Intra-cardiac echocardiography (ICE) has become an important tool for catheter ablation. Adoption of ICE imaging is still limited because of its prohibitively high cost. Our aim was to study the safety and feasibility of ICE catheters reprocessing and its environmental and financial impact..This was a single center retrospective analysis of all consecutive electrophysiology procedures in which ICE catheters were used from 2015 to 2022. In total, 1128 patients were studied . The majority of procedures were related to atrial fibrillation ablation (84.6%)For the whole cohort, 57 new ICE catheters were used. Consequently one catheter could be used for 19.8 procedures. New catheters were only used when the image obtained by reused probes was not satisfactory. There were no cases of ICE probe steering mechanism malfunction, no procedure related infections and no allergic reactions that could be attributed to the resterilization process. In total, there was 8.6% of complications not related to ICE imaging. Financially, ICE probe reprocessing resulted with 90% cost reduction (> 2 millions of Euros savings for the studied period) and 95% waste reduction .Our data suggests that ICE catheter reprocessing is feasible and safe. It seems that risk of infection is not increased. Significant economic and environmental savings could be achieved by ICE catheters reprocessing. Furthermore, ICE reprocessing could allow more extensive ICE usage resulting in safer procedures with a potential reduction of serious complications. Intra-cardiac echocardiography (ICE) is increasingly used in several percutaneous interventional and electrophysiology (EP) procedures. It has become the essential tool which offers real-time, high-quality evaluation of cardiac anatomy. ICE imaging markedly increases the safety of various cardiac interventions, especially the trans-septal punctures (TSP) and related procedures . FurtherHowever, ICE catheters are designated for a single use only and their high cost precludes its widespread adoption. Similar to other EP catheters that are officially single use devices, ICE catheters can be resterilized and reused in several countries that permit reprocessing . ICE resData were retrospectively collected from patients having undergone EP procedures using ICE catheters between February 2015 (when we started using ICE imaging) and December 2022 at the University Hospital Centre Zagreb, Croatia. There were no exclusion criteria, and all consecutive procedures when ICE was used were included regardless of arrhythmia and ablation type. Intracardiac echocardiography was consistently used for all atrial fibrillation (AF) procedures to guide TSP. The same was true for all other transseptal procedures including left sided accessory pathway ablations, left sided atrial flutters (Aflu), and structural ventricular tachycardia (VT) ablations. Furthermore, selected typical flutter ablations (such as “redo” cases) and premature ventricular contractions (PVC) ablation cases in specific locations such as aortic cusps and papillary muscles were also performed with the help of ICE. All procedures were performed by 3 different experienced operators and all patients provided written informed consent for the ablation which included the information about catheter resterilization. The research was conducted in accordance with the principles of the Declaration of Helsinki.A transthoracic echocardiogram was performed within six months prior to any ablation procedure to assess the left ventricular ejection fraction and to determine any structural heart and/or valvular disease. A transesophageal echocardiogram was performed before the procedure to exclude left atrial appendage thrombi only in patients who presented with persistent AF or AFlu before procedure. If the patients were anticoagulated, we used the uninterrupted anticoagulation strategy in all patients in accordance with recent recommendations .All procedures were performed under conscious sedation combining fentanyl and diazepam or midazolam. Two types of ICE catheters were used, 8 F and 10 F Accuson Acunav phase array catheters (Siemens AG). For AF ablation, cryoballoon (CB)  was most commonly used. Between 2015–2016 radiofrequency (RF) point by point AF ablations were performed usually with double transeptal punctures using circular mapping catheters. Later on, we adopted a single transeptal puncture strategy with high density (HD) mapping catheters that were exchanged for irrigated, contact sensing ablation catheters. Two different 3D mapping systems were used: Carto 3 (Biosense-Webster) and Ensite Precision (Abbott). Three-D mapping systems were consistently used in all AF: atypical atrial flutter, PVC and VT ablation procedures, except when AF ablation was performed with cryoballoon. In some instances, a 3D mapping system was also used in typical flutter and accessory pathway (AP) ablations, especially after 2019 when we adopted “zero fluoro” strategy for the selected EP procedures. For typical flutters, ICE was most commonly used in redo procedures. For PVC ablations ICE was used in all transeptal procedures and for selected aortic root ablations. Usually, HD mapping catheters were used beside the irrigated contact sensing ablation catheters. For structural VT ablations (ischemic and nonischemic), ICE was consistently used for TSP, guiding the ablation and monitoring for possible complications. Intracardiac echo catheters were always reused while new catheters were opened when acquired images were suboptimal . We did not count the exact number or resterilization processes per single catheter. Rather, the catheters were used as long as echo imaging was satisfactory or the catheter did not have clear visible damage to the material. Also, we did not perform any special electrical testing of the catheters. At the beginning of the procedure the resterilized catheter was connected to the echocardiography machine and if the image was present, the catheter was used. In the case that echo machine did not recognize the resterilized catheter, it was discarded.The primary endpoint of the study was the safety of the ICE catheter resterilization. We particularly took note of possible infections and allergic reactions related to resterilization. Besides, we searched for all other possible related complications like material embolization or the need for reused catheter extractions (possible “knotting” of reused catheters). Secondary endpoint included the durability of ICE catheters, as we wanted to determine the mean number of resterilization processes before the catheter was deemed unusable.Categorical variables are expressed as absolute and relative frequencies. Continuous variables followed normal distribution and were expressed as mean ± standard deviation. Statistical analyses were conducted using the SPSS software .A total of 1128 patients were included in our retrospective analysis. Overall, 70.6% of patients were male and the mean age was 57.9 ± 13.2 years. The majority of patients suffered from atrial fibrillation (84.5%), and atrial flutter was the least common (0.8%). More than half of the population had arterial hypertension (64.4%), and significant portion suffered from heart failure (16.1%). Anticoagulant therapy was highly prevalent in the whole cohort (78.4%). None of the patients had known chronic infections such as hepatitis C virus, hepatitis B virus or human immunodeficiency virus. The detailed baseline clinical characteristics of the study population are provided in Table For the total of 1128 procedures, 57 new ICE catheters were used. Thirty-four probes were 10 F in size (59.6%) and twenty-three sized 8 F (40.4%). Consequently, one ICE catheter could be used for an average of 19.8 different procedures. There was no case of an ICE probe steering mechanism malfunction, even after multiple reprocessing cycles. We generally used reprocessed catheters, and a new catheter was opened when operator decided acquired images were suboptimal or there was a significant visual damage to the catheter. Vast majority of all interventions included transseptal punctures (99.4%) and all were successfully performed with the help of ICE. Most procedures were AF ablation, and 789/954 (82.7%) of these were performed by the means of cryoballoon ablation. For the remaining procedures, RF energy was used. Three-D mapping system was used in 235 procedures (20.8%). Among them, Carto 3 system was used in 93.1% of cases and Ensite Precision in the remaining 6.9% of procedures. In the whole cohort, mean procedure time and fluoroscopy time were 88.7 ± 47.9 and 11.6 ± 9.4 min, respectively. Acute procedural success was achieved in 97.1% of procedures.There were no procedure-related infections and no allergic reactions which could be attributed to the resterilization process. Furthermore, we did not have cases of material embolization or the need for the special extraction procedures related to resterilized ICE catheters. Also, there were no deaths following the ablation procedures. In total, 97 complications (8.6%) occurred with no correlation with ICE catheters. Vascular complications were the most common, occurring in 42 (3.7%) patients. They included large (> 5 cm) groin hematoma, deep vein thrombosis, arterial pseudoaneurysms and arterio-venous fistulae. The second most common was pericardial effusion without a need of intervention, which was present in 23 (2.0%) patients. There were 8 cases of cardiac tamponade that required drainage (0.7%), 6 cases of pericarditis without sequelae (0.5%) and 13 cases of persistent phrenic nerve palsy (PNP) (1.1%). All PNPs recovered during the follow up and tamponades were treated with pericardial puncture, without a need for surgical intervention. There were 2 cases (0.2%) of gastroparesis that were spontaneously resolved. One case of air embolism was noted (0.1%), one case of mild allergic reaction to protamine (0.1%), and one case of periprocedural stroke (0.1%) related to a VT ablation occurred. The detailed procedural characteristics and complications rates are provided in Table New ICE catheter cost in our institution was 2208.84 Euros. If we had always used new catheters for 1128 procedures that would have accounted for 2,491,571.52 Euros in total. On the other hand, catheter resterilization cost was 103.50 Euros per catheter. We resterilized each ICE probe for almost 19 times which adds up to 110,848.50 Euros. Therefore, cumulative cost (new catheters + resterilization cost) of ICE imaging in our institution in the aforementioned period was 236,752.38 Euros. Hence, ICE probe reprocessing resulted with the total savings of 2,254,819.14 Euros in nearly 8 years. Furthermore, one whole ICE catheter package weights 596 g. In total, by reprocessing, 639.5 kg less waste was produced. The ICE catheter itself weights 130 g and it mainly consist of synthetic, nondegradable materials. Therefore, in that time frame, 139.2 kg less, complex, non-degradable medical waste was produced.The main findings of this study are i) one ICE catheter could be used for up to 20 different EP procedures, ii) ICE catheter reprocessing does not result in increased risk of infections or possible other resterilization-related complications, iii) significant economic and environmental savings could be achieved by ICE catheter reprocessing.There are two different advantages of EP/ICE catheters reprocessing that have to be taken into account. Firstly, catheter reprocessing could allow higher number of safer and more efficient ablation procedures for the growing number of patients. Secondly, material reprocessing might significantly limit the negative environmental impact of healthcare industry . WorldwiCardiac electrophysiology is a medical field with a heavy hi-tech burden, characterized by single use tools that consist of plastics, common and rare metals, microchips and circuit boards that are discarded after only few hours of use (or even less). This practice is high environmentally unfriendly and unsustainable, especially when one takes in to the account that more than 1,100,000 EP procedures are performed yearly worldwide and the numbers are expected to grow . Thus, cSimilar to other surgical tools, until 1980 EP catheters were customarily re-used in the United States. Later on, the new regulation was passed that required that manufacturers provide evidence of reusability or classify the device as disposable . New legReprocessed EP catheter functionality is another issue. There is a number of older studies that showed reprocessed EP catheters are safe and functional if routine visual inspection, electrical continuity testing, and confirmation of manual deflection ability are employed –17. A moReprocessing of ICE probes could lead to a higher percentage of transeptal punctures performed with the help of ICE imaging, consequently increasing the safety of the procedures, especially when considering cardiac tamponade. In a large German AF ablation registry that included more than 20000 patients, the overall rate of tamponade that required intervention was 0.9%. The proportion of patients with tamponade went up to 2% in low volume centers that used RF energy. The exact proportion of ICE guided TSP was not reported in that study . CatheteFinancially, ICE probe reprocessing resulted with more than 90% of cost reduction for intracardiac imaging. Similar economic effects were already reported for EP and other cardiac single use devices , 22. By This was a retrospective, single center study with all inherent limitations of this type of research. The number of studied procedures with resterilized ICE catheters was significant but we might miss some very rare complications that might occur in < 1:1000–10000 procedures. Although we advised all patients to come to our ER in the case of any possible complication of ablation procedures, some complications might be missed. Because of the retrospective nature of the study, we could have also missed some procedure-related infections. Furthermore, we do not have the exact count of resterilization processes per one particular catheter, rather, we have calculated the mean resterilization number for all catheters in total. Still there is a significant number of countries in EU that does not allow resterilization. Furthermore, even the counties that allow resterilization have different reprocessing policies. This significantly limits the generalizability of our study.Our data suggests that ICE catheter resterilization and reuse is feasible and safe. Good quality of ICE probe manufacturing allows a high number of reuse cycles. It seems that it does not increase the risk of infection or allergic reactions. Significant economic savings could be achieved by ICE catheters reprocessing and even more important is the potential to reduce negative environmental impact of cardiac electrophysiology. Moreover, ICE reprocessing could increase the proportions of ICE-guided EP procedures worldwide which might result in a lower number of serious procedure-related complications.


