for 10 min, at room temperature. Citrated platelet-poor plasma and EDTA plasma were then aliquoted and stored at -80°C for analysis.ROTEM experiments (EXTEM test) were conducted on citrated whole blood, according to the manufacturer’s instructions. The EXTEM test explores the tissue-factor coagulation pathway. Experiments were performed as previously described. In EXTEM test, 20 μL of Star-tem, 20 μL of r ex-tem and 300 μL whole blood were added into the cup. In addition to CT and MCF, the lysis index (LI) 30 was measured. LI30 is the residual clot firmness at 30 minutes from CT and reflects the fibrinolysis phase.Platelet count was performed in EDTA whole blood with a MS9-3 veterinary hematology analyzer .Fibrinogen level was measured on citrated platelet-poor plasma using the standard Clauss method. It was performed on STA-R Max automated coagulation analyzer system  using Dade Thrombin reagent .VWF antigen was measured on citrated platelet-poor plasma using the ELISA Asserachrom VWF:Ag assay (Stago). Results were normalized using a normal rat citrated pool plasma.ADAMTS13 activity was measured on citrated platelet-poor plasma by an in-house FRETS-VWF73 assay  using coTGA was performed on ST Genesia analyzer system (Stago), a fully automated system enabling quantitative standardized TG assessment derived from Hemker’s fluorescence method, using dedicated reagents, calibrator and quality controls . Thrombi2. Turbidimetry was recorded at 350 nm at 37°C every 6 s. Five parameters were analyzed: lag time , time to peak , start tail  and peak height ; maximal turbidity value). Data were normalized for determining lag time, time to peak, and start tail.Fibrin polymerization and fibrinolysis kinetics were assessed by turbidimetry in a TECAN Infinite M200Pro spectrophotometer  as previously described . BrieflyMultiplex assays using the Luminex Magpix platform  were performed with the Rat Adipokine Magnetic Bead Panel  for the measurement of interleukin (IL)-1ß, IL-6, tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor type-1 (PAI-1), and the Rat Vascular Injury Magnetic Bead Panel for the measurement of soluble E-selectin and soluble intercellular adhesion molecule-1 (ICAM-1) in EDTA plasma. These kits were processed according to the manufacturer’s instructions.in vitro experiments, ROTEM parameters had a normal distribution and were compared between the three experimental conditions  using one-way analysis of variance (ANOVA), followed by Tukey’s multiple tests. Distribution of measured values were represented as boxplots for CT and MCF. The median effective concentrations (EC50s) were determined visually using GraphPad PRISM 9.5.0.Analyses were performed using GraphPad PRISM 9.5.0 . All results are expressed as mean ± standard deviation for quantitative data and % of reference values for qualitative data. Data were tested for normality by visual inspection and Shapiro-Wilk tests. For ex vivo experiments, parameters were compared between B. atrox, B. lanceolatus, and saline groups at each sample time using one-way ANOVAs  followed by Tukey’s multiple tests if parameters were normally distributed. If parameters were not normally distributed, comparisons were performed using Kruskal-Wallis’s tests followed by Dunn’s multiple tests. A p-value ≤ 0.05 was considered statistically significant.For B. atrox and B. lanceolatus venoms presented dose-dependent procoagulant activities with significant decrease in CT compared to the negative control (p = 0.0008) but no significant difference was observed when compared to one another (p = 0.1)  while B. lanceolatus venom showed a slight hypercoagulable profile (p = 0.02). There was no significant difference between the two venoms for MCF (p = 0.7)  exhibited a CT of 261.2 ± 66.5 s and a MCF of 75.7 ± 4.6 mm. Positive control (with r ex-tem) exhibited a CT of 32.5 ± 5.5 s and a MCF of 78.2 ± 2.4 mm. .1) Figs  and S1. ox venom . In comp.7) Figs  and S1.B. atrox venom-treated rats was significantly lower than controls , H6 (p = 0.02), and H24 (p < 0.001) while B. lanceolatus venom had no effect on platelet count (B. atrox and p = 0.005 for B. lanceolatus) and at H6 (p = 0.03 for B. atrox and B. lanceolatus). At the opposite, plasma fibrinogen was significantly increased at H24 with B. lanceolatus venom  and only for B. atrox at H6 (p = 0.019) (B. atrox and p = 0.006 for B. lanceolatus) (B. atrox venom at H3 (p = 0.02)  . Similareolatus) . In paraeolatus) . The end = 0.02) .B. lanceolatus venom significantly increased the start tail and the peak height at H24   Figs  and S3.B. lanceolatus venom (p = 0.006) and at H6 with the two venoms (p = 0.04 for B. atrox and p = 0.02 for B. lanceolatus), before returning to non-significantly different values at H24 in comparison to the control group (B. atrox venom-treated rats and one B. lanceolatus-treated rat due to insufficient sample volume. No modification in soluble E-selectin was observed whatever the venom at H3 and with B. atrox venom at H6 and H24 (B. lanceolatus venom at H6 (p = 0.006) and H24 (p = 0.02). No modification in soluble ICAM-1 was observed whatever the venom or sampling time was  and at H6 and H24 with B. lanceolatus venom  increased endogenous thrombin potential of human platelet-poor plasma in vitro [B. jararaca-related coagulopathy [Bothrops envenoming. The presence of such activators has been already reported with B. atrox [B. lanceolatus venom yet despite in vitro data showing its ability to reduce coagulation time [TGA showed a decrease in lag-time and time to peak and an increase in thrombin peak at H3 with the two venoms while ETP was only increased with than ETP . Therefoin vitro . SVMP-inulopathy . DecreasB. atrox –32 but nion time ,46.B. atrox venom, starting at H3 and being maximal at H24. Because fibrinogen was corrected at that time, the decrease in ROTEM MCF at H24 was more related to platelet count impairment. Indeed, systemic bleeding has been attributed to thrombocytopenia in B. atrox-envenomated patients [B. jararaca-bitten patients, systemic bleeding is more frequent in the presence of acute thrombocytopenia than the blood hypocoagulability [B. atrox-envenomated patients on admission, suggesting peripheral platelet destruction, which tends to increase the mean platelet volume [B. atrox venom, induce washed human platelets aggregation in a less potent manner than thrombin [B. atrox venom. Moreover, in a mouse model of B. jararaca envenomation, pretreatment with warfarin that decreases the prothrombin level thus the thrombin generation, did not prevent the occurrence of thrombocytopenia, suggesting a SVMP-independent mechanism. Finally, thrombocytopenia could be explained by platelet sequestration in the multiple bleeding sites caused by hemorrhagic SVMPs. Therefore, thrombocytopenia should be considered more as a consequence rather than a cause of venom-induced hemorrhages, although it contributes to bleeding.A gradual decrease in platelet count was only observed with patients ,64. In Blability ,66. A net volume . Thrombothrombin ,68. Nevethrombin . The incthrombin . HoweverB. lanceolatus venom in our rat model, nevertheless of little contribution to explain the mechanisms of thrombosis in this envenomation. Based on postmortem data in a B. lanceolatus-bitten patient, direct endothelial damage was hypothesized [B. jararaca-envenomated rat [2-EDTA suggested that its decrease was due to SVMPs and thrombin/plasmin generation [B. jararaca-bitten patients, suggesting more endothelial activation by inflammation than a real endotheliopathy directly linked to the venom [B. lanceolatus venom did not increase ICAM-1 or E-selectin in vitro [Bothrops envenomations in Brazil, angiopoietin-1 and vascular cell