The reverse torque value (RTV) was measured at 0° for each specimen. The second part consisted of three groups: a 0° access channel with a zirconia crown (ASC-0) (n = 5); a 15° access channel with a zirconia crown (ASC-15) (n = 5), and a 25° access channel with a zirconia crown (ASC-25) (n = 5). The manufacturer’s recommended torque was applied to each specimen, and baseline RTV was measured before cyclic loading. Each ASC implant assembly was cyclically loaded at 0 to 40 N with 1 million cycles at 10 Hz. RTV was measured after cyclic loading. Kruskal–Wallis test and Jonckheere–Terpstra test were used for statistical analysis. All specimens were examined under a digital microscope and scanning electron microscope (SEM) to observe the wear of the screw head before and after the whole experiment. A significant difference in the different percentages of straight RTV (sRTV) between the three groups was found (p = 0.027). The angle of ASC to the different percentages of sRTV showed a significant linear trend (p = 0.003). No significant differences were found in RTV difference after cyclic loading among the ASC-0, ASC-15, and ASC-25 groups (p = 0.212). The ASC-25 group had the most serious degree of wear based on a digital microscope and SEM examination. The ASC angle will affect the actual preload acting on a screw: the larger the ASC angle, the smaller the preload. The performance of the angled ASC groups in RTV difference was comparable to that of 0° ASC after cyclic loading.This in vitro experiment aimed to understand the difference in preload acting on an abutment screw under different angles of angulated screw-retained crown and the performance after cyclic loading. In total, thirty implants with angulated screw channel (ASC) abutments were divided into two parts. The first part consisted of three groups: a 0° access channel with a zirconia crown (ASC-0) ( The reconstruction of edentulous areas with implants is a highly predictable approach with high success and survival rates ,2. A cohTraditionally, a cement-retained implant prosthesis is often used in the aesthetic area due to anatomical conditions, better mechanical behavior, and fewer mechanical complication such as screw loosening ,5,6, butThe most common complications of implant-supported prostheses are screw loosening, screw fracture, and implant fracture. Among them, screw loosening is the most common with a rate of 2.1% ; it can In total, 30 implants  with a full zirconia crown . To be closer to the clinical situation and the standardized reproduction of all the zirconia crowns, a wax pattern replicating a maxillary right central incisor was scanned  to a standard tessellation language file.n = 5). The crown of the 15° ASC (sASC-15) group allowed the screwdriver to input a torque value at either 0° or 15° angle (n = 5). The crown of the 25° ASC (sASC-25) group allowed the screwdriver to input a torque value at either 0° or 25° angle (n = 5); the implant was fixed to a specially designed metal jig using angle vice . To understand the difference in RTV changes at different ASC angles after cyclic loading, the samples were fixed in a universal testing machine  A. The mePrior to the experiment, the side of the screw head in each group of ASC screws was marked to obtain images before and after cycling at exactly the same position using a digital microscope (Keyence VHX-900F) and SEM  with a 20 kV accelerating voltage at ×100 magnification.After the whole experiment, the specimens were untightened using a torque device. The ASC screw hex was inspected under a digital microscope at ×100 magnification and SEM at ×100 magnification to observe whether the screw hex was worn or not. Kruskal–Wallis test was used to analyze the change percentage in sRTV at different ASC angles and torque value loss after cyclic loading. Jonckheere–Terpstra test was used to analyze whether there is a linear trend between different groups. The statistical software used was SPSS . p = 0.027) (p = 0.003).The sRTV of all samples was lower than the input torque value, and there was a significant difference among sASC-0, sASC-15, and sASC-25 (= 0.027) . Table 1The mean bRTV values of the ASC-0, ASC-15, and ASC-25 groups were 28.84 ± 0.91, 28.87 ± 1.43, and 27.14 ± 0.80 Ncm, respectively . The RTVp = 0.212; After cyclic loading, the RTVs of all samples were lower than the input torque value, and the RTV differences of the ASC-0, ASC-15, and ASC-25 groups were −2.41 ± 1.68, −2.70 ± 1.44, and −1.38 ± 1.40 Ncm, respectively. However, there was no significant difference between the three groups (No crown mobility, crown fractures, screw fractures, or implant fractures were noted after the whole experiment. Based on digital microscope investigation, the inner wall of the screw head had obvious irregular depressions, and the hexagon was also worn to a flat obtuse angle. This wear condition was found in all used ASC screws, and it became more severe as the ASC angle increased . CompariEven though a higher risk of mechanical issues is reported in the literature, the screw-retained prosthesis is a reliable treatment and may have some biological advantages over the cement-retained prosthesis ,21,22,23There are two main methods to measure preload at present. One is using strain gauges to measure the tension when attaching the abutment screw to the implant fixture. However, it is time-consuming and effort-intensive ,31,32. AIn theory, when a screwdriver is aligned along the long axis of the abutment screw, 100% of the applied force is transferred to the screw . Opler eMany studies pointed out that RTV will decrease significantly after cyclic loading, the possible reason being that loading at an angle will generate a force of screw loosening ,41. HeinNo crown mobility or fracture occurred in all samples, but wear was found on the inner wall and hex of the ASC screwhead after repeated tightened and loosening . Future Within the limitations of the method and based on the results of this in vitro study, the following conclusions were drawn:The ASC angle will affect the actual preload acting on the abutment screw and the larger the ASC angle, the lower the preload.The RTV of the ASC of all groups decreased after cyclic loading, and there was no significant difference between the 0° and angled groups. Thus, the performance of angled ASCs after cyclic loading was comparable to that of 0° ASC.


Mental disorders, characterized by the National Institute of Mental Health as disruptions in neural circuitry, currently account for 13% of the global incidence of such disorders. An increasing number of studies suggest that imbalances between excitatory and inhibitory neurons in neural networks may be a crucial mechanism underlying mental disorders. However, the spatial distribution of inhibitory interneurons in the auditory cortex (ACx) and their relationship with excitatory pyramidal cells (PCs) remain elusive. In this study, we employed a combination of optogenetics, transgenic mice, and patch-clamp recording on brain slices to investigate the microcircuit characteristics of different interneurons  and the spatial pattern of inhibitory inhibition across layers 2/3 to 6 in the ACx. Our findings revealed that PV interneurons provide the strongest and most localized inhibition with no cross-layer innervation or layer specificity. Conversely, SOM and VIP interneurons weakly regulate PC activity over a broader range, exhibiting distinct spatial inhibitory preferences. Specifically, SOM inhibitions are preferentially found in deep infragranular layers, while VIP inhibitions predominantly occur in upper supragranular layers. PV inhibitions are evenly distributed across all layers. These results suggest that the input from inhibitory interneurons to PCs manifests in unique ways, ensuring that both strong and weak inhibitory inputs are evenly dispersed throughout the ACx, thereby maintaining a dynamic excitation–inhibition balance. Our findings contribute to understanding the spatial inhibitory characteristics of PCs and inhibitory interneurons in the ACx at the circuit level, which holds significant clinical implications for identifying and targeting abnormal circuits in auditory system diseases. In the past decade, a global decline in the prevalence of numerous chronic diseases has been observed. However, mental disorders have exhibited a contrasting trend, emerging as the predominant burden on families and societies worldwide. The ambiguous pathological mechanisms underlying many mental illnesses often render treatments ineffective, emphasizing the urgency to investigate the root causes of these conditions. An increasing body of research suggests that the pathogenesis of several mental disorders—including epilepsy, schizophrenia, depression, and autism spectrum disorder—may be closely associated with an imbalance in excitation and inhibition within the brain.This excitation–inhibition imbalance has been implicated in abnormal neural circuitry within the central auditory pathway, which has been linked to various forms of hearing loss. For instance, auditory verbal hallucinations (AVHs) have been connected to hyperactivity in the auditory cortex, as demonstrated by functional imaging studies. This aberrant disinhibition of the auditory cortex culminates in self-generated speech, resembling the experience of AVH episodes . In the These discoveries highlight the significance of maintaining equilibrium between excitation and inhibition for the precise encoding of intricate information. Both overexcitability and diminished inhibition can contribute to atypical cortical network activity, leading to the emergence of brain disorders. In this study, we utilized a multidisciplinary approach, incorporating optogenetics, transgenic mice, and patch-clamp recording on brain slices, to examine the microcircuit characteristics of distinct interneurons  and their inhibitory input to PCs across layers 2/3 to 6 in the ACx. Our study enhances the understanding of the spatial inhibitory characteristics between PCs and inhibitory interneurons in the ACx at the circuit level, offering significant clinical insights for the identification and targeted intervention of aberrant circuits in auditory system diseases. In the neural circuits of the cerebral cortex, although excitatory pyramidal neurons (PC) are the main components, the inhibition mediated by GABAergic neurons plays a more important role in the stability of the neural circuits. In the neocortex, the vast majority of inhibitory neurons are PV -, SOM (somatostatin)- and VIP -expressing neurons, which account for nearly 100% of neocortical GABAergic neurons ,9. PV neThe mammalian sensory cortex is typically divided into six layers, meaning that the type and function of neurons in each layer may be different. In different layers of the primary auditory cortex (A1), PV, SOM and VIP interneurons have completely different distribution characteristics, and their different connections with PC result in different auditory responses and behaviors ,22,23. IGuangzhou, China. Transgenic mouse lines aged 7–11 weeks  used in this study were C57BL/6-Tg(pv-cre)Smoc (PV-Cre), Ssttm2.1(cre)Zjh/J (SOM-Cre), and B6J.Cg-Viptm1(cre)Zjh/AreckJ (VIP-Cre) [B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J  reporter line.All experimental procedures used in this study were approved by the Animal Care and Use Committee of Southern Medical University, VIP-Cre) . To visuv/v), a small incision was made in the skin covering the ACx, and a craniotomy of a small hole (0.5 mm diameter) was drilled . AAV2/9.EF1α. DIO. hChR2 (H134R). EYFP virus  was delivered to the ACx with a beveled glass micropipette of a tip size of 30–40 μm. For each injection, 40 nL of the viral solution was injected at a rate of 20 nL/min. We injected the virus into two locations  and at two depths (300 and 600 μm). The pipette was allowed to rest for 7 min before withdrawal after each injection. Finally, we sutured the scalp and injected 0.1 mg/kg buprenorphine subcutaneously and returned the mice to the cage.Viral injections were performed as previously described . Adult t2PO4, 25 mM NaHCO3, 115 mM sucrose, 10 mM glucose, 7 mM MgCl2, 0.5 mM CaCl2; bubbled with 95% O2 and 5% CO2; pH = 7.4; 285–295 mOsm). A vibrating microtome  was used to cut 350 μm thick slices of the coronal cortex from the infected cerebral hemisphere, including the ACx area. The slice was transferred to the recording chamber at room temperature after 30 min of incubation in warm (35 °C) artificial cerebral spinal fluid .Slice recording was performed four weeks after viral injection. Cortical slices were prepared as our previous study ,29. Afte2, and 8 mM phosphocreatine sodium, adjusted to pH 7.3 with KOH). The pipette and whole-cell capacitances were completely compensated, and the initial series resistance was compensated for 50% at a 100 μs lag. Inhibitory postsynaptic currents (IPSCs) were recorded with the membrane potential voltage clamped at 0 mV. Signals were filtered at 2 kHz and sampled at 10 kHz.Electrophysiological recording was performed as previously described . Recordi2. For each stimulus, we repeated 10 trials and averaged the responses. The pia surface of the brain slice was set as 0 μm, stratifying the ACx based on our previous research , which delivered 470 nm blue light square of a 3 ms pulse duration to activate ChR2. A facula, which is set as a 30 × 30 μm blue square, was randomly illuminated around the recorded PC until the entire ACx area was covered (including the location of the patched cell). There is no gap among the facula. Before each experiment, we calibrated the blue illumination to ensure that the stimulus was at the target position. The light intensity measured at the focal plane (measured at the tip of the fiber) is 2.3 mW/mml., 2016 ).Only data with the virus in the target were analyzed. The response was considered to be evoked only if the response amplitude exceeded the average baseline level by 2 standard deviations (SDs) of the baseline fluctuations and if the probability of consistent responses after repetitive stimulation was greater than 50%.For the analysis of the inhibitory strength and range, we took the patched cell as the center and divided all the stimulus blue squares into different areas according to the number of circles. For example, the first lap consists of all the closest blue squares around the patched cell. The one outside of the first lap is the second one, and so on. Lap 0 means the stimulus square is on the patched cell. The farthest range is the tenth lap . For the statistics of the average amplitude, each IPSC amplitude was normalized from averages of multiple (at least three) IPSCs when the stimulus square was on the recorded cell.p value < 0.05 was considered as statistically significant.Custom-made MATLAB programs  were used to analyze the offline data. OriginPro 8.0 and Excel 2007 were used to calculate the values of relevant parameters. One-way ANOVA (LSD’s tests for multiple comparisons) was used to compare means, and a To study the morphological structure of neurons, we used biocytin in this experiment to label soma, dendrites and axons of neurons by fluorescent staining. During the whole-cell recordings, 0.1% biocytin  was added to the internal solution and stably recorded for at least 30 min. Then, the glass pipette was removed slowly from the recorded neuron to prevent leakage of the biocytin and to maintain the normal morphology of the neuron. Brain slices were immersed in 4% PFA and left at 4 °C overnight. Then, the slices were equilibrated at room temperature for 1 h and transferred to 1× PBS. We added 500 μL 0.3% Triton X-100 to each brain slice and incubated it for 2 h with shaking at room temperature. After rinsing with 1× PBS, each brain slice was immersed in 500 μL streptavidin-Cy3 (dilution ratio of 1:200) and incubated for 4 h in the dark at room temperature. After cover slipping, we used a confocal microscope to observe the morphology of the neurons.To confirm the infection efficiency of the virus, we made the quantification of virus-marker colocalization firstly. We injected an AAV vector encoding Cre-dependent hChR2 fused with EYFP into the ACx of the adult transgenic mouse lines PV-Cre-tdTomato, SOM-Cre-tdTomato and VIP-Cre-tdTomato  see Sec. After f+-based internal solution. PV neurons displayed the greatest number of action potentials (AP) [To re-confirm the labeled inhibitory cell type of transgenic mice and assure a high infection efficiency work of the virus, the basic spiking properties of the three inhibitory neurons were examined. Whole-cell recording in current-clamp mode was performed in fluorescence-labeled cells with a Kals (AP) , high-frals (AP) ,38. The Here, we used the inhibitory strength and range to measure the inhibitory efficiency of different interneurons–PCs. To explore the inhibition in each layer of ACx, a 30 × 30 µm blue square was applied to randomly stimulate different areas of different transgenic ACx slices, and the IPSCs of the patched PCs were recorded until the entire ACx was stimulated ). This result suggests that different inhibitory neurons show diverse inhibitory efficiency to L2/3 PCs. PV neurons have the strongest inhibition, and VIP neurons have the widest inhibitory range.To compare the inhibitory range of interneurons–L2/3 PCs in each layer for the three subtypes of interneurons, we analyzed the probability of IPSCs by calculating the area of the rectangle corresponding to each stimulus lap, respectively. Following the same method, we explored the inhibitory efficiency of the three subtypes of interneurons to PCs in other layers. 2 for PV; 0.27 ± 0.05 mm2 for SOM; 0.26 ± 0.05 mm2 for VIP; mean (±SD)), SOM neurons have the most extensive inhibitory range for L5 ) and L6 PCs ). These results indicate that PV neurons supply the strongest and most local inhibition, while SOM and VIP neurons supply the weaker and wider inhibition, which is the common characteristic across all layers of ACx, but the inhibitory source of the broadest inhibitory range is different in each layer.However, when we analyzed the area with more than 10% IPSCs probability, it is found that SOM and VIP neurons have the most extensive inhibitory range for L4 PCs . We employed whole-cell recordings from pyramidal cells (PCs) in transgenic mice expressing green fluorescent protein in specific inhibitory neuron populations combined with optogenetics. As layer 1 contains few PCs, it was not included in our study.Our findings demonstrate that parvalbumin (PV) neurons, the most potent inhibitory input source for all layers of PCs, provide the strongest inhibition to adjacent PCs among the three interneuron subtypes. The inhibitory range of PV-PCs is localized, inhibiting PCs within the same or adjacent layers without cross-laminar inhibition. These characteristics are consistent with other brain regions and are similar across all layers of the ACx. PV neurons have no distinct spatial inhibitory preferences, ensuring an evenly distributed inhibition in each layer. PV neurons thus exert precise regulation to each layer of PCs evenly, with strong and local inhibition and no layer specificity throughout the ACx. This is crucial for the processing of fine sounds in the ACx, enabling the rapid and precise processing of auditory information and discrimination of new auditory stimuli. The reduced expression or functional abnormalities in PV neurons may lead to deficits in rapid auditory processing, such as autism spectrum disorder (ASD) , generalSimilar to SOM, vasoactive intestinal peptide (VIP) neurons also exhibit weak inhibitory input and a wide range. Unlike the other two interneuron subtypes, VIP neurons are primarily distributed in layer 2/3 of the barrel cortex . VIP neuIt is currently widely believed that the imbalance between excitatory and inhibitory influences  in the brain has been proposed as a potential mechanism. GABA may play an important role in pathophysiology ,7,57,58.Due to the different targeting patterns of various interneuron subtypes on excitatory cells, this may affect the connectivity between different cell types, which is a limitation of current experimental methods. Therefore, here, we emphasize more on the spatial pattern of inhibition. Our study combined transgenic mice with optogenetics to selectively control specific neuron types, analyzed the spatial inhibitory characteristics of VIP neurons to PCs, and proposed that inhibitions of SOM and VIP neurons exhibit complementary spatial inhibitory preferences. This ensures the excitation–inhibition dynamic balance of the entire ACx column, contributing important theoretical information for the study of pathological brain circuits and the discovery of potential therapeutic targets.Accumulating evidence supports the significant involvement of PV, SOM, and VIP circuit abnormalities in diseased brains. Considering the neural circuits of these neurons in both healthy and pathological conditions may help tailor specific interventions to restore dysfunctional host circuitry . Additio


Chemical anomalies in polar ice core records are frequently linked to volcanism; however, without the presence of (crypto)tephra particles, links to specific eruptions remain speculative. Correlating tephras yields estimates of eruption timing and potential source volcano, offers refinement of ice core chronologies, and provides insights into volcanic impacts. Here, we report on sparse rhyolitic glass shards detected in the Roosevelt Island Climate Evolution (RICE) ice core (West Antarctica), attributed to the 1.8 ka Taupō eruption —one of the largest and most energetic Holocene eruptions globally. Six shards of a distinctive geochemical composition, identical within analytical uncertainties to proximal Taupō glass, are accompanied by a single shard indistinguishable from glass of the ~25.5 ka Ōruanui supereruption, also from Taupō volcano. This double fingerprint uniquely identifies the source volcano and helps link the shards to the climactic phase of the Taupō eruption. The englacial Taupō-derived glass shards coincide with a particle spike and conductivity anomaly at 278.84 m core depth, along with trachytic glass from a local Antarctic eruption of Mt. Melbourne. The assessed age of the sampled ice is 230 ± 19 CE (95% confidence), confirming that the published radiocarbon wiggle-match date of 232 ± 10 CE (2 SD) for the Taupō eruption is robust. Volcanic signals related to aerosol deposition and physical traces in ice cores provide the opportunity to date, constrain, and link palaeoarchives, enabling the assessment of environmental and climatic impacts across regional and global scales9. The volcanically-produced sulfur (sulfate) measured in ice cores  is used as a record of regional and global volcanism10, and provides insight into any potential volcanogenic climate forcing, as well as eruption plume extent and dynamics11. However, volcanic sulfur anomalies, irrespective of their apparently close temporal coincidence with documented eruptions, cannot unambiguously pinpoint the source eruptions, particularly in the prehistoric record. Unsupported linkages may have consequences for the precision and accuracy of ice core chronologies and evaluation of volcanic impacts8.Ice cores recovered from Antarctica contain exceptional continuous climate records extending back to ~800 ka12. In ice cores from polar areas in both hemispheres, correlated tephras and (more recently) cryptotephras are used as valid high-precision climate-independent chronological markers, where annual layers can and cannot be resolved16. Englacial tephra records from Antarctic ice cores are dominated by regional volcanic sources, which have produced numerous explosive eruptions, typically of alkalic compositions, over the past 500,000 years17. This dominance in part reflects the challenge of finding and identifying tephra particles derived from non-Antarctic sources, and in part