adhesion protein-1 (VCAM-1) were only increased in acute kidney injury (AKI) patients whereas other biomarkers suggestive of endotheliopathy  were not significantly increased, even in AKI patients [B. jararaca-bitten patients, suggesting endothelial damage at least of late-onset and/or in this snake species [A non-significant trend to MCF increase at H24 and a slight hypercoagulable profile were observed with thesized . Howeverated rat . Partialneration . Intereshe venom . Here, whe venom . Consistin vitro , supportpatients . By cont species .B. lanceolatus envenomation model. An increase in fibrinogen level during the follow-up and/or a late onset of hyperfibrinogenemia was often reported in patients with thrombosis in Martinique, also sometimes despite antivenom [B. lanceolatus venom, although considered as the most abundant family of proteins in this venom [B. jararaca venom upregulated hepatic mRNA synthesis of fibrinogen chains, which may explain the fast fibrinogen level recovery, even in the absence of antivenom [B. lanceolatus venom is able to activate factor XIII: in B. atrox venom, thrombocytin is able to activate factor XIII by limited proteolysis and increase procoagulant activity of factor VIII similarly to thrombin [B. lanceolatus venom could increase fibrin generation: once bound to fibrin, the capacity of batroxobin, a TLE from B. atrox venom, to promote fibrin accretion was found 18-fold greater than that of thrombin [Venom-induced thrombotic complications could be explained, at least in part, by quantitative changes in fibrinogen. An increase in plasma fibrinogen at H24 was remarkable in our ntivenom ,47,62. Eis venom . In micentivenom . In our thrombin . Moreovethrombin .B. lanceolatus venom injection. This result confirms an ex vivo model based on human whole blood, in which this venom elicited an inflammatory reaction combining pro-inflammatory interleukin production , chemokine upregulation , complement activation and eicosanoid release  [2 isolated from B. lanceolatus venom [B. lanceolatus-bitten patients [B. lanceolatus envenomation but may rarely occur with B. atrox bite [Bothrops envenomation patients, including by B. jararaca [B. venezuelensis [B. erythromelas [B. venezuelensis bite, hyperfibrinogenemia began at day 2 and persisted over 2 weeks despite antivenom administration 4.5 h after the bite [B. atrox venom despite an increasing trend in inflammatory biomarkers. Systemic inflammation has been demonstrated with Bothrops venoms [B. atrox-bitten patients [B. atrox envenomation in the Brazilian Amazon [Bothrops envenomation, even after antivenom administration.Thrombosis may be due to coagulation-induced inflammatory activity of the venom. Here, we observed a systemic inflammatory response involving IL-6, TNF-α, and MCP-1 after xane B2) . These eus venom . In varius venom . Consistpatients ,62. Howerox bite ,80. In arox bite . Finallyjararaca ,82, B. vuelensis  and B. ehromelas , presentthe bite . Here, ws venoms ,86. An ipatients ,87. No sn Amazon . HoweverBothrops venoms on hemostasis, we used a rat model supporting envenomation features with this genus. However, extrapolation to humans should remain cautious, particularly for thrombin generation as peaks are twice lower in the rats than in humans [B. lanceolatus venom-induced thrombosis [B. atrox venom depends on various factors, notably the geographical origin of the specimens collected [B. atrox venom as that used for B. lanceolatus.Our study has limitations. First, to understand the effects of n humans . Likewisn humans . Secondlrombosis ,91. Insuollected . The venBothrops envenomation-induced bleeding and thrombosis are not distinct but represent the two sides of the same coin (B. atrox venom (as shown at H24 with ROTEM data) or persistent hypercoagulability with B. lanceolatus venom (as shown with fibrinography data), while these two venoms both have a similar proteomic composition with a predominance of SVMPs and procoagulant activity in vitro. By analogy to the Greek myth of Charybdis and Scylla, Bothrops envenomation may thus follow two opposite clinical expressions: in the event of major platelet and fibrinogen consumption promoting bleeding, hemorrhage will occur early, whereas in case of minor consumption or early restoration of platelet and fibrinogen limiting bleeding, venom-induced systemic inflammation will expose to thrombotic complications of delayed onset. Our experimental findings also help understanding why B. atrox envenomation could be complicated with thrombosis lately, as sometimes reported in patients with moderate initial consumption coagulopathy.Our global investigation of hemostasis suggest that mechanisms involved in ame coin . An initS1 FigB. atrox venom added at various concentrations: 100 μg/mL (red trace), 10 μg/mL (green trace), 1 μg/mL (blue trace), 100 ng/mL (pink trace); (b) B. lanceolatus venom added at various concentrations: 100 μg/mL (red trace), 10 μg/mL (green trace), 1 μg/mL (blue trace), 100 ng/mL (pink trace); (c) B. atrox venom at 10 ng/mL (blue trace), B. lanceolatus venom at 10 ng/mL (red trace), 0.9% NaCl (pink trace) and r ex-tem (green trace).Representative thromboelastometry trace with non-treated rat whole blood in presence of (a) (TIF)Click here for additional data file.S2 FigB. atrox (blue line) or B. lanceolatus venom (red line) injection in rats.Representative thromboelastometry trace at H3 (a), H6 (b) and H24 (c) after 0.9% NaCl (green line), (TIF)Click here for additional data file.S3 FigB. atrox (blue line) or B. lanceolatus venom (red line) injection in rats.Representative turbidimetry curve generating by fibrinography at H3 (a), H6 (b) and H24 (c) after 0.9% NaCl (black line), (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Hipparion as part of the evidence. We show here that, while there is good anatomical and embryological evidence for the proximal portions of all the accessory digits  being retained in the feet of modern horses, evidence is lacking for the retention of any distal portions of these digits. There is also good ichnological evidence that many tridactyl equids possessed a frog, and that the frog has been part of the equid foot for much of equid evolutionary history.The traditional story of the evolution of the horse (family Equidae) has been in large part about the evolution of their feet. How did modern horses come to have a single toe (digit III), with the hoof bearing a characteristic V-shaped keratinous frog on the sole, and what happened to the other digits? While it has long been known that the proximal portions of digits II and IV are retained as the splint bones, a recent hypothesis suggested that the distal portion of these digits have also been retained as part of the frog, drawing upon the famous Laetoli footprints of the tridactyl (three-toed) equid  Such distal mud adhesions often leave an imprint behind the hoof with an irregular posterior border . Another hypothesis is that the posterior impressions were made by the interphalangeal area of the equid limb during extreme dorsiflexion of the fetlock joint at mid-stance in fast gaits .Solounias et al.  also advet al.  had origEquus-type of spring foot  ([a). It is difficult to envisage what a footpad might look like in an equid that had reduced side toes and an enlarged central toe, as in Hipparion. An extensive pad projecting behind the ungual phalanx has been envisaged ([f), which would account for the length of the footprint impression; but this would require the retention of a tapir-like metapodial pad that seems unlikely with the side digits being so reduced. By contrast, a digital pad of the main digit     would bEquus, and the phalanx is pointed rather than rounded (fig. 9e in [f