the large uncertainties in the age of prehistoric eruptions and distribution of their products20. While some tephra horizons derived from non-Antarctic eruptions are present and recognisable, small particle sizes (<10 µm) usually limit quantitative geochemical analysis and subsequent correlation to an eruptive source21. Nevertheless, recent progress in analytical capabilities and innovative preparation procedures has supported the isolation and geochemical fingerprinting of eruptive material associated with volcanic signals in polar ice cores23. These developments allow further direct correlations between different intercontinental sedimentary records to be made and provide valuable independent time-stratigraphic markers for ice core chronologies25.To overcome this problem, the geochemical characterisation and fingerprinting of visible (tephra) and microscopic (cryptotephra) layers of volcanic ash is evolving as an invaluable tool for correlating palaeoarchives , that can enable volcanic glass deposits to be attributed to a specific time-controlled eruptive source27 and is one of several potential distal Southern Hemisphere sources for cryptotephra in Antarctic ice core records . In particular, the most recent explosive eruption of Taupō volcano . The six-stage eruption sequence lasted between several days to several weeks, producing five physically distinct fall deposits that were widely dispersed by strong south-westerly to westerly winds over a large portion of the North Island, with additional material dispersed over the southwest Pacific Ocean28. The eruption culminated with a voluminous , extraordinarily energetic pyroclastic flow that devastated an area of c. 20,000 km229.Located in the southwest Pacific Ocean, New Zealand hosts two large rhyolitic caldera volcanoes that have been frequently active through the late Quaternary30. This assignment was seemingly confirmed by a volcanic sulfate spike in the 180s CE in the Greenland Ice Sheet Project Two (GISP2) ice core record31 but has since been disproven. Matching a 14C chronology from trees killed by the eruption at the Pureora buried forest  by wiggle-matching new high-precision radiocarbon dates from trees at the same site to a regional calibration curve data set for the Southern Hemisphere34. This age was disputed, however, by Holdaway et al.36 who proposed that the reported eruption date was inaccurate due to magmatic carbon contamination and instead occurred decades to two centuries later. Validating the age of the Taupō eruption via other chronological methods, such as presented here, is crucial for multiple stratigraphic frameworks and testing the reliability of existing radiocarbon age data around volcanoes.As a major time-stratigraphic marker horizon in New Zealand, and a large enough event to have had potentially global impacts, the age of the Taupō eruption has been the subject of marked interest and debate. Initially, the eruption was postulated to have occurred just prior to 186 CE based on historical accounts of atmospheric phenomena from Roman and Chinese sources6; 236 ± 3 CE7; 236 ± 33 CE15 [all at a 95% confidence interval]), but have not yet been verified by the geochemical fingerprinting of insoluble eruptive material . Here, we investigate volcanic signals from specific depth intervals in three Antarctic ice cores , the model ages of which temporally overlap with the Taupō eruption radiocarbon wiggle-match date of 232 ± 10 CE; and report the identification and geochemical characterisation of Taupō-derived glass shards in the Roosevelt Island Climate Evolution (RICE) ice core.Given the magnitude and stratospheric plume heights of the Taupō eruption, it is puzzling as to why this event has been so challenging to detect and validate in Antarctic ice cores. To date, reported volcanic anomalies in ice cores from Greenland and Antarctica linked to the Taupō eruption have been assigned varied ice core ages  and increased particle counts over core sections that spanned the reported date of the Taupō eruption34, using the WD201437, SPC14-02, and RICE1715 chronologies . Particle spikes were often observed down-core from volcanogenic glaciochemical anomalies, consistent with previous studies38. Note that since sampling, the SP19 chronology39 has replaced SPC14-02, with new intervals in the time period of interest subsequently identified down-core for further investigation.Samples of the West Antarctic Ice Sheet (WAIS) Divide (WDC06A), South Pole SPC14), and RICE ice cores  and are followed by an elevated nss cond signal . All three approaches  thus converge to an age for this RICE ice core interval of 230 CE with age uncertainties between ± 3 years  and ± 19 years (95% confidence) tephra comparisons22. However, there are measurable Na2O variations between SEM–EDS and EMPA-WDS data for the rhyolitic and trachytic glass shards  and within populations measured using the same instrument. The SEM–EDS population variability, notably in Kwt% Fig. , which lwt% Fig. A–C. The 37). There was also a trachyte population of volcanic glass identified in the SPC14 ice core at 176.54 m to 176.64 m depth , which has a geochemical affinity to other englacial cryptotephra derived from Mt. Berlin , which is close in composition to Taupō-derived glass except for significantly lower CaO wt% , not only offering chronological constraints on annual layer counting but also providing insights into volcanogenic forcing of climate9. Identification of volcanic glass in ice cores provides a geochemical fingerprint to determine the source volcano, and is particularly important at depths where annual layer counting becomes challenging, and ice age uncertainties greatly increase42. In turn, well-defined ice core chronologies, particularly in ice <2000 years old, provide a means to verify eruption age if an eruptive source can be uniquely identified57.Detection of glaciochemical and physical traces of volcanic eruptions in polar ice core records helps to construct, constrain, and correlate chronologies41  supports the conclusion of Di Roberto et al.52 that the Mt. Melbourne volcanic complex was highly active in historical times and, in the case of the Taupō eruption, may obscure records of distal cryptotephra.Our analysis of rhyolite glass particles from RICE strongly imply that Taupō is the source volcano, with major element compositions that are indistinguishable from groundmass glass previously analysed from juvenile Taupō pumice clasts from all phases of the eruption34. However, this age estimate was challenged by Holdaway et al.36 who proposed that 14C measurements on material from close to the volcano may be contaminated by magmatic infinite-age carbon introduced into the growing trees from groundwater. Holdaway et al.36 instead asserted that the Taupō eruption occurred at an unknown date, decades to two centuries later, bringing into question the use of the Taupō Tephra as a reliable time-stratigraphic marker. Their proposal has significant implications for both the timing of the Taupō eruption and also for other proximally-dated, unobserved, eruptions such as the Minoan eruption of Thera59 (Santorini) and the 1314 ± 12 CE (2 SD) Kaharoa eruption60 .The current age estimate of the Taupō eruption is 232 ± 10 CE (2 SD), based on wiggle-matching high-precision radiocarbon dates from trees buried and preserved by the climactic pyroclastic flow34 is here validated by geochemical fingerprinting of Taupō-derived volcanic glass in the RICE ice core. In addition, further examination of the record for the RICE ice core, including the section containing the Taupō-derived shards, permits a more precise estimate to be made of the age of the ice at this time. For this section , the RICE17 age model is an independent annual-layer-counted chronology15 that was augmented using the StratiCounter layer-counting algorithm61 to interpret annual signals based on the full suite of continuous-flow analysis (CFA) records  in parallel, as necessitated by changing properties and availability of data with depth. The RICE17 annual-layer marks   and is at a maximum within a region corresponding to late summer/early autumn. This specific time of year is remarkably consistent with the independently proposed timing  for the climactic phase of the Taupō eruption from records in the Pureora buried forest Fig.  on the b29. Injection into the stratosphere and subsequent atmospheric circulation of volcanic aerosols and particles over polar surfaces13 via the Antarctic Polar Vortex suggests that this valuable time-stratigraphic marker should be present in more Antarctic ice cores. Travel distances for eruptive clouds depositing (crypto)tephra have been reported to exceed 7,000 km along dominant wind dispersal directions for moderate10, and large explosive volcanic eruptions64, including the Ōruanui event13. In the case of the Taupō eruption, the predominant dispersal path of ash over the Pacific Ocean to the east of the vent is consistent with the prevailing tropospheric winds and strengthening westerly flow in the stratosphere during early autumn65. Neff and Bertler66 also demonstrated that there are several potential synoptic tropospheric transport pathways for dust particles to travel from New Zealand to Antarctica. We therefore infer that particles from Taupō may have been feasibly transported to, deposited, and will be recorded in other Antarctic ice core records .The deposition of glass particles from the 1.8 ka Taupō eruption in Antarctica ~5000 km away was likely related to the exceptionally powerful eruption plume associated with the climactic emplacement of the Taupo ignimbrite15. The seasonal peaks in biogenic nssS are on the same order of magnitude as the expected sulfate deposition from large volcanic eruptions, inhibiting identification of these events using traditional volcanic ice core tracers. Instead, detection of volcanic horizons in the RICE ice core relies on direct measurements of total acidity and estimated nss cond15. For this study, we selected intervals in the RICE ice core based on particle counts and nss cond15 and found Taupō glass in a section of core that would usually be overlooked for the subtle nature of the eruption signature. While sulfate data are not available for the interval of interest in the RICE ice core, the likely corresponding interval of the WDC06A ice core  contains a broad nssS anomaly, which has previously been attributed to the Taupō eruption by Sigl et al.6 at ~ 231/232 CE and later revised to 236 ± 3 CE (95% confidence)7. The glass particles are either absent from this section of the core, or they are present in bracketing ice not selected for melting, highlighting the challenges of pinpointing this event. The stratospheric sulfur injection calculated for this attributed sulfate signal using a total of 12 ice cores (10 from Antarctica and 2 from Greenland) of 5.8 ± 1.2 Tg of sulfur67 is comparable to that calculated using petrological techniques of ~6.7 Tg of sulfur68. This is a relatively low sulfur emission given the size of the eruption and reflects the lower concentration of sulfur in the rhyolite magma when compared to other smaller volume, but sulfur-rich events68. Although while during the Taupō event, the maximum values of sulfate in each annual cycle from the WDC06A and NEEM S1 ice cores were only moderately enhanced compared to other volcanic signals, the annual minimum values of sulfur exceeded the natural background for up to 7 consecutive years6. This moderate but longer-lasting flux of volcanic sulfur has been suggested to result from the high stratospheric plume and subsequent size-dependent fallout of volcanic aerosols from the stratosphere. These findings highlight that other large volume eruptions of sulfur-poor magmas may be missed in ice core records and that nssS anomalies are not necessarily indicative of eruption size but rather plume height and aerosol transport pathways5.A major challenge with precisely locating the Taupō event is the