which illustrates the supposed claw on Hipparion). A similar ungual phalangeal anatomy can be seen in most tridactyl equids, and in more basal tridactyl or monodactyl Equini such as Pliohippus pernix (e.g. F:AM 60803) and Astrohippus stocki . However, in the somewhat more derived Dinohippus (e.g. F:AM 87203) the ungual phalanx is rounded and lacks an anterior cleft, resembling that of Equus. The large anchitheriins such as Hypohippus (e.g. F:AM 60606) and Megahippus (e.g. USNM 175375) also have more rounded ungual phalanges and lack this cleft, and so the presence or absence of a cleft is probably simply related to phalanx shape (see later discussion). The issue of a claw in any equid, neonatal or adult, is further discussed in §5.2 below.Finally, it was proposed  that theg. 9e in , and theEquus , rather than the presence of an anterior ‘claw’, that would be expected to exert higher pressures as the foot toes off, resulting in deeper portions of the track. The issue of the possible retention of a fetal claw is discussed further in §5.2.Moreover, other footprints at Laetoli show a more rounded hoof impression anteriorly as in Equus . Based o. 4.3c) that may act to transfer force of impact to the digital cushion (or subcutaneous tissues) and/or as mechanisms of venous return [Equus, but to the plantar process in Hipparion. But this bony ‘wing’ is not a natural part of an Equus hoof: rather, it is a pathological condition of ossification of a portion of the lateral cartilage known as sidebone, seen mainly on the lateral side of the front hoof in certain modern domestic horses  of the ungual phalanx support collateral cartilages  that m horses , where the collateral cartilage attaches to the plantar process and extends posteriorly would be capable of making the lateral impressions seen in Laetoli print B7 (f), the ungual phalanges of Equus and Hipparion are depicted as placed flat on the surface, which is not how the ungual phalanx is positioned within the hoof capsule . As is clear in X-rays, the phalanx in Equus is at an angle of approximately 30 degrees to the ground (c); it is supported ventrally by the digital cushion, and the frog lies distal to the cushion. Thus, these processes and cartilages are elevated dorsally , away from any possibility of contact with the substrate during normal locomotion.It might still seem possible that the ‘wings’ shown in pparion . This occurs in a few hipparionin trackways of the Miocene and Pliocene. Other hipparionin trackways, however, show side toe impressions that occur well behind and clearly separated from the central hoof , reflecting the loss of the pad foot that freed up the side digits. Similar trackways, with posterior side toe imprints, can be seen in extant reindeer . FurtherThus, the ichnological evidence supports the hypothesis that digits II and IV were indeed able to make contact with the ground during tridactyl equid locomotion, at least in certain substrates, such as soft ones. This presents three challenges to the interpretation presented in . First, . 4.5b), and it is continuous posteriorly with the bulbs of the heel, forming their anterior extension [The frog is a wedge-shaped structure comprising of soft, elastic horn that forms part of the plantar surface of the hoof; the remainder of the plantar surface is covered by the sole . It is lfigure 2xtension . Anterioxtension . Althougxtension  that thextension ,67,76), The frog is heavily cornified, like the hoof wall and sole, but the structure and properties of the keratinous layers of these parts differ, as pointed out in ; howeverThe frog acts primarily as a shock absorber that transmits forces to the digital cushion located deep in it and helps dissipate force during initial ground contact –86. SecoEquus condition in figure 2b,c) commonly extend anteriorly onto the plantar surface of the foot to merge with the sole in other perissodactyls and artiodactyls. The frog and heel bulbs have been envisaged as being homologous with the digital pad of pigs and canines, the footpad of tapirs and rhinos, and the bulb of the finger in humans . Moreover, the frog frequently does not leave an impression at all, due to it being filled up or covered over with sediment, or due to collapse of the substrate after the foot is withdrawn (figure 6b). Sometimes the print is an outline of the hoof shape alone (as on dry sand or dirt), sometimes just the hoof wall, sole, V-bars and collateral sulci, but not the frog, leave an impression , and on occasion the frog itself will make an impression . Additional figures of the footprints of extant horses, showing the presence or absence of a frog impression, can be found in the electronic supplementary material (figures S10 and S1b). The same pattern occurs in Pleistocene equid footprints. Occasionally a frog is clearly visible and ‘easily distinguished’ at the back of a footprint  horses when the hoof is clean and the substrate apposite , the impfigure 6see also , pp. 313see also , p. 5, fsee also , p. 11; see also , pp. 204see also , p. 217,Hence, the absence of frog impressions in fossil equid trackways by itself need not indicate the absence of a morphological frog on the hoof. The fact that no frog is present in the Laetoli equid impressions may be more related to substrate and preservation than to the lack of a frog on the hoof. Indeed, the hominid footprints at Laetoli fail to preserve much detail regarding toes or grooves in human feet, and so it is reasonable to assume that the sediment either adhered to the feet, reducing fidelity of tracks, or responded to indentation in such a way as to not preserve subtle detail.Hippipeda parva), formed in marine sand, bears the clear trace of a well-developed frog at the back of the hoof [Anchitherium, which matches well with its size and geological dating, but could also be from a hipparionin equid , as anchitheres are considered to have had a footpad. Whatever the identity of the maker of this trackway, it shows an indisputable frog in a tridactyl equid, as monodactyl equids were unknown in the Old World until the Pleistocene.There are also ichnological indications of a frog in the footprints of some tridactyl Miocene and Pliocene equids. Some of these are often rather equivocal and open to interpretation and are detailed further in the electronic supplementary material. But a clear frog can be seen in a hoof print from the late middle Miocene outside of Bila Tserkva, Ukraine . This small—2.8 cm long by 2.2 cm wide—hoof print . Moreover, a few of the Hoya de la Sima prints display a small indentation running parallel to the direction of travel posterior to the V-bars that greatly resembles those formed by the central sulcus of the frog in modern equine prints (figure 8b), further suggesting the presence of a frog. Other trackways of probable tridactyl equids evidencing frogs are further discussed in the electronic supplementary material.In addition, a V-shape (uve) occurs at the rear of footprints made by a tridactyl equid at the late Miocene (Messinian) Hoya de la Sima site mentioned above , then it must be a feature of all of the family Equinae, at least the ones more derived than the basal species of ‘Merychippus’: Hipparionini plus Protohippini diverged from Equini in the late early Miocene approximately 17.5 Ma [Archaeohippus, Desmatippus and Parahippus, which first appeared in the latest Oligocene . The in. 4.7Equus and many other species in the Equinae, . Domestic horses  have very variable phalanx size and comprise the data points that diverge the most from the isometric line that in the main separates the tridactyl species from the monodactyl ones  have a mean OI of 1.19 while the monodactyl equines have a mean OI of 0.88 (n = 96). With the removal of the tetradactyl (at least in the forefoot) hyracotheres, which also tend to have very narrow ungual phalanges, the tridactyl anchitheriines or equines (n = 110) have a mean OI of 1.15 for the ungual phalanx III. Restricting the tridactyl forms further to just the equines (n = 65), so spring-footed forms are being compared with each other, their mean OI is still 1.15. A final analysis compared this same grouping, but without the individuals of the domestic horse, Equus ferus caballaus, as this species can have highly variable ungual phalanges, some of them being very wide (n = 78) was only slightly greater (0.91) and the difference between tridacyl and monodactyl equines was still highly statistically significant .The contrast in ungual phalanx shape is borne out by statistical analysis comparing the OI values (maximum length/maximum width) for the ungual phalanx III of tridactyl versus monodactyl equid taxa . All the analyses yielded a statistically significant two-sample Welch's ery wide , and it n = 18, mean OI = 1.12; monodactyl, n = 17, mean OI = 0.85; p = 0.000001 in a Welch's t-test comparing these two groups) or hind foot ungual phalanges III alone  of the same individuals, or in combination .The hind ungual phalanges of digit III do tend to be slightly narrower and longer than those of the front from the same individual in many equids, and so overall have a slightly larger OI value . However, a statistically significant large difference in mean OI between equids still occurs when comparing forefoot ungual phalanges III alone  compared with all monodactyl forms  does not show a significant difference . However, if the heminones and the South American monodactyl (sub)genera of Hippidion and Amerhippus are excluded, both of which tend to have hooves that are longer than wide, then a statistically significant result is obtained  when performing a two-sample Welch's t-test comparing the OI of tridactyl hoof prints with the remaining monodactyl forms .A statistical analysis of the OI of the hoof prints of all tridactyl forms . At. AtDinoh kg  middle to late Miocene anchitheriin genera halanges , and poshalanges . Some ofAmerhippus and Hippidion, as well as some extinct hemione-like Equus specimens . The limbs are completely formed by day 107. Although they do not specifically address the issue of side digits, they do not mention any evidence of these in the specimens they examined. Barreto et al. [a illustrates a cross-section through the hoof at 45 days, clearly showing the corneal tissue of the walls, sole and the region where the frog will develop. By day 55, the hoof is well developed. Again, there is no evidence of any distal segment of the side digits.In examining the developmental evidence, it is critical to identify the stage of embryonic/fetal development. Kavanagh et al.  examinedet al.   that th [et al.  examinedo et al.  examinedet al. [The equine embryos examined by Solounias et al.  were at et al. [An additional difficulty with their interpretation of the ‘folds’ is that Solounias et al.  examinedet al. ,119,120.et al.  examinedet al. ,122.. 5.2Hipparion [It has been claimed that horses are born with a claw on the front of their hooves ,123 thatipparion . Where dd), as it is stated that the ‘claw’ is not worn off until sometime after birth [During later stages of fetal development, as the hoof wall, sole and frog continue to grow and begin to produce hard keratin, the soft keratin is displaced distally, where it forms a soft mass covering the ends and plantar surface of the hoof by the time of birth ,121,122.er birth ,123.These soft keratinous structures in foals appear to be highly variable and are generally described as leaf-like masses. The hooked structure observed by  and inteHipparion, the ungual phalanx is somewhat less rounded anteriorly and has a small notch (cleft or fissure) on the anterior border that Solounias et al. [Thoatherium, although we do not know of any instances in extant taxa, and its function is unknown. However, chalicotheres (large fossil perissodactyls) have fissured ungual phalanges that do appear to have supported claws, but the entire phalanx is elongated and arched [Thus, we do not consider it appropriate to refer to this structure as a ‘claw’; not only is it part of a variable soft keratin deciduous structure, but it also lacks the characteristics of a true mammalian claw, and there is no mechanism to maintain this shape in the equine hoof after birth. Mammalian claws, like hooves, consist of a hard keratin sheath, which takes its shape from the underlying dermal layer, which in turn follows the contours of the supporting ungual phalanx and is attached to the periosteum ,125–127.s et al.  believe d arched . The fis. 5.3As described above in §4.4, the frog is a specialization of the anterior projection of the bulbs onto the plantar surface of the posterior hoof (heel); the heel bulbs project anteriorly into the sole in a wide range of ungulates. Only the degree of specialization in equids is unique, suggesting that there is no need to invoke incorporation of additional digits to explain its evolutionary origin. Bragulla  examinedAdditional evidence that only digit III contributes to the frog is provided by some so-called polydactyl horses (variants of regular domestic horses) that possess at least one additional digit see ,134–137)–137134–1There is also no developmental evidence to support the hypothesis that the plantar processes and collateral hoof cartilages (hoof wings) are derived from digits I and V, since the distal portions of those digits are not reported in any developmental study ,117,118.. 5.4et al. [Solounias et al.  consideret al. ,76. Theset al. ,76.The plantar digital arteries give off numerous branches supplying the phalanges and interphalangeal joints ,76. We b. 6et al. [et al. [Solounias et al.  present  [et al. . HoweverHipparion possessed a footpad is also refuted by the osteology of the foot of derived tridactyl equids (i.e. derived Anchitheriinae plus hipparionin and protohippin Equinae) which clearly shows an Equus-like spring foot with an unguligrade foot posture and loss of the footpad .Further confirmation of the frog being formed independently of any distal remnants of side digits is found in the occasional modern horse born with an additional vestigial toe or toes, so-called polydactyl horses . In someet al. [While the developmental studies of Kavanagh et al.  and otheet al.  that disEquus foot can be shown to be present in fossil hoof prints of tridactyl equids. Small frogs may also have been present on the hooves of the side digits of tridactyl equids, as sometimes seen today in the accessory digits of polydactyl domestic horses. The original equid footpad was certainly lost in the spring-footed forms (Equinae and derived Anchitheriinae) and may have been lost in more basal anchitherines, as suggested at least by one fossil footprint. The equid frog and bulbs of the heel may well represent a remnant of the original footpad, retained in a modified form as a shock-absorbing structure and has probably been a feature of the hoof for much of equid evolutionary history. The feet of modern horses may technically be polydactyl proximally, but they are definitely monodactyl distally.The composition of the hoof of modern horses was probably also the form of the hoof of the central toe (digit III) in many of the extinct tridactyl forms. The frog that characterizes the sole of the AM or AMNH = American Museum of Natural History, New York, USA. F:AM = Frick collection, American Museum of Natural History. USNM = United States National Museum, Washington, USA. MCZ = Museum of Comparative Zoology, Harvard University, USA.