subtle nature of any associated volcanogenic glaciochemical signals in available ice core records. Of the three ice cores investigated here, only the RICE ice core contains a population of rhyolite glass that can be confidently linked to the Taupō eruption. With its coastal location and low altitude, the site of the RICE ice core receives a significant seasonal influx of sulfur-bearing compounds from biological oceanic sources, with peak values of up to 200 ppb of nssS34, and with previous inferences that the eruption occurred in late summer/early autumn . An accurate age estimate offers a new time period within which possible global impacts  could be sought, particularly in palaeoclimate records from the Northern Hemisphere. Six of the seven rhyolite glass shards analysed match published glass chemistry of the Taupō eruption products, while a seventh shard matches the composition of the ~25.5 ka Ōruanui eruption51, also from Taupō volcano and previously identified in the WDC06A ice core by Dunbar et al.13. The combined presence of these two distinctive compositions thus absolutely pins the horizon in which the shards were found to a Taupō volcano source. In this instance, eruptive particles were not found in association with glaciochemical proxies for volcanism previously attributed to the Taupō eruption in the RICE ice core15, providing an important opportunity to improve the accuracy of the RICE17 chronology. Furthermore, while populations of Taupō-derived cryptotephra were not located in the WDC06A and SPC14 ice core samples examined in this study, promising chronological links may be developed following identification and correlation to the temporally coincident Mt. Melbourne cryptotephra. The relationship between these two geochemically distinct eruptive deposits in the RICE ice core may lead to recognition of eruptive material from the Taupō eruption in other Antarctic ice cores. Both eruptions combined provide a valuable, precisely-dated chronostratigraphic marker for the developing Antarctic tephrochronological framework.In conclusion, we have identified rhyolite glass shards from the 1.8 ka Taupō eruption in Antarctic ice for the first time, providing an eruption date estimate of 230 ± 19 CE (95% confidence). This date is consistent with the radiocarbon wiggle-match date estimate of 232 ± 10 CE (2 SD) from material buried by the eruption deposits69 and Iverson et al.22. Large and small volume samples were prepared differently to maximise particle extraction, summarised in a sample preparation flow chart .Large volume samples (>10 ml) of the WDC06A and SPC14 ice cores were cut at the National Science Foundation Ice Core Facility  and processed at New Mexico Tech (NMT). Small volume filtered meltwater samples (<10 ml) from the outer section of the RICE ice core were obtained from the New Zealand National Ice Core Research Facility . To account for volumetric differences between samples of the WDC06A, SPC14, and RICE ice cores, we modified the sample preparation procedures developed by Dunbar et al.22 found maximises particle recovery, improves the polishing quality of exposed grains, and minimises any material loss associated with over-polishing. After the samples were imaged and analysed, a tape dam was adhered around the disk and filled with EpoThin epoxy . Following curing, the EpoThin round was separated from the disk using a razor blade and, where necessary, prepared for EMPA-WDS . Once cleaned the disk could also be reused.The WDC06A and SPC14 ice core samples were prepared at NMT using the large volume procedures . Each sample was melted at room temperature and filtered through a 0.2 µm nucleopore polycarbonate membrane. A folded piece of polyimide tape was then wiped across the upper surface of the filter to pick up particles deposited during the filtering process, concentrating them along the fold. The tape was then flattened, pressed onto a 25-mm diameter aluminium disk, which had been coated with a thin layer of light adhesive (Mikrostik™), and peeled back leaving some of the particles on both the disk and tape . The disk was then attached, with double-sided carbon tape to a cylindrical aluminium specimen stub for uncoated SEM–EDS characterization. Up to four samples could be adhered to the same disk. The disks were manufactured by drilling and dremeling aluminium hard disk platters (HDPs) removed from used computer hard drives. They provide a conductive, ultra-flat surface that Iverson et al.Particles remaining on the polyimide tape were mounted in epoxy by affixing the tape to the bottom of a polished (with 30 µm and 15 µm grits) and 4-hole epoxy round, which was then backfilled with low viscosity Spur 4-part epoxy and cured in an oven at 80 °C for 4 hr . To ensure that the tape did not wrinkle during curing, a lead weight was placed on top of the mount. By using the same epoxy for the round and the samples, the surface of the backfilled holes was found to remain flatter, making the mounts easier to polish and reducing the potential for material loss by over-polishing. Mounts prepared using either of these procedures were then polished with diamond powder suspended in deionised water . Typically, samples were polished for 2 min with 6 µm and for another 2 min with 1 µm grits. Polishing times vary depending on the sample and it was considered important to check the sample during polishing at 30 sec intervals on a petrographic microscope using both transmitted and reflected light to ensure that the grains had been exposed then to preclude them from being polished away. Once polished, the sample was carbon coated and made ready for quantitative WDS analysis. The parallel sample preparation method allowed for multiple microanalytical methods to be performed on a single sample and provided a backup in case one of the two preparation procedures was unsuccessful.22 for ‘cryptotephra’ samples . Samples were melted at room temperature in 15 ml polycarbonate vials and then centrifuged at 3,000 rpm for 5 min. Liquid from the bottom of the test tube was pipetted onto the surface of an aluminium disk, which was then heated to 75 °C on a hot plate. Three to nine 40 µl aliquots of particle-bearing liquid were evaporated on the hot plate, concentrating the particles on the upper surface of the HDP. This process was observed under a stereoscopic microscope and the outer boundaries of each aliquot were outlined with permanent marker. Once cooled, the disk was attached to a specimen stub with double-sided carbon tape and the sample material concentrated on the upper surface of the disk was ready for SEM–EDS without carbon coating. If further quantitative analysis was required, a tape dam was adhered around the disk, which was then filled with EpoThin epoxy, cured and polished following the same process as described above for large volume samples . The remaining meltwater was then filtered through a 0.2 µm filter, which was archived and can be prepared for future imaging and geochemical analysis following the parallel preparation procedures outlined above for filtered large volume samples .The RICE ice core samples were prepared at the New Zealand National Ice Core Research Facility using the small volume procedures, which were similar to those above and developed by modifying the methods of Iverson et al.N EDS detector, respectively, using an accelerating potential of 15 kV and a probe current of 75 nA. Due to the low number and small size of analysable particles, SEM–EDS analysis of unpolished samples was critical to locate and document the geochemical signature, size, and shape of microscopic volcanic glass shards.Unpolished samples were imaged and characterised by SEM–EDS. Images and geochemical measurements, particularly of small grains (<10 µm in diameter), were obtained at NMT with a JEOL JSM-IT700HR SEM equipped with an Oxford Instruments X-Max22. All analyses were normalized to 100 wt% to allow comparison of all particles. This technique was effective at minimising the unavoidable Na loss associated with analysing small particles and did not adversely affect analytical accuracy; however, the precision for a set of points may decrease22. A set of secondary standards, including rhyolitic glass VG568, were analysed during analytical sessions to monitor accuracy and precision of both unpolished SEM–EDS and polished EMPA-WDS analyses.Quantitative geochemical analyses of polished samples were performed by EMPA-WDS on a 4 WDS spectrometer Cameca SX-100 Electron Probe Microanalyzer at NMT using a 15 kV accelerating potential and 10 nA probe current, with beam diameters of 20, 15 or 10 µm, depending on particle size. In some instances, beam sizes slightly larger than the glass particles were used resulting in low analytical totals due to the overlap on epoxy following the broad beam overlap method of Iverson et al.Supplementary Information 1.Supplementary Information 2.


Under many kinds of stress, eukaryotic cells rapidly decrease the overall translation level of the majority of mRNAs. However, some molecular mechanisms of protein synthesis inhibition like phosphorylation of eukaryotic elongation factor 2 (eEF2), which are known to be functional in animals and yeast, are not implemented in plants. We suggest that there is an alternative mechanism for the inhibition of protein synthesis in plant cells and possibly, in other eukaryotes, which is based on the discrete fragmentation of 18S rRNA molecules within small ribosomal subunits. We identified four stress-induced small RNAs, which are 5’- and 3’-terminal fragments of 18S rRNA. In the present work, we studied the induction of 18S rRNA discrete fragmentation and phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) in germinated wheat embryos in the presence of glyphosate, which imitates the condition of amino acid starvation. Using northern and western blotting, we have shown that stress-induced 18S rRNA fragments started to accumulate in wheat embryos at glyphosate concentrations that did not evoke eIF2α phosphorylation. It was also found that cleavage of 18S rRNA near the 5’-terminus began much earlier than eIF2α phosphorylation, which became noticeable only at higher concentration (500 μM) of glyphosate. This result suggests that discrete fragmentation of 18S rRNA may constitute a regulatory mechanism of mRNA translation in response to stress and may occur in plant cells in parallel with and independently of eIF2α phosphorylation. The identified small 5’- and 3’-terminal fragments of 18S rRNA that accumulate during various stresses may serve as stress resistance markers in the breeding of economically important plant crops. Protein biosynthesis is a very energy-intensive process,so under stress conditions, the translation of most cellularmRNAs is inhibited in order to save energy and resources andto ensure preferential synthesis of stress proteins. Several molecularmechanisms of protein synthesis inhibition have beendescribed in mammalian and yeast cells. One of these mechanismsis the eukaryotic translation elongation factor 2 (eEF2)phosphorylation, which is carried out by a highly specific proteinkinase in response to a sharp decrease in cytosolic ATPconcentration levels. Phosphorylation inactivates mammalianeEF2 by preventing it from binding to the ribosome . However, plants do not exhibit endogenous kinaseactivity for eEF2 either under normal conditions , or under stress .The second mechanism known in animals to reduce the levelof mRNA translation is triggered under conditions of aminoacid starvation and is mediated by eIF4E-binding proteins(4E-BPs), which prevent eIF4E from binding to the m7Gcapstructure of mRNA . However,no clear homologs of these eIF4E-BPs have yet been foundin plants , nor were anyorthologues of the 4E-BPs genes found in plant genomes.Another important mechanism of eukaryotic protein synthesisinhibition is the phosphorylation of the α-subunit ofeukaryotic initiation factor 2 (eIF2α) by specific protein kinases.This process