A combination of phosphorylated peptide enrichment and mass spectrometry was then applied to study the cGMP-PKG-dependent phosphoproteome. We identified a host of novel potential cGMP-PKG downstream substrates and related kinases using this approach and selected the RAF1 protein, which may act as both a substrate and a kinase, for further validation. This showed that the RAS/RAF1/MAPK/ERK pathway may be involved in retinal degeneration in a yet unclarified mechanism, thus deserving further investigation in the future.Retinitis pigmentosa (RP) is a frequent cause of blindness among the working population in industrial countries due to the inheritable death of photoreceptors. Though gene therapy was recently approved for mutations in the RPE65 gene, there is in general no effective treatment presently. Previously, abnormally high levels of cGMP and overactivation of its dependent protein kinase (PKG) have been suggested as causative for the fatal effects on photoreceptors, making it meaningful to explore the cGMP-PKG downstream signaling for more pathological insights and novel therapeutic target development purposes. Here, we manipulated the cGMP-PKG system in degenerating retinas from the  There ise cells . The degse cells , respectse cells ,9. It woPKG1, and PKG2, with PKG1 having two isoforms, PKG1alpha and PKG1beta [There are two forms of PKG, PKG1beta . cGMP-PKPKG1beta . The impPKG1beta ,12 and aPKG1beta . DespiteCertain analogs of cGMP, a category of chemically modified versions of the native cGMP ,15,16, erd1 retinal explants, and via a phosphoproteomic analysis, which combined phosphopeptide enrichment with mass spectrometry (MS) [rd1 retina and, as such, provide more insights into the retinal degeneration mechanisms in RP.In this study, we have therefore used a PKG inhibitor on try (MS) , comparet-test showed that 299 phosphosites of 108 proteins had decreased phosphorylation, while 184 phosphosites of 83 proteins had increased phosphorylation in retinas with PKG inhibition for 2 h compared to their untreated counterparts  or 4 days (long term) PKG inhibition. . For proPKG1), general transcription factor IIi (GTF2I), and protein phosphatase 1 catalytic subunit alpha (PPP1CA), were involved in the cGMP-PKG pathway. This supported that PKG signaling in explants was appropriately inhibited during culturing. By contrast, the proteins of the increased phosphorylation were mostly related to pathways connected with proteoglycans, ErbB signaling, and tight junctions (A protein annotation in the Kyoto Encyclopedia of Genes and Genomes (KEGG), with the targets mentioned above, was performed to address the biological pathways in which the cGMP-PKG-dependent substrates were involved. Proteins with decreased phosphorylated sites after PKG inhibition were annotated in RNA transport, Spliceosome, and Rap1 signaling pathway A. A partunctions B. In thihttps://phosphomatics.com/). The outcomes are given as kinome trees  and cAMP-dependent protein kinase catalytic subunit alpha (PRKACA), whereas 10 kinases came up as having increased activity at both time points. Of the latter, four kinases were related to the mitogen-activated protein kinase (MAPK) family  family . There w) family , such asRAF1) was selected for additional analysis. As mentioned above, several of the upstream kinases with increased activities belonged to the MAPK family. The rationale for selecting RAF is thus that MAPK1 is expressed in photoreceptors and is linked to photoreceptor death, at any rate in Drosophila [RAF1 are required to satisfy photoreceptor differentiation [RAF1 was proposed as having higher kinase activity after 2 h of PKG inhibition , where the photoreceptor cell bodies and processes reside , followed by an analysis of possible RAF1 alterations in photoreceptors after treatment. RAF1 expression was higher in the ONL after PKG inhibition, regardless of the different lengths of treatment.Next, the potential link between RAF1 expression was in any way also reflected in the phosphorylation state of RAF1, the PLA technique for the combination of RAF1 protein and phosphorylated serine [RAF1 phosphorylation profile was applied. The PLA uses antibodies against two different antigens, as well as certain secondary antibodies containing complimentary “primers” of a reaction product. If the two antigens are in close proximity (<40 nm), the method then allows a reaction product to be formed. Thus, if the RAF antibody is in close contact with the phosphoserine antibody, it is likely that both antibodies sit on the same protein, namely RAF1, and thus identify phosphorylated RAF1. We focused on the RAF1 phosphorylation in ONL since RAF1 was higher expressed in the rd1 ONL  it identified potential retinal PKG substrates that could be involved in retinal degeneration, (2) it gave information on the kinase and pathway networks in operation during the degenerative events, and (3) it showed a correlation between the cGMP-PKG system and the The upstream kinase and KEGG analyses both picked up PKG or the cGMP-PKG signaling pathway as being affected by the treatment, which corroborated the usefulness of the approach. In addition, the two treatment regimes, i.e., 2 h or 4 days of PKG inhibition, gave data on proteins whose phosphorylation was decreased as well as such that displayed increased phosphorylation in either or both of these regimes.TOP2B) and capicua transcriptional repressor (CIC). Interestingly our previous transcriptome study revealed the association between cGMP-PKG and these two targets; hence, there is reason to keep TOP2B and CIC as candidates for links between PKG activity and events during retinal degeneration. Proteins that instead showed persistent increased phosphorylation after PKG inhibition are, by contrast, more likely to be dependent on kinases under direct or indirect negative regulation by PKG and were represented by as many as 16 proteins. Some deserve to be mentioned here, such as ankyrin 2 (ANK2), with a possible role in maintaining ion balance, via anchoring of ion transporters to the photoreceptor cell membrane [MAP) family, including MAP1B, MAP2, and MAP4, were also seen. MAPs could be critical for photoreceptors, and at least one of these may associate with retinal degeneration [The assumption that increased phosphorylation by PKG is part of the degeneration mechanism makes the proteins with reduced phosphorylation at both time points of PKG inhibition particularly interesting with respect to a pathologic involvement. Among the five proteins of this category, we noticed DNA Topoisomerase II Beta  and control C3H wild-type (wt) were kept and bred in-house under standard white cyclic lighting, with free access to food and water [The C3H/nd water . The ani2 atmosphere, and the medium was replaced every two days. No additions were made to the cultures for the first 2 days. One group of the cultures, which at this point were at an age corresponding to P7, were then exposed to 50 µM Rp-8-Br-PET-cGMPS  for the following 4 days, with the endpoint thus equivalent to P11 (n = 4), with their corresponding controls (n = 4) receiving the same amount of distilled water. Another group (n = 4) was exposed to the Rp-8-Br-PET-cGMPS with the same concentration but only for 2 h before the end of the protocol, with controls (n = 4) similarly exposed to distilled water. All retinal explants were either collected for protein extraction, followed by phosphoproteomic analyses. In parallel, another four groups of rd1 explants, reaching a total of 13 explants, were generated. One group of retinas (n = 4) was exposed to the 50 µM Rp-8-Br-PET-cGMPS for 4 days, while their corresponding untreated controls (n = 3) received the same amount of distilled water. The remaining two groups included one (n = 3) that was treated with 2 h of PKG inhibition with the same concentration and one with its untreated peers (n = 3). These explants were fixed, sectioned, and used for microscopy-based studies.Retinas from P5 mice were used to generate explants according to our standard protocol . InsertsAnimals were randomly assigned to the experimental groups, and the experimenters were aware of the conditions of the animals during retinal explant culture. No statistical method was applied to pre-determine the sample size of the experimental groups. The sample size was chosen based on the relevant literature in the field as well as on ethical considerations. The study was not pre-registered. The study was exploratory without exclusion criteria being pre-determined.2PO4, 1 mM DL-Dithiothreitol (DTT)), supplemented with phosphatase inhibitors  using a homogenizer . The homogenate was then centrifuged at 10,000× g for 5 min at 4°C. The soluble fraction was collected after centrifugation, with the concentration measured by Bio-Rad Protein Reagent Assay Kit .A total of 16 retinal explants were used for MS measurement, and 4 explants were used per group. The experimenter was unaware of the explant’s group during experimentation. Each explant was homogenized separately in buffer . Digestion was performed by adding trypsin  in a ratio of 1:50 to the samples and incubated overnight at 37 °C. The digestion was stopped by the addition of 5 µL 10% trifluoroacetic acid (TFA).For each separated sample, proteins were reduced with DTT to a final concentration of 10 mM and heated at 56 °C for 30 min, followed by alkylation with iodoacetamide for 30 min at room temperature in the dark to a final concentration of 20 mm. Subsequently, samples were precipitated with ice-cold ethanol overnight at −20 °C followed by centrifugation at 14,000× The Pierce High-Select Fe-NTA Phosphopeptide Enrichment Kit  was used to enrich phosphopeptides according to the manufacturer’s protocol. The phosphopeptides were run in a Speed Vac to dryness and resolved in 2% acetonitrile (ACN) and 0.1% TFA to a peptide concentration of 0.25 µg/µL.−1. In the first step, a gradient of 10 to 30% of buffer B was run for 90 min, followed by a 30 to 45% gradient of buffer B in 20 min. One full MS scan  was followed by MS/MS scans . The precursor ions were isolated with 1.3 m/z isolation width and fragmented using higher-energy collisional-induced dissociation at a normalized collision energy of 28. Charge state screening was enabled, and singly charged ions as well as precursors with a charge state above six were rejected. The dynamic exclusion window was set to 10 s. The automatic gain control was set to 3 × 106 for MS and 1 × 105 for MS/MS, with ion accumulation times of 45 ms and 60 ms, respectively. The intensity threshold for precursor ion selection was set to 1.7 × 104.The peptide analyses were performed on a Q Exactive HFX mass spectrometer (Thermo Scientific) connected to an EASY-nLC 1200 ultra-high-performance liquid chromatography system (Thermo Scientific). Peptides, 1 µg, were separated on an EASY-Spray column  operated at a constant pressure of 800 bar. A two-step gradient of buffer B  in buffer A (aqueous 0.1% formic acid) was applied at a flow rate of 300 nL minThe raw DDA data were analyzed with Proteome Discoverer™ Software . Peptides were identified using both SEQUEST HT and Mascot against the UniProtKB mouse database (UP000000589 plus isoforms). The search was performed with the following parameters applied: static modification: cysteine carbamidomethylation and dynamic modifications: N-terminal acetylation. Phosphorylation  was set as a variable for the phosphopeptide analysis. Precursor tolerance was set to 10 ppm and fragment tolerance to 0.02 ppm. Up to 2 missed cleavages were allowed, and Percolator was used for peptide validation at a q-value of a maximum of 0.05. Peptides with different amino acid sequences or modifications were considered unique. Extracted peptides, with a quality q-value lower than 0.05, and modification of S, T, and Y phosphorylation detected were used to identify and quantify them by label-free relative quantification. The extracted chromatographic intensities were used to compare peptide abundance across samples.https://phosphomatics.com/; [t-test (two-tailed) was performed to compare phosphorylated site levels between the rd1 explants with 2 h of PKG inhibition vs. untreated control and 4 days of PKG inhibition vs. untreated controls. A p-value of 0.05 was defined as the cutoff. Two lists of the phosphorylated sites identified previously were used to perform a further upstream kinase analysis in Phosphomatics. The biological pathways that might be affected between rd1 untreated and rd1 with PKG inhibition samples were determined in Enrichr (https://maayanlab.cloud/Enrichr/), where differentially phosphorylated sites mentioned above were used as an input list to perform pathway-based analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG).The MS results were processed via Perseus software (version 1.6.0.7) . The procs.com/; ). In Phord1 and wt in vivo at P9, as well as P11 cultured explants from rd1, were treated with 4% formaldehyde for 2 h, washed 3 × 15 min in phosphate-buffered saline (PBS), cryoprotected in PBS + 10% sucrose for overnight at 4 °C and subsequently with PBS + 25% sucrose for 2 h. After embedding in a medium with 30% bovine serum albumin  and 3% gelatine  mixed in H2O, 12 μm thick retinal cross-sections were cut and collected from an HM560 cryotome . The sections were stored at −20 °C for later usage. Cryosections were then used for immunostaining and PLA. Totally 10 retinal samples were used, with 5 each in either rd1 or wt strains, while 13 rd1 explants were used, with 4 in the group of retinas with 4 days of PKG inhibition and 3 each in the other 3 groups.Retinal tissues from RAF1  was diluted with 1% BSA and 0.25% Triton X100 in PBS (PTX) and incubated at 4 °C overnight; a no primary antibody control ran in parallel. Sections were washed 3 × 5 min each in PTX and incubated with a donkey anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor™ Plus 488  at 1:800 dilution in PTX. After 3 × 5-min PBS washes, the sections were mounted with Vectashield DAPI .For immunostaining, briefly, the cryosections were dried at room temperature for 15 min and rehydrated in PBS. Then they were blocked with 1% BSA + 0.25% Triton X100 + 5% goat serum in PBS at room temperature for 45 min. The primary antibody anti-RAF1  and anti-Phosphoserine  were incubated overnight at 4 °C. PLA Probe anti-rabbit PLUS, and anti-mouse MINUS were incubated for 1 hr at 37 °C. The ligation and amplification steps were performed using the Detection Reagents Red, followed by the addition mounting of Vectashield DAPI .The PLA, which detects if two antigens are in close proximity (<40 nm) with each other, was performed on cryosections, using Detection Reagents Red kit , the PLA Probe anti-rabbit PLUS , and anti-mouse MINUS . The procedure followed the manufacturer’s instructions. In short, the retinal sections were blocked with blocking solution (provided in PLA Probe kit) for 45 min at 20 °C, and primary antibodies antibody anti-A Zeiss Imager Z1 Apotome Microscope  with a Zeiss Axiocam digital camera was used for microscopy observations. Image generation and contrast enhancement were performed identically for all images via the ZEN2 software (blue edition). The immunostaining was analyzed for staining differences via three sections, each from three to five animals for each condition, after which the fluorescent intensities of positive cells were randomly distributed within the area of interest  were assessed. Fluorescence intensity was captured and analyzed by the ImageJ software . The freehand selection function was used to target the ONL, after which the fluorescence intensity was calculated with the measure function. The values of all sections from the same animal were averaged. For PLA, the images were also generated with the aid of a Zeiss Imager Z1 Apotome Microscope and the ZEN2 software (blue edition). The punctuations of ONL, which represent reaction products from when the two antigens are in close proximity, were counted manually in three sections, each from three to five animals for each condition, with values from the same animal averaged. The experimenter was unaware of the groups of the samples during image processing.t-test was used to compare the values of immunostaining intensities and PLA punctuations in different conditions in R, and the p-value cutoff was defined as 0.05. Visualization of KEGG analysis was also performed in R.The Student’s


Atlantic Niño, the dominant climate mode in the equatorial Atlantic, is known to remotely force a La Niña-like response in the Pacific, potentially affecting seasonal climate predictions. Here, we use both observations and large-ensemble simulations to explore the physical mechanisms linking the Atlantic to the Pacific. Results indicate that an eastward propagating atmospheric Kelvin wave from the Atlantic, through the Indian Ocean, to the Pacific is the primary pathway. Interaction of this Kelvin wave with the orography of the Maritime Continent induces orographic moisture convergence, contributing to the generation of a local Walker Cell over the Maritime Continent-Western Pacific area. Moreover, land friction over the Maritime Continent dissipates Kelvin wave energy, affecting the strength of the Bjerknes feedback and thus the development of the La Niña-like response. Therefore, improving the representation of land–atmosphere–ocean interactions over the Maritime Continent may be fundamental to realistically simulate Atlantic Niño’s impact on El Niño-Southern Oscillation. Equatorial Atlantic sea-surface temperature anomalies force an eastward propagating atmospheric Kelvin wave, enabling the Atlantic to impact the Pacific, with the interaction of the Kelvin wave and the Maritime Continent critical in this teleconnection. The current skill and reliability of seasonal climate forecasts, however, leave much room for improvement2. Since the key paradigm for seasonal climate forecasts is El Niño-Southern Oscillation (ENSO)—the most prominent