in mammalian and yeast cells leads tothe blocking of GDP → GTP exchange protein eIF2B and toa sharp inhibition of the mRNA translation initiation . However, recycling of the ternary complex inplant cells can occur without the participation of eIF2B , and eIF2α phosphorylation in plant systemsin vitro does not lead to strong inhibition of protein synthesis. In addition, of the four protein kinases that phosphorylate theeIF2α in mammalian cells, only pGCN2-kinase was foundin plants, and the phosphorylation of eIF2α in plants is not auniversal response to all stress types .Thus, the mechanisms of protein biosynthesis suppressiondue to the phosphorylation of translational factors, which arewell described for mammals and yeast, are either used to alimited extent or are not realized at all in plant cells. We suggestthat another mechanism of protein synthesis inhibitioncan function in plants, which is triggered by certain abiotic andbiotic stresses. In our understanding, this mechanism is associatedwith the cleavage at certain sites of the 18S rRNA as partof 40S ribosomal subunits (40S RS). Previously, we describedthe process of 18S rRNA cleavage, leading to 5′-terminal fragmentsformation of 132–134 nt. and of 54–57 nt. , as well as a 3′-terminalfragment of 100 nt. . Our data arequite consistent with the data of full-transcriptome analysis,which showed that breaks in 28S-, 18S-, and 5.8S-rRNA donot occur randomly, but discretely, which leads to the fact thatsome fragments of ribosomal RNA are detected in the cell significantlymore often than other fragments .The process of RNA cleavage is widely used by cells duringthe processing of ribosomal RNA from their precursor duringribosome biogenesis . In addition, in proandeukaryotic organisms, the mechanism of protein biosynthesissuppression is realized due to the cleavage of the 28SrRNA molecule from the large (60S) ribosomal subunit alongthe sarcin-ricin loop with the cleavage of the 3′-terminal EndorRNA-fragment . Toxins of plants , fungi (α-sarcin) and bacteria (Shiga toxin) actthis way . Possibly similar endonucleasesand/or glycosylases  are activatedin plant cells during stress, but targeting 18S rRNA in 40S RSinstead of 28S rRNA in 60S RS, and thus leading to temporaryor permanent suppression of mRNA translation.In this work, we have shown that in the case of glyphosatemediatedamino acid starvation, when the only specific eIF2αkinase of plants (pGCN2-kinase) is activated, in addition toplant eIF2α phosphorylation, another protective mechanismis triggered in plant cells, namely, discrete fragmentation of18S rRNA. It was shown that the accumulation in plants of18S rRNA 5′-terminal fragments of 75 nucleotides (75nt-5′18S) and 134 nucleotides (134nt-5′18S) begins earlier thanthe activation of pGCN2 kinase and becomes noticeable atrelatively low concentrations of glyphosate when plant eIF2αphosphorylation does not occur at all.Plant material and treatment. Wheat (Triticum aestivum L.cv. Kazakhstanskaya 10) seeds were sterilized in 70 % (v/v)ethanol for 2 min, then in 2 % (w/v) NaOCl for 20 min, andwashed thoroughly with sterile water. Seeds were germinatedat 26 ºС on sterile filter paper soaked in water. After 18 hours,viable embryos were isolated by spatula from swollen seedsand placed in 1 % glucose solution containing 50 U/ml penicillin,50 μg/ml chloramphenicol, and 50 μg/ml nystatin. Afterthis, embryos were divided into equal portions (1 g), whichwere subjected to treatment with glyphosate (simulation ofamino acid starvation) or without any additives (control).Synthesis of probes. DIG-labeling of de novo synthetizedoligodeoxyribonucleotides 5′18S (5′-ACAAGCATATGACTACTGGCAGGATCAACCAGGTA) and 3′18S (5′-CAATGATCCTTCCGCAGGTTCACCTACGGAAACCT)was carried out using DIG Oligonucleotide 3′-End LabelingKit (Roche) according to the manufacturer’s manual. Probes(5′18S-DIG and 3′18S-DIG) were used for northern blottingNorthern blotting. Total RNA was extracted from planttissues with Tri-reagent (Sigma Aldridge) and analyzed on10 % PAGE with 8 M urea in Tris-borate buffer . RNAs were blottedto a nylon membrane (Roche) equilibrated in 0.1x TBEusing a semi-dry blotter (Sci-Plas) at 250 mA for 30 min.The membrane was dried and irradiated with UV light for2 min at 10 mJ/cm2 in a crosslinker (UVP). Hybridization ofDIG-labeled probes and subsequent chemiluminescent banddetection was performed with DIG Luminescent DetectionKit for Nucleic Acids (Roche) according to the manufacturer’sprocedure. The hybridization temperature was 55 °C.Anti-Digoxigenin-AP Fab fragment conjugates (Roche) wereused to detect bound DIG-labeled probes. The blots weredeveloped using a commercial alkaline phosphatase substrateCSPD (Roche).SDS-polyacrylamide gel electrophoresis (SDS-PAGE)and immunoblotting. Frozen embryos were ground toa powder in a mortar and then homogenized in Laemmlisample buffer . Proteins were separated by12.5 % SDS-PAGE with 0.1 % SDS. The separated proteinswere transferred to a nitrocellulose membrane (GVS) thatwas afterwards stained with Ponceau S (Sigma-Aldrich). Theantibodies against human phospho-eIF2α (S51) produced inrabbit  were used for theimmune-detection of phosphorylated T. aestivum (Ta) eIF2α(TaeIF2(αP)). Then horseradish peroxidase-conjugated antirabbitsecondary antibodies produced in donkey  were used.The effect of glyphosate concentration on 18S rRNA fragmentationin wheat embryos. Since the mechanisms ofmRNA translation inhibition mediated by 4E-BPs and eEF2Kare not implemented in plants, it is believed that the mainresponse in plants to amino acid starvation is eIF2α phosphorylationwith pGCN2 kinase . To testwhether the process of discrete fragmentation of 18S rRNAis also induced under these conditions, the herbicide glyphosatewas used. Glyphosate targets 5-enolpyruvoylshikimate3-phosphate synthase, which catalyzes the key penultimatereaction in the shikimate pathway .Therefore, it inhibits the synthesis of many aromatic plantmetabolites including the amino acids tryptophan, tyrosine,and phenylalanine and leads to pGCN2 kinase activationand phosphorylation of the plant eIF2α .Germinated wheat embryos were treated with glyphosate atvarious concentrations, after which the content of 18S rRNAsmall fragments and the phosphorylation status of TaeIF2αwere assessed in their cells. The results are present in Figure 1.Phosphorylation of TaeIF2α becomes noticeable only atrelatively high concentrations (0.5 and 5 μM) of glyphosate, at which wheatembryos stopped to grow . Theappearance of 3′-terminal fragments 100nt-3′18S and 70nt-3′18S was observed at the same concentrations of glyphosate. At the same time,5′-terminal fragments of 18S rRNA, 134nt-5′18S and 75nt-5′18S, began to accumulate in noticeable amounts even atvery low concentrations (5 μM) of glyphosate .The results of semi-quantitative optical densitometry analysisfor this experiment are presented in Table 1. Since the 134nt-5′18S fragment can be a precursor of 75nt-5′18S, and the100nt-3′18S fragment can act as a precursor for 70nt- 3′18S,it is reasonable to estimate the sum of these small 18S rRNAfragments.The dynamics of glyphosate influence on 18S rRNA fragmentationin wheat embryos. Then, we assessed how quicklywheat embryos respond to glyphosate treatment by measuringthe time dependence of TaeIF2α phosphorylation and of discretefragmentation of 18S rRNA. For this, a glyphosateconcentrationof 500 μM was chosen, which induced quite effectivephosphorylation of TaeIF2α, as well as a significantincrease in the content of 18S rRNA small fragments: 134nt-5′18S, 75nt-5′18S, 100nt-3′18S and 70nt-3′18S . The results of the experiment are shown in Figure 2.The results of semi-quantitative optical densitometry analysisof the data presented in Figure 2 are shown in Table 2.Data presented in Figure 2 and Table 2 show that TaeIF2αphosphorylation begins 45 min after the start of glyphosatetreatment , and TaeIF2αP becomes quite noticeable after 60 minof such treatment .The 3′-terminal fragmentation of 18S rRNA is observedafter 3 hours after the start of glyphosate treatment: fragments100nt-3′18S and 70nt-3′18S become detectable as faintlyvisiblebands on track 7 of Figure 2, b (right panel). Theamount of these 3′-coterminal fragments is significantly lowerthan after 10 hours of the same treatment with glyphosate.As for fragmentation from the 5′-terminus of 18S rRNA,the amount of both 5′-coterminal fragments, 134nt-5′18Sand 75nt-5′18S, is significantly increased as early as by the15th min after the start of treatment of wheat embryos withglyphosate . Notably, the amountof the fragment 134nt-5′18S is higher than that of 75nt-5′18Sfragment during 30–45 min of incubation with glyphosate.By 60 min of incubation their amounts become almost equaland after that, the amount of 75nt-5′18S fragment becomeshigher (by 90 min) and even obviously prevalent by 180 min. Similar interrelation can be seenin Figure 1, b (right panel) regarding the applied concentrationsof glyphosate.These observations suggest that cleavage at 134th nucleotidemay happen more quickly and this site is more susceptibleat the beginning of stress. The cleavage site at 75th nucleotidebecomes more prevalent with an increase of stress durationand severity. The cleavage sites at the 3′-terminal segmentof 18S rRNA occur only at very high severity and durationof stress. Therefore, there seemingly exist several differentmechanisms for the cleavage at 5′- and 3′-termini of 18SrRNA, which may result in several different consequencesfor the functioning of 40S RS.No phosphorylation of eIF2α was observed in plants underosmotic and oxidative stresses , heatshock and during unfolded protein response in plants . At the same time, during these stresses a significantdecrease in the translation level of most mRNAs is observedwith exception only for those templates that are responsiblefor the synthesis of stress proteins . Most likely, in plants, other mechanismsof protein biosynthesis suppression are realized,than eIF2α phosphorylation . In addition,eIF2α phosphorylation is not the only possible mechanism ofresponse to some types of stress in different eukaryotic cells.For example, when yeast cells are exposed to harsh ultravioletlight, phosphorylation of eIF2α is observed, as well as a suppressionof the overall level of protein synthesis. However,inhibition of mRNA translation upon exposure to UV lightoccurs even in cells containing a mutant form of eIF2α that isnot capable of phosphorylation .We postulate that the process of discrete fragmentationof 18S rRNA observed under glyphosate mediated amino acid starvation may lead to a decrease in the level of mRNAtranslation. This molecular mechanism can be realized inparallel with the known mechanism of translational regulationmediated by eIF2α phosphorylation and independentlyof it.Understanding the molecular mechanisms of plant adaptationto stresses can make it possible to increase the efficiencyof breeding work to obtain genetic lines and varieties ofeconomically important plant species that are characterizedby increased resistance to certain stresses.This paper presents data indicating that in plant cells the imitationof amino acid starvation induces, in addition to eIF2αphosphorylation, another cellular response that involves thecleavage of the 18S rRNA molecule with the formation ofdiscrete 5′- and 3′-terminal fragments. At the same time,3′-terminal fragments of 18S rRNA appear only at lethalconcentrations of glyphosate and after a prolonged period ofstress (3 hours or more). In contrast, 5′-terminal fragments of18S rRNA began to accumulate in wheat embryos at relativelylow glyphosate concentrations, at which wheat embryos couldcontinue development, and already 15 min after the start ofglyphosate treatment. Thus, the process of 18S rRNA fragmentationin wheat embryo 40S RS is triggered even underconditions where eIF2α phosphorylation does not occur. Wesuggest that such cleavage of the 18S rRNA molecule, whichis activated during amino acid starvation, may result in eitherglobal or selective suppression of mRNA translation.The authors declare no conflict of interest.Altschuler M., Mascarenhas J.P. Heat shock proteins and effects of heatshock in plants. Plant Mol. Biol. 1982;1(2):103-115. DOI 10.1007/BF00024974.Baird Th.D., Wek R.C. Eukaryotic initiation factor 2 phosphorylationand translational control in metabolism. Adv. Nutr. 2012;3:307-321.DOI 10.3945/an.112.002113Ballard D.J., Peng H.