coupled atmosphere–ocean phenomenon on seasonal-to-interannual timescales3, it is paramount that we continue to improve our understanding of ENSO dynamics and its predictability.As seasonal climate forecasts continue to improve, the use of these forecasts has increased across a range of application sectors, including agriculture, health, water management and others4. In particular, studies have shown that Atlantic Niño or Atlantic Zonal Mode5—a phenomenon in the equatorial Atlantic similar to ENSO, albeit weaker amplitude and more sporadic, can remotely force a La Niña-like response in the Pacific16. The unfolding 2021/22 La Niña event is consistent with this Atlantic-forcing-Pacific premise: following the exceptionally strong Atlantic Niño in the boreal summer of 2021, a La Niña peaked in the Pacific during the winter of 2021 . However, this description is based on a fully developed response of the atmosphere to Atlantic Niño sea-surface temperature (SST) forcing and does not explain how the anomalous Walker Circulation is first developed, which leaves a gap in our understanding of the fundamental mechanism in the Atlantic-to-Pacific teleconnection. Some of the specific questions remaining to be examined include: how does the anomalous Walker Circulation develop outside the EA? Where does the development start and how? What dynamical processes are responsible for the development? Understanding these questions can have important potential implications for improving ENSO simulations and predictions. This study focuses on exploring these questions by combining observational analyses with large-ensemble climate simulations.Mechanisms of Atlantic Niño remotely forcing La Niña in the tropical Pacific have been described in terms of an anomalous Walker Circulation with an ascending branch over the equatorial Atlantic (EA) where SST is anomalously warm and a descending branch over the Western and Central Pacific (WP/CP) . Figure iña Fig.  coupled iña Fig. . This de21, with a fast, eastward propagating atmospheric Kelvin wave, carrying easterly wind anomalies across the Indian Ocean into the Pacific. This Kelvin wave mechanism linking Atlantic Niño to the Pacific has been proposed22, but not fully validated in a coupled modeling framework. To further validate this mechanism, we conducted large-ensemble Community Earth System Model23 (CESM) simulations (hereafter CTRL), where an observed Atlantic Niño SST anomaly is prescribed  , we removed all the topographic features over the MC  while keeping everything else the same as CTRL  ATL3 index, which was defined by averaging detrended equatorial Atlantic SST anomalies over the region 20°W–0° and 3°S–3°N, after linearly regressed out December-January-February (DJF) Niño3.4 index from all the variables to remove ENSO’s influence on Atlantic Niño. Here the Niño3.4 index was defined by averaging equatorial Pacific detrended SST anomalies over the region 170°W–120°W and 5°S–5°N, and both the ATL3 and ENSO indices were normalized by dividing by their respective standard deviations. Note that we did not use the commonly used Jun-Jul-Aug (JJA) ATL3 index in Fig. 19. The partial linear lag-regression against the Feb-Mar-Apr (FMA) ATL3 index is also shown in Supplementary Fig. In Fig. −1J0F0 (−1 indicates the previous year and 0 indicates the current year) Niño3.4 index exceeded its standard deviation. The results are shown in Supplementary Fig. To test the robustness of the results, we also used a different method to remove ENSO’s influence, in which we first excluded the years of strong ENSO from the data record and then performed lag-regression analysis against the MJJ ATL3 index. A strong ENSO year was defined from current January to December if the D26 during 1959–2021, and the winds at various isobaric levels from 175 to 1000 hPa from ERA5 Reanalysis27 (1959–2021), ERA-Interim Reanalysis28 (1979–2018) and JRA55 Reanalysis29 (1959–2021). To fully utilize the three reanalysis products and minimize internal variability, we first averaged the wind fields from the three reanalysis datasets and then performed the lag-regression analysis  with a nominal ~1° horizontal resolution for all model components that include ocean, atmosphere, land, sea ice, land ice, and river components. The ocean component model—Parallel Ocean Program version 2 (POP2) has 60 vertical levels, the atmosphere component model—Community Atmospheric Model version 5.2 (CAM5.2) adapts 30 hybrid vertical pressure levels, and the land component model—Community Land Model version 4 (CLM4) shares the same horizontal grid with atmosphere component, while the sea-ice model—Community Ice Code version 4 (CICE4) shares the same horizontal grid with the ocean component. Three ensembles of the CESM1.1.2 simulations were performed, each of which consists of two sets of simulations—one with positive (+) Atlantic Niño SST anomaly  has no modifications made to MC and follows the standard pacemaker experiment design15, in which model SST is restored to the prescribed +/− Atlantic Niño SST plus the daily-mean model climatology over the region 69°W–18°E and 24°S–24°N with a restoring timescale of 5 days and 8° of buffer zones in the meridional direction at the northern and southern boundaries . A brief summary of the three ensembles is given in Table All the large-ensemble experiments were conducted using Community Earth System ModelSince we combined the observed Atlantic Niño SST anomaly with model climatology, the model SST bias is retained in the simulations. The advantage of such a setup is that model mean states in each tropical basin are dynamically consistent, but the disadvantage is that the model bias may have an impact on simulating Atlantic Niño teleconnection. However, CESM1 biases are relatively small with SST bias of less than ±1 °C over much of the tropical oceans , and d is the displacement height (m). ρatm is the density of atmospheric air (Kg m−3), ratm is the aerodynamic resistance to momentum transfer (s m−1).Momentum fluxes over land grid points are determined using sub-grid roughness lengths (m) and displacement heights (m) appropriate for the land surface heterogeneity. The momentum fluxes between the land surface and atmospheric reference height are written in the form31, the dimensionless mean horizontal wind speed profile gradients depend on the similarity function of 32, where Z is the atmosphere height (m), and L is the Monin–Obukhov length scale (m) that accounts for buoyancy effects resulting from vertical density gradients . The momentum aerodynamic resistances can then be written as30:k is the von Kármán constant (0.4), Va is the wind velocity at reference height, including the convective velocity, and ϕm is a function of ζ that relates the constant fluxes of momentum to the mean profile gradients of winds.Based on the Monin–Obukhov similarity theoryZ0m is a constant (0.01 m) for soil, glaciers and wetland non-vegetated surfaces. For vegetated surfaces, Z0m depends on plant height and adjusts for canopy density (10−1−101 m)30. In NFRC simulation of this study, Z0m is set to an artificially small value of 1 × 10−10 m to reduce land surface friction.In CLM4, 34 (Kg s−1), which can be calculated by:a is the radius of the Earth, Δφ is the width of 10°S–10°N in radians proportional to Earth’s radius, g is the gravity constant, and uD is the divergent part of zonal winds averaged between 5°S–5°N in the meridional direction.The intensity of the Walker Circulation can be estimated by the zonal mass stream-functionIn Fig. Supplementary InformationPeer Review File


At seasonal-to-interannual timescales, Atlantic hurricane activity is greatly modulated by El Niño–Southern Oscillation and the Atlantic Meridional Mode. However, those climate modes develop predominantly in boreal winter or spring and are weaker during the Atlantic hurricane season (June–November). The leading mode of tropical Atlantic sea surface temperature (SST) variability during the Atlantic hurricane season is Atlantic Niño/Niña, which is characterized by warm/cold SST anomalies in the eastern equatorial Atlantic. However, the linkage between Atlantic Niño/Niña and hurricane activity has not been examined. Here, we use observations to show that Atlantic Niño, by strengthening the Atlantic inter-tropical convergence zone rainband, enhances African easterly wave activity and low-level cyclonic vorticity across the deep tropical eastern North Atlantic. We show that such conditions increase the likelihood of powerful hurricanes developing in the deep tropics near the Cape Verde islands, elevating the risk of major hurricanes impacting the Caribbean islands and the U.S. Atlantic Niño, the Atlantic counterpart of the Pacific El Niño, increases the likelihood of powerful hurricanes developing near the Cape Verde islands, elevating associated risks for the Caribbean islands and the U.S. The positive phase of the Atlantic Meridional Mode (AMM), characterized by warm SSTAs and low-level westerly wind anomalies over the tropical North Atlantic, also decreases the atmospheric static stability and vertical wind shear over the MDR and thus increases Atlantic TC genesis3. Conversely, El Niño, the positive phase of ENSO, and the negative phase of AMM both tend to increase the atmospheric static stability and vertical wind shear over the MDR and thus suppress Atlantic TC genesis8.Seasonal outlooks for Atlantic tropical cyclone (TC) activity are issued by the National Oceanic and Atmospheric Administration (NOAA), the European Center for Medium-Range Weather Forecasts and other agencies to provide a general guide to the expected overall activity during the upcoming hurricane season. These outlooks are largely based on foundational relationships between observed oceanic and atmospheric states. For instance, during La Niña, the negative phase of El Niño–Southern Oscillation (ENSO), cold equatorial Pacific sea surface temperature anomalies (SSTAs) produce a fast tropical teleconnection (via atmospheric Kelvin waves) that decreases the atmospheric static stability and vertical wind shear over the TC main development region (MDR) and thus increase Atlantic TC activity16. The West African summer monsoon is also directly connected to the seasonal development of the West African westerly jet in the deep tropical eastern North Atlantic centered around 10°N18. The westerly jet produces positive low-level relative vorticity  along its northern flank, promoting the formation of TCs in the deep tropical eastern North Atlantic17.Seasonal TC activity in the Atlantic basin is also closely tied to the West African summer monsoon intensity. Specifically, the West African summer monsoon promotes synoptic-scale low-pressure disturbances known as African Easterly Waves (AEWs) that often provide the mechanical uplift of surface air parcels above the level of free convection and thus can trigger TC genesis19. During the 2008 Atlantic hurricane season, however, both ENSO and AMM remained in neutral conditions. Coincidentally, the equatorial Atlantic was dominated by Atlantic Niño conditions, typically characterized by warm SSTAs and deeper thermocline in the eastern basin and low-level westerly wind anomalies in the western basin22.These climate modes of variability and the associated atmosphere-ocean processes are well-established predictors or indicators for seasonal Atlantic TC activity. While the use of potentially predictable climate modes has made skillful seasonal Atlantic TC outlooks possible, there is still much uncertainty in these forecasts. For instance, the 2008 Atlantic hurricane season was one of the deadliest and costliest Atlantic hurricane seasons, with 16 named storms and five major hurricanes  including a powerful category-4 hurricane (Ike) that resulted in 164 casualties and $30 billion damage in the Caribbean islands and the U.S19. Conversely, the 1992 hurricane season had only four named storms with an Accumulated Cyclone Energy of 76.2 × 104 kt2, both significantly below average19. During that season, ENSO and AMM were both in neutral phases, while Atlantic Niña conditions developed, characterized by cold eastern equatorial Atlantic SSTAs and low-level easterly wind anomalies in the western equatorial Atlantic. These cases during 1992, 2003, and 2008 suggest that Atlantic Niño/Niña is potentially linked to Atlantic TC activity.Similarly, during the 2003 hurricane season, Atlantic Niño conditions developed in the eastern equatorial Atlantic as early as July and continued until January 2004, while both ENSO and AMM remained in near-neutral conditions. The 2003 hurricane season was highly active with 16 named storms, including three major hurricanes. Among those, a tropical depression developed near the Cape Verde islands in early September, triggered by a strong AEW from the West African coast. It later strengthened to a powerful category-5 hurricane (Isabel), made landfall in North Carolina, and resulted in 51 fatalities and $3.37 billion in damage30. Based on the well-established relationships between Atlantic Niño/Niña, the ITCZ and the West African rainfall, we hypothesize that Atlantic Niño/Niña modulates Atlantic TC activity through its impact on atmospheric convection from the deep tropical eastern North Atlantic to sub-Sahel West Africa and the associated changes in regional atmospheric circulations and AEW activity. Consistent with this hypothesis, the number of TCs generated over the tropical North Atlantic  is significantly higher during Atlantic Niño (5.1 ± 0.58 TC/year) and lower during Atlantic Niña (2.9 ± 0.61 TC/year)  rainband, with the ITCZ and sub-Sahel West African rainfall enhanced during Atlantic Niño and suppressed during Atlantic Niñaar) Fig. . This diThe main objectives of this study are to investigate further the links between Atlantic Niño/Niña and seasonal Atlantic TC activity and explore the associated physical mechanisms by analyzing observational and reanalysis datasets. We show the critical role of Atlantic Niño/Niña in the development of major Atlantic hurricanes in the deep tropics near the Cape Verde islands.The spatial patterns of Atlantic TC genesis and track density linked to Atlantic Niño and Niña are first examined using a composite analysis for 74 years  during the Atlantic hurricane season (JJASON) is virtually zero (r = −0.03). However, while the correlation between ATL3 and NIÑO3.4 indices in JJASON is statistically insignificant based on a two-tailed Student’s t-test (r = −0.15), some Atlantic Niño/Niña events are directly forced by ENSO in boreal winter and spring, and Atlantic Niño/Niña may influence ENSO evolution in boreal summer and fall38. Therefore, to properly address the potential interbasin interactions and their impact on Atlantic TC activity, it is necessary to separate the impact of Atlantic Niño/Niña on Atlantic TCs from that of ENSO. To do so, we carried out partial regressions of Atlantic TC genesis and track density onto ATL3 and NIÑO3.4 indices  is significantly higher during Atlantic Niño years (2.16 ± 0.56 per year) and lower during Atlantic Niña years (0.92 ± 0.51 per year). This difference is significant at the 95% confidence level based on a two-tailed Student’s Figure 49, Atlantic Niño/Niña is significantly linked to the number of major hurricanes generated over the tropical North Atlantic. This relationship is even stronger during the post-satellite period, suggesting that not all major hurricanes generated in the tropical North Atlantic are accounted for in IBTrACS during the pre-satellite period50. The influence of Atlantic Niño/Niña on Atlantic TC activity is quite different from that of ENSO, which is predominantly linked to TCs that form in the southern Gulf of Mexico, Caribbean Sea, and central tropical North Atlantic  in the deep tropics (5°–15°N) east of 40°W. The enhanced ITCZ also reinforces the West African westerly jet, strengthening positive low-level relative vorticity in the deep tropical eastern tropical North Atlantic. These atmospheric anomalies lead to an increase in the formation of deep tropical Atlantic TCs in the eastern tropical North Atlantic  and thus potentially increase the probability of TC landfall in the Caribbean islands and the area around Florida Fig. . Since Tic Figs. b and 3c.Although Atlantic Niño/Niña was found to significantly influence the genesis of Cape Verde hurricanes, the overall seasonal Atlantic TC activity is still determined predominantly by ENSO and AMM  and AMM (r = −0.03), two well-established predictors of Atlantic seasonal TC activity. As such, Atlantic Niño/Niña may serve as an additional predictor to improve seasonal Atlantic hurricane outlooks, especially when ENSO and AMM are in near-neutral phases, as occurred during the 1992, 2003, and 2008 hurricane seasons.Finally, Atlantic Niño/Niña is a potentially predictable climate mode of variability that prevails during the Atlantic hurricane season. It is also statistically independent from ENSO 52 is used to derive monthly atmospheric circulation fields. Monthly precipitation is obtained from NOAA’s Gridded Precipitation Reconstruction dataset53. SST is derived from the Hadley Centre Global Sea Ice and Sea Surface Temperature version 1 (HadISST1)54. All analyses carried out in this study are for the Atlantic hurricane season  spanning 74 years (1948–2021). The results are largely consistent and more robust during the post-satellite period  during JJASON or are less than negative one standard deviation, respectively. In total, 19 Atlantic Niño and 14 Atlantic Niña events were identified 16. Following a previous study55, we calculate EKE asEddy kinetic energy (EKE) at 700 hPa is often used as a measure of AEW activity Eq. 16. Follot-test.In order to objectively separate the impact of Atlantic Niño/Niña on Atlantic TC from that of ENSO, we computed partial regressions of Atlantic TC genesis and track density onto ATL3 and NIÑO3.4 indices as followsions Eq. . \documeSupplementary informationPeer Review File