-Y., Das J.K., Kumar A., Wang L., Ren Y.,Xiong X., Ren X., Yang J.-M., Song J. Insights into the pathologicroles and regulation of eukaryotic elongation factor-2 kinase. Front.Mol. Biosci. 2021;8:839. DOI 10.3389/fmolb.2021.727863.Browning K.S., Bailey-Serres J. Mechanism of cytoplasmic mRNAtranslation. Arabidopsis Book. 2015;13:e0176Chen Z., Sun Y., Yang X., Wu Z., Guo K., Niu X., Wang Q., Ruan J.,Bu W., Gao S. Two featured series of rRNA-derived RNA fragments(rRFs) constitute a novel class of small RNAs. PLoS One. 2017;12:e0176458. DOI 10.1371/journal.pone.0176458Echevarria-Zomeno S., Yanguez E., Fernandez-Bautista N., Castro-Sanz A.B., Ferrando A., Castellano M.M. Regulation of translationinitiation under biotic and abiotic stresses. Int. J. Molec. Sci. 2013;14:4670-4683. DOI 10.3390/ijms14034670.Endo Y. Mechanism of action of ricin and related toxins on the inactivationof eukaryotic ribosomes. Cancer Res. Treat. 1988;37:75-89.DOI 10.1007/978-1-4613-1083-9_5.Gallie D.R., Le H., Caldwell C., Tanguay R., Hoang N.X., BrowningK.S. The phosphorylation state of translation initiation factorsis regulated developmentally and following heat shock in wheat.J. Biol. Chem. 1997;272:1046-1053. DOI 10.1074/jbc.272.2.1046.Gallie D.R., Le H., Caldwell C., Browning K.S. Analysis of translationelongation factors from wheat during development and followingheat shock. Biochem. Biophys. Res. Comm. 1998;245:295-300. DOI10.1006/bbrc.1998.8427.Henras A.K., Plisson-Chastang C., O’Donohue M.F., Chakraborty A.,Gleizes P.E. An overview of pre-ribosomal RNA processing ineukaryotes.Wiley Interdiscip. Rev. RNA. 2015;6(2):225-242. DOI10.1002/wrna.1269.Hernandez G., Altmann M., Lasko P. Origins and evolution of themechanisms regulating translation initiation in eukaryotes. TrendsBiochem. Sci. 2010;35(2):63-73. DOI 10.1016/j.tibs.2009.10.009.Immanuel T.M., Greenwood D.R., MacDiarmid R.M. A critical reviewof translation initiation factor eIF2α kinases in plants – regulatingprotein synthesis during stress. Funct. Plant Biol. 2012;39(9):717-735. DOI 10.1071/FP12116.Kamauchi S., Nakatani H., Nakano C., Urade R. Gene expression inresponse to endoplasmic reticulum stress in Arabidopsis thaliana.FEBS J. 2005;272(13):3461-3476. DOI 10.1111/j.1742-4658.2005.04770.x.Kast A., Klassen R., Meinhardt F. rRNA fragmentation induced bya yeast killer toxin. Mol. Microbiol. 2014;91(3):606-617. DOI10.1111/mmi.12481.Knutsen J.H., Rødland G.E., Bøe C.A., Håland T.W., Sunnerhagen P.,Grallert B., Boye E. Stress-induced inhibition of translation independentlyof eIF2α phosphorylation. J. Cell Sci. 2015;128(23):4420-4427. DOI 10.1242/jcs.176545.Laemmli U.K. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature. 1970;227(5259):680-685.DOI 10.1038/227680a0.Lageix S., Lanet E., Pouch-Pelissier M.N., Espagnol M.C., Robaglia C.,Deragon J.M., Pelissier T. Arabidopsis eIF2α kinase GCN2 is essentialfor growth in stress conditions and is activated by wounding.BMC Plant Biol. 2008;8:134. DOI 10.1186/1471-2229-8-134.Padgette S.R., Kolacz K.H., Delannay X., Re D.B., LaVallee B.J., TiniusC.N., Rhodes W.K., Otero Y.I., Barry G.F., Eichholtz D.A., PeschkeV.M., Nida D.L., Taylor N.B., Kishore G.M. Development, identificationand characterization of a Glyphosate-tolerant soybean line.Crop Sci. 1995;35(5):1451-1461. DOI 10.3389/fpls.2016.01009Ruberti C., Kim S.J., Stefano G., Brandizzi F. Unfolded protein responsein plants: one master, many questions. Curr. Opin. Plant Biol.2015;27:59-66. DOI 10.1016/j.pbi.2015.05.016.Shaikhin S.M., Smailov S.K., Lee A.V., Kozhanov E.V., Iskakov B.K.Interaction of wheat germ translation initiation factor 2 with GDPand GTP. Biochimie. 1992;74(5):447-454. DOI 10.1016/0300-9084(92)90085-s.Smailov S.K., Lee A.V., Iskakov B.K. Study of phosphorylation oftranslation elongation factor 2 (EF-2) from wheat germ. FEBS Lett.1993;321(2-3):219-223. DOI 10.1016/0014-5793(93)80112-8.Zhang Y., Wang Y., Kanyuka K., Parry M.A., Powers S.J., Halford N.G.GCN2-dependent phosphorylation of eukaryotic translation initiationfactor-2alpha in Arabidopsis. J. Exp. Bot. 2008;59(11):3131-3141. DOI 10.1093/jxb/ern169Zhanybekova S.S., Polimbetova N.S., Nakisbekov N.O., Iskakov B.K.Detection of a new small RNA, induced by heat shock, in wheat seedribosomes. Biochemistry (Moscow). 1996;61:862-870.Zhigailov A.V., Alexandrova A.M., Nizkorodova A.S., Stanbekova G.E.,Kryldakov R.V., Karpova O.V., Polimbetova N.S., Halford N.G.,Iskakov B.K. Evidence that Phosphorylation of the α-subunit ofeIF2 does not essentially inhibit mRNA translation in wheat germcell-free system. Front. Plant Sci. 2020;11:936. DOI 10.3389/fpls.2020.00936.Zhigailov A.V., Polimbetova N.S., Borankul R.I., Iskakov B.K. Investigationof discrete fragmentation of 18S rRNA within 40S ribosomalsubparticles of plant cells. Vestnik KazNU. Biological Series.2013;2:81-87. (in Russian)Zhigailov A.V., Polimbetova N.S., Doshchanov Kh.I., Iskakov B.K.Detection in plant cells of a new 75-nucleotide cytoplasmic RNAcorresponding to the 5′-terminal fragment of 18S RNA. VestnikKazNU.Biological and Medical Series. 2014;1:191-194. (in Russian)Yu Ch.-Y., Cho Y., Sharma O., Kanehara K. What’s unique? The unfoldedprotein response in plants. J. Exp. Botany. 2021:erab513.DOI 10.1093/jxb/erab513.


There is growing interest in using corrosion inhibitors and protective treatments to limit the degradation of mild steel, leading to the development of numerous Schiff bases as cutting-edge inhibitors. In this study, the effectiveness of a Schiff base, 3-imino)indolin-2-one (MTIO), to prevent mild steel corrosion in HCl was investigated using weight loss measurements, potentiodynamic polarization measurements, electrochemical impedance spectroscopy techniques, and surface characterization. The experimental results showed that 0.5 mM MTIO exhibited a satisfactory inhibitor efficiency of 96.9% at 303 K. The MTIO molecules physically and chemically adsorbed onto the mild steel surface following the Langmuir model, forming a compact protective film attributed to the presence of a thiazole ring in the MTIO structure. Theoretical calculations were combined with experimental techniques to investigate the anticorrosion performance and mechanism of inhibition. Therefore, ongoing research aims to develop corrosion inhibitors5 for industrial applications, especially in the oil and gas industries9. An efficient inhibitor requires a heterocyclic ring and/or heteroatoms such as nitrogen, oxygen, sulfur, and pi-systems to coordinate with the d-orbital of iron and form coordination bonds12. Organic inhibitors are environmentally safe and exhibit good anti-corrosion characteristics15. The aromatic thiadiazole, containing heteroatoms of sulphur and nitrogen, along with isatin, which contains oxygen and nitrogen, serve as electron donors. Previous research reported that 0.01 M of 2-amino-5-mercapto-1,3,4-thiadiazole achieved 99% inhibition efficiency for mild steel corrosion in 1 M HCl16. Al-Amiery et al. investigated the corrosion protection of a novel Schiff base, 5,5'--)bis(azanylylidene))bis (PBB), containing an imine bond and a phenyl ring, and achieved 95.16% inhibition efficiency for mild steel in 1 M HCl solution17. Comparing the two studies reveals that the chemical structures of the inhibitors used in both studies contain thiadiazole, but the addition of an imine bond and a phenyl ring in PBB resulted in slightly lower inhibition efficiency than that achieved with 2-amino-5-mercapto-1,3,4-thiadiazole. Anticorrosion behavior has been evaluated, but it is still unclear which substituents contribute most to corrosion inhibition. Experimental research is costly and time-consuming, so theoretical approaches, currently supported by sufficient software and technology, have been adopted to overcome such issues. A particle's capability to prevent corrosion is dependent on its charge distribution, which can be precisely determined through theoretical research, as the adsorption site during corrosion inhibition can be predicted through the application of quantum chemical simulations18. Issues regarding analytical outcomes relating to the interactions of natural compounds with metallic surfaces can be answered using quantum chemistry calculations19. Density functional theory (DFT) can be used to provide a complete description of inhibitor behavior concerning its orientation and structure, as well as how the inhibitor adsorbs to the metal surface20. For example, Hadisaputra et al. used DFT to forecast the effectiveness of coumarins and caffeine as metallic anticorrosion compounds21. The degree to which organic corrosion inhibitors interact with metallic surfaces depends on the donor- and electron-withdrawing sites, as well as the position22.Mild steel is commonly used to make structural components13, such as organic compounds with structural moieties including tetrazoles, imidazoles, triazoles, quinolones, pyridines, Schiff bases, quaternary ammonium salts, and Mannich bases30. A particularly significant class of corrosion inhibitors utilized in the highly concentrated acidic environment is Schiff bases made from thiadiazoles and aldehydes32. The production of a protective barrier on a steel surface is significantly influenced by the Schiff base produced from thiadiazole molecules. Although numerous studies of corrosion inhibitors for mild steel have been published, most are performed in strong acid environments, such as hydrochloric acid, sulfuric acid, and phosphoric acid34. By establishing a protective coating, becoming adsorbed, or producing an insoluble compound on the steel surface, the inhibitor blocks the active site to prevent corrosion35, so the inhibitor efficiency is influenced by the inhibitor structure. The adsorption of the inhibitor to the steel surface is determined by the presence of phosphorus, sulfur, oxygen, and nitrogen atoms, as well as pi-electrons (double bonds)36. Simplified correlations between molecular electronic parameters and corrosion inhibition efficiencies are often evaluated. In many studies, a relationship has been found between these electronic parameters and the corrosion inhibition efficiencies of an organic corrosion inhibitor. But some researchers have a different opinion depending on their research results, where 12 various molecular electronic parameters were examined, and no one of the tested parameters showed any significant association with protection performance. The observed correlations between these characteristics and inhibitory performance, established for only a few compounds and widely published in the publications, are thus called into question by their findings37.There is much research on mild steel anticorrosion compounds in corrosive environmentsCorrosion of metallic substrates is a major challenge faced in various industrial processes. Schiff bases have emerged as promising candidates for corrosion inhibitors due to their excellent adsorption and film-forming properties. In particular, the Schiff base 3-imino)indolin-2-one (MTIO) has shown great potential as a corrosion inhibitor for metallic substrates in acidic media. Although several other Schiff bases have been reported in the literature, the corrosion inhibition efficiency of MTIO has not been extensively studied using both experimental and theoretical approaches. Therefore, in this study, we investigated the corrosion inhibition performance of MTIO using weight loss measurements, potentiodynamic polarization (PDP) measurements, electrochemical impedance spectroscopy (EIS) techniques, and surface characterization. Additionally, we used density functional theory (DFT) calculations to gain insights into the molecular mechanism of corrosion inhibition. The results of this study provide new insights into the effectiveness of MTIO as a corrosion inhibitor and contribute to the development of more effective corrosion prevention strategies for metallic substrates in acidic environments. Herein, we report the investigation of the corrosion prevention properties of MTIO, a Schiff base-type anticorrosion agent depicted in Fig. 38, and a series of silicon carbide plates (Grade 320–1200) was used to scrape the surfaces. The specimens were rinsed in double-distilled water followed by acetone and oven-dried.An X-Ray fluorescence (XRF) spectrometer was employed to analyse the chemical composition of the mild steel coupons with the following weight ratios: 0.21 C, 0.05 Mn, 0.09 F, 0.05 S, 0.01 Al, 0.38 Si, and balance Fe. Specimens with a cubic area of The corrosive environment was HCl 1 M, prepared by the dissolution of 37% hydrochloric acid in double-distilled water. Various concentrations of the inhibitor  were prepared by suitable dilution in 1 M HCl.39. The manufactured metallic substrate coupons were weighed and placed in 500 mL beakers with 400 mL of 1 M HCl with the addition of different concentrations of tested inhibitor  at a resolution of 0.8 nm (15 kV/1.6) in STEM mode at the Electron Microscopy Unit of Universiti Kebangsaan Malaysia in Selangor. After exposure to the acidic media with and without the inhibitor, the coupons were washed with distilled water, dried, and analyzed by SEM.20. The B3LYP functional was used to optimise the organic molecule structure in the gaseous state, with the justification set to “6-31G +  + ” . Relations   20. The In order to determine the electronegativity (χ), softness (σ) and hardness (η), Eqs.  to 11)  11) were42:Equation  was applFe) was equal to 7 eV, whereas the hardness of iron ηFe was equal to 0 eV as in Eq.  to evaluate the effect of temperature on the inhibitory performance of MTIO, as illustrated in Fig. 45  and activation entropy (∆S) were calculated for the activation complex formation in the transition state relation using the experimental values of the d in Eq. :15\docum47. The ∆S values vary negatively with and without the inhibitor, suggesting that the association rather than dissociation, which indicates greater ordering, produces the activated complex in the rate-determining phase. These results are consistent with the previous findings from weight loss measurements, where the inhibition efficiency increased with increasing temperature and concentration, indicating that the inhibitor molecules adsorb onto the metallic substrate's surface to form a protective layer. Additionally, the positive ∆H* values suggest that the adsorption of the inhibitor molecules onto the metallic substrate's surface is an endothermic process, indicating that the adsorption occurs via physisorption rather than chemisorption.Figure entclass1pt{minima48.MTIO molecules adsorption onto the metallic surface protect it from corrosion, therefore, understanding the inhibition process of MTIO makes the research of the adsorption isotherm extremely important. The percentage surface covering was determined and three adsorption isotherms, namely Frumkin, Temkin, and Langmuir were evaluated to identify the adsorption mechanism. After analyzing the experimental data, it was found that the Langmuir isotherm was the most appropriate model to fit the adsorption data Fig. . The Lan49. The tested inhibitor had the greatest 50, the The ated Eq. :Table 3T52. The ads value alone makes it impossible to distinguish between chemisorption and physisorption. Furthermore, the physical adsorption of inhibitors on the metal surface occurs before their chemical adsorption54. The values were positive and increased with an increase in temperature. With the increase in concentration the free energy of activation increases and ascribed to the formation of unstable activated complex in the rate determining transition state.Typically, physisorption  occurs between inhibitor molecules and the mild steel surface when the free energy change of adsorption  was computed using Eq. .EIS provides data on the solution/metal interface capacitive and resistive behaviour without and with different concentrations of the investigated inhibitorsing Eq. 59:17\doc60:max.CPE can indicate impedance  over time. The main parameter utilized to evaluate the kinetic efficacy of protective coatings is the Figure 65. In an acid medium, MTIO successfully stopped or retard the corrosion of metallic substrate68. Following are the metallic substrate and cathode reactions in HCl, and relation , boosts the inhibitory performance of the MTIO. If other compound global softness (σ) is greater, MTIO can interact with them in increasingly intricate ways. The dipole moment is a significant additional factor that contributes to enhanced electrostatic relationship between two interrelated components. The stronger the electrostatic contact between MTIO and the various metal ions substrate, the larger the MTIO dipole moment.The MTIO molecules' mixed-type inhibiting properties can therefore be described by a straightforward two different electrical transmission. A passive metal ion-inhibitor coating forms on the surface of metallic substrate specimen as a result of MTIO molecules' binding of Fe-atoms to form a metallic complexes, which prevents further corrosion. The heterocyclic ring may have a substantial impact on the whole adsorption process even if it is not intimately implicated in charge transfer during adsorption. Although the energy gap (∆E) is normally expanding, the EHOMO is significant and the ELUMO is remarkably low. As shown by the electronic donation fraction (∆N), the impact of donating electrons from the MTIO molecules to the Fe is comparatively quite pervasive than that of the retroactive donation from the steel to the inhibitor. According to Elnga and colleagues., whenever ∆N is positive, electrons donating to the ironIt is regular procedure to quantify atomic charges within compound and find inhibitor active site using Mulliken charges. Additionally, a heteroatom's potential for donation adsorption onto a metal substrate increases with the negative charge of the atoms. Because of their significant atomic charges (S17) = −0.862, N(1) = −0.123, N(14) = −0.377, O(10) = −0.467), the nitrogen, and oxygen, atoms are thought to be responsible for adsorbing metal. Mulliken charges of the MTIO are displayed in Table  = −0.86273 also confirmed the assumptions of the Langmuir adsorption model for how the MTIO particles adsorb to the metallic substrate. Chemical adsorption onto the metallic substrate is facilitated by electron pairs of heteroatoms in the MTIO. Figure The adsorption of inhibitor molecules onto the metallic surface is influenced by several factors, including their chemical structure, atomic charges, behavior in the acid medium, and interaction with the surface properties of the metallic substrate. EIS, PDP and weight loss techniques revealed that the MTIO particles considerably retard/inhibit the metallic substrate corrosion. The adsorption isotherm explorations75.This work aimed to investigate the inhibitory effect of a specific compound, MTIO, on the corrosion of mild steel in hydrochloric acid solution. The study explored the impact of temperature and concentration of the inhibitor on the corrosion behavior using various methods such as weight loss, electrochemical impedance spectroscopy, and potentiodynamic polarization techniques. The results indicated that MTIO showed promising inhibition properties, significantly reducing the corrosion rate of mild steel. Therefore, one of the main problems that this work solved is the development of an effective inhibitor for the corrosion of mild steel in hydrochloric acid solution77. Second, it applies various analytical techniques, such as electrochemical impedance spectroscopy, potentiodynamic polarization, and weight loss measurements, to comprehensively evaluate the inhibitive efficacy of MTIO. Third, the study provides insights into the kinetic and thermodynamic aspects of the inhibition process, which can contribute to the development of new and effective corrosion inhibitors. Finally, the results of this study could potentially have practical implications for the selection and application of corrosion inhibitors in various industries, such as oil and gas, construction, and transportation.First, it investigates the inhibitive properties of MTIO on mild steel corrosion in acidic solutions, which is a relevant topic in the field of corrosion science and engineeringThe importance of this work in real industries lies in the development of new, effective, and environmentally friendly inhibitors for the protection of mild steel in acidic media. Mild steel is widely used in various industrial applications, and its corrosion can result in significant economic losses. By developing effective inhibitors, the industries can prevent corrosion and extend the service life of mild steel, resulting in cost savings and improved operational efficiency. Additionally, the use of environmentally friendly inhibitors aligns with the growing demand for sustainable and eco-friendly practices in the industry. Overall, this work can contribute to the development of more efficient and sustainable corrosion protection strategies in the real industries.The use of a new inhibitor, MTIO, to protect mild steel from corrosion in acidic media.The investigation of the inhibitory properties of MTIO using a combination of weight loss, electrochemical, and surface analysis techniques.The determination of the kinetic-thermodynamic parameters of the corrosion process and inhibition mechanism using Tafel polarization curves.dl and rise in Rct in electrochemical impedance spectroscopy (EIS) tests.The observation of MTIO particles adhering to the surface of the specimen through the drop in CThe comparison of the inhibitory properties of MTIO with other known inhibitors, such as benzimidazole, imidazole, and thiourea.The evaluation of the effect of temperature and concentration on the inhibition efficiency of MTIO.The investigation of the surface morphology and composition of the mild steel specimens before and after exposure to corrosive media using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX).The novel contributions and advancements made in the new work are:These contributions represent advancements in the field of corrosion inhibition and provide new insights into the use of MTIO as an effective inhibitor for the protection of mild steel in acidic media.In the corrosive media, MTIO demonstrated excellent metallic substrate anticorrosion efficacy with an inhibitor efficiency of 96.9% at 303 K, as determined by the weight loss method.MTIO particles chemically adsorb and weakly bind to metal substrates, with the inhibiting performance diminishing as the temperature increases. The inhibiting performance increases with rising MTIO concentration and reduces with increasing temperature. At 303 K in a 1 M HCl solution, the greatest inhibitory performance was 96.9%.The The decrease in the double-layer capacitance (Additionally, SEM pictures show that the MTIO prevents the metal surface from corrosive attack.The quantum chemical computations show that MTIO uses oxygen, sulphur, and nitrogen to adsorb onto the tested specimen surface. The outcomes of the simulations analysis were in agreement with the laboratory observations and gave understanding into the electronic structure of MTIO particles and their adsorption behaviour on the metal substrate.MTIO exhibits significant barrier properties for metallic substrate specimens in corrosive media due to the highly efficient and effective adsorption centers present, such as O, N, S, and pi-bonds. These centers impede the active locations on the surface of the specimen. The following are the main conclusions: