S)-5′,8-cyclo-2′-deoxyadenosine (ScdA) present in the oligonucleotide sequence lowered BsmAI activity more than (5′R)-5′,8-cyclo-2′-deoxyadenosine (RcdA). Interestingly, in the case of 5′,8-cyclo-2′-deoxyguanosine (cdG), both 5′S and 5′R diastereomers inhibited BsmAI activity (up to 55% more than cdA). The inhibition was weaker when cdG was present at the recognition site rather than the cleavage site.Restriction endonucleases (REs) are intra-bacterial scissors that are considered tools in the fight against foreign genetic material. SspI and BsmAI, examined in this study, cleave dsDNA at their site of recognition or within a short distance of it. Both enzymes are representatives of type II REs, which have played an extremely important role in research on the genetics of organisms and molecular biology. Therefore, the study of agents affecting their activity has become highly important. Ionizing radiation may damage basic cellular mechanisms by inducing lesions in the genome, with 5′,8-cyclo-2′-deoxypurines (cdPus) as a model example. Since cdPus may become components of clustered DNA lesions (CDLs), which are unfavorable for DNA repair pathways, their impact on other cellular mechanisms is worthy of attention. This study investigated the influence of cdPus on the elements of the bacterial restriction–modification system. In this study, it was shown that cdPus present in DNA affect the activity of REs. SspI was blocked by any cdPu lesion present at the enzyme’s recognition site. When lesions were placed near the recognition sequence, the SspI was inhibited up to 46%. Moreover, (5′ DNA is affected by numerous factors, which may induce up to a million DNA lesions per day, per cell ,2. Prokaeference .R and 5′S diastereomers of cdPus may change the spatial structure of the DNA helix, especially 5′ to the lesion [CdPus are formed as a result of •OH action on the H5′-atom of the sugar moiety of 2′-deoxyadenosine or 2′-deoxyguanosine ,9. 5′R ae lesion . CdPus me lesion . CDLs are lesion ,12. Whene lesion ,9,13,14.Restriction endonucleases (REs) require specific nucleotide sequences to recognize, bind, and cleave DNA ,16. BaseSspI recognizes palindromic sequences of 5′-AATATT-3′ and cleaves both DNA strands between T3 and A4 of the sequence, generating blunt ends . It is wBsmAI, a type of IIS endonuclease, recognizes asymmetric DNA sequences and cleaves the strand downstream . This distance coincides with the distance covered by CDLs, which may indicate a possible impact of CDLs on enzyme activity. BsmAI recognizes and cleaves both strands of the dsDNA N1/N5 downstream from the specific sequence of 5′-GTCTC-3′ using two separate catalytic sites within the protein structure . After DREs can cut DNA molecules in regions with a well-defined sequence . Owing tS)-5′,8-cyclo-2′-deoxyadenosine (ScdA), (5′R)-5′,8-cyclo-2′-deoxyadenosine (RcdA), (5′S)-5′,8-cyclo-2′-deoxyguanosine (ScdG), and (5′R)-5′,8-cyclo-2′-deoxyguanosine (RcdG) on the activity of BsmAI and SspI. This study used dsDNA with cdPus in one or both DNA strands located within or next to the recognition sites of REs.The present study examined the influence of the cdPus (5′R)/(5′S) cdA and cdG were synthesized according to Romieu et al. [The oligonucleotides were synthesized and purified in the Bioorganic Chemistry Department, Polish Academy of Science, Lodz, Poland, using a Geneworld synthesizer  and nucleotide phosphoramidites . The phosphoramidite derivatives of  and the data processing was performed with Waters MassLynx 4.1 software . The mass spectra and obtained and found masses of analyzed oligonucleotides are available in the Oligonucleotides were analyzed in the negative-ion mode on a Waters Synapt G2-Si HDMS quadrupole time-of-flight hybrid mass spectrometer . Samples were dissolved in 10 mM ammonium acetate with 50% acetonitrile to obtain a final concentration of 0.1 OD/mL. Samples were injected into the source of the mass spectrometer by a syringe pump with a flow rate of 10 μL/min. Other parameters of the analysis were as follows: capillary voltage 2.6 kV, cone voltage 40 V, source temperature 120 °C, desolvation temperature 400 °C, cone gas 30 L/h, and desolvation gas 600 L/h. The data were collected in full-scan negative-ion mode  in 20 μL of buffer  for 30 min at 37 °C. The protein denaturation was obtained by heating the samples to 95 °C for 5 min. Radiograms showing the efficiency of oligo labeling are available in the The 40-mer ss oligonucleotides (230 pmol) were 5′-end-labeled using 5 U of polynucleotide kinase T4  with 2 μCi (0.2 μL) [γ-2O. After 10 min at 90 °C, the samples were cooled to room temperature for 3–4 h. Obtained duplexes were precipitated with ice-cold ethanol (250 µL) for 30 min and centrifuged . After ethanol removal, samples were dried under reduced pressure at room temperature. The hybridization efficiency and the purity of dsDNA were examined on 15% denaturing polyacrylamide gel. Radiograms showing the efficiency of oligo labeling and hybridization are available in the The radiolabeled oligonucleotides were hybridized with a 1.2-fold excess of the purified, non-radiolabeled complementary strand in pure HEach dsDNA fragment was prepared two times to obtain two different samples with opposing strands radiolabeled. A list of 15 duplexes (A–O) with indicated radiolabeled strands (1–30) is available in BsmAI and SspI were purchased from NEB . The radiolabeled ds oligos (2.3 pmol) were incubated in 5 µL of reaction buffer  with 0.5 U BsmAI at 55 °C or 1.5 U SspI at 37 °C for each reaction time . The cleavage of each strand was observed. To ensure no incisions of ssDNA were made by REs, control cleavage assays for both enzymes were also performed. All 30 radiolabeled ds oligonucleotides  were treThe reactions were stopped by placing the samples in an ice/water bath and adding 7 µL of denaturing loading dye . The samples were analyzed for 120 min at 45 W, on 15% denaturing polyacrylamide gel containing 8 M urea in 1x TBE . The results were visualized by autoradiography and quantified using Quantity One 1-D analysis software (Bio-Rad). All experiments were performed three times to ensure repetitive and consistent data. To obtain a percentage value of DNA cleavage, the intensity of each band was calculated as a percentage of the total intensity of all bands within one lane.In the case of each strand and enzyme, values of DNA cleavage for 60 min were taken into account, unless otherwise indicated. If the strand cleavage value is given in relation to the control, it means that the final cleavage values (after 60 min) for both strands are compared. Complete raw numerical data is shown in In the present study, the influence of single- and bi-stranded CDLs on the activity of BsmAI and SspI were examined. The radiolabeled ds oligonucleotides containing cdPus  were treated with both enzymes separately . Lesions in substrate ds oligos were located within or outside restriction and/or cleavage sites. Double-stranded DNA fragments were designed to show the cleavage efficiency of each strand  and cleaved strand, which differed in length because of different incision sites. For SspI, 14-mer and 26-mer products were obtained after incision of the leading and complementary strands, respectively. BsmAI created 23-mer and 13-mer products after incision of the leading and complementary strands, respectively. Control assays for BsmAI showed 99% cleavage (strand 1) and 98% cleavage (strand 2) after 60 min; the cleavage efficiency for SspI was 86% for strand 1 and 82% for strand 2 . The indThe SspI incision site is located within its recognition site. First, the impact of lesions located within the recognition site was considered. The distance in bp between the incision site of the enzyme and the lesion varied and were as follows: +1 toward the 3′-end of the leading strand (strands 3 and 5), −1 toward the 3′-end of the complementary strand (strands 12 and 14), and –2 toward the 5′-end of the leading strand . For all substrates, only one row of bands representing intact dsDNA was observed . TherefoLesions located outside the recognition site were also evaluated. The distance between the incision site of the enzyme and the lesion varied and were as follows: +10 toward the 3′-end of the leading strand  when compared with native strands (50% of cleavage achieved in approximately 10 min for duplex A). Moreover, these results were comparable for 5′ereomers . This inIn the case of lesions adjacent to the cleavage site of the enzyme  may appear due to the greater distance between the lesion (ScdG) and the recognition site of the enzyme. In the case of duplex D containing ScdA, the distance was shorter, and the enzyme remained inactive.The influence of cdG on BsmAI activity was also evaluated . CleavagIn the case of dsDNA containing lesions in both strands, ScdG and RcdG showed a lower impact on the activity of BsmAI when present within the recognition site .The presence of cdG (both diastereomers) next to the incision site of BsmAI lowered its activity more than cdA (both diastereomers).CdG showed a lower impact on the activity of BsmAI when it was located within the recognition site than next to the incision site.Numerous studies have indicated the biological significance of CDLs and cdPus ,31,32,33Restriction endonucleases play an extremely important role in bacterial defense against foreign genetic material. Therefore, agents affecting their activity have become a point of interest. This study showed the potential of cdPus to diminish RE activity. Since it is known that cdPus may affect eukaryotic DNA repair ,13,14, i


Omalizumab, a human immunoglobulin (Ig)G1 antibody against IgE, is a therapeutic agent for bronchial asthma. The Global Initiative for Asthma guidelines indicate that the use of omalizumab should be considered as an option in step 5 of treatment for patients with the most severe type of bronchial asthma. In patients with atopic asthma who are at a high risk of exacerbation, and in whom symptoms are poorly controlled despite treatment with inhaled corticosteroids, omalizumab is one of the few drugs that improves symptoms, reduces the risk of exacerbation, and improves the quality of life while offering a high level of safety. On the other hand, the associated treatment costs are high, and there are no clear methods to identify responders. A recent study suggested that evaluating the therapeutic effects and monitoring the pharmacokinetics of omalizumab could improve the success of omalizumab therapy. This review outlines the relationship between IgE-targeted therapy and the serum level of IgE to enhance the current understanding of the mechanism of omalizumab therapy. It also describes the clinical significance of measuring serum free IgE levels and monitoring omalizumab therapy. A previous study showed that IgE is an important regulator of FcεRI expression in DCs . OmalizuHigh-affinity FcεRI exhibits cell-specific expression primarily on mast cells and basophils, whereas CD23 is expressed in various cells including B cells, DCs, basophils, eosinophils, epithelial cells, and smooth muscle cells. It is thought that in tissues expressing CD23, IgE binds to CD23/FcεRII, thereby forming an IgE tissue pool . CD23 isIn contrast to these findings, a soluble form of CD23 (sCD23) cleaved from membrane CD23 (mCD23) on the cell surface by proteases  colligates both mIgE and mCD21 on the surface of IgE-producing B cells to upregulate IgE synthesis. In clinical trials of inhibitory antibodies against CD23 in humans, it was shown that the anti-human CD23 monoclonal antibody, lumiliximab, inhibits human serum IgE in a dose-dependent manner . Under nα antibodies. From another point of view, this means that monitoring the suppression of IgE production in the use of dupilumab may be useful in predicting the therapeutic effect. In particular, the serum IgE would be an important indicator for evaluating how dupilumab changes the atopic status of patients. In case of patients treating with dupilumab, total serum IgE is the same as free IgE, so total serum IgE is a clinical biomarker for dupilumab. However, total IgE levels may also be affected by IgE bound to the low-affinity IgE receptor CD23 . Therefore, as with free IgE when using omalizumab, it remains questionable whether total IgE sensitively reflects IgE production levels. Further consideration will be needed in the future.As described above, the production of human IgE is basically dependent on IL-4. This can also be confirmed by the fact that IgE production is suppressed by the use of dupliumab, which is an anti-IL-4 receptor α-1,3-core fucose , we concluded that the working ranges for this assay were as follows: lower limit of quantitation (LLOQ) = 9.38 ngml and upper limit of quantitation (ULOQ) = 600 ngml. On this method, we were able to measure serum free IgE level in the patients with omalizumab  that secreted rsFcεRIα . To evallizumab (.in vivo measurements of IgE and FcεRI (in vivo can be accurately measured in patient serum.We were able to obtain near nd FcεRI . MoreoveUsing the method described above, we measured serum free IgE levels in patients who had received at least 1 month of omalizumab therapy at our institution  Baker e. OverallSome Free IgE measurement kits are only available for research use, but there are no commercially available kits that can be used in biomarker grade for clinical settings. A free IgE measurements produced by Genentech, which have been used in many clinical studies to date, are not commercially available and cannot be used clinically, at least in Japan. The allergy center of our hospital in Japan, has accept samples from other facilities for measurements of free IgE using our method. It means that our free IgE test is the only way that is currently clinically used. One of the advantages of our measurement method that it can measure not only free IgE in the patients with omalizumab, but also total IgE in the patients without omalizumab. Since this method does not require different measurement methods for patients using omalizuamb and those not using it, the cost of the test should be reduced, and clinical application might be expected as a cost-effective test.Serum free IgE decreases rapidly following the administration of omalizumab, and this decrease is maintained with continuous therapy. In a clinical study of 240 patients with allergic rhinitis who were administered omalizumab for 12 weeks, although symptoms improved in the groups with serum free IgE levels of <20 ng/ml and >40 ng/ml, symptoms improved more markedly in the <20 ng/ml group. Furthermore, there was no difference observed between the <40 ng/ml, > 100 ng/ml, and <100 ng/ml groups.Based on these results, the clinical target level of a patient’s total serum IgE in omalizumab therapy is ≤30 ng/ml . The omaPrevious reports have discussed whether serum free IgE levels should be monitored during omalizumab therapy. For example, In contrast, in a prospective study utilizing our IgE measurement method and FcεRIα recombinant protein with the human glycosylation structure, the findings indicated the significance of measuring the serum free IgE level . Tajiri In addition to the value of using serum free IgE as a predictor of omalizumab’s therapeutic effects, it has been suggested that quantifying serum free IgE would be beneficial . HamiltoOmalizumab is an effective molecularly targeted drug for patients with severe asthma. However, the associated healthcare costs with omalizumab are high, and there is no means to discern between responders and non-responders prior to usage. Therefore, we believe that future clinical studies that examine treatment applications of omalizumab are required. With respect to omalizumab therapy, we believe that the level of serum free IgE should be measured, and that in patients who receive long-term treatment or exhibit an insufficient response, the dosage should be reassessed by examining whether the level of serum free IgE is within the acceptable therapeutic range.


Atlantic Niño is the Atlantic equivalent of El Niño-Southern Oscillation (ENSO), and it has prominent impacts on regional and global climate. Existing studies suggest that the Atlantic Niño may arise from local atmosphere-ocean interaction and is sometimes triggered by the Atlantic Meridional Mode (AMM), with overall weak ENSO contribution. By analyzing observational datasets and performing numerical model experiments, here we show that the Atlantic Niño can be induced by the Indian Ocean Dipole (IOD). We find that the enhanced rainfall in the western tropical Indian Ocean during positive IOD weakens the easterly trade winds over the tropical Atlantic, causing warm anomalies in the central and eastern equatorial Atlantic basin and therefore triggering the Atlantic Niño. Our finding suggests that the cross-basin impact from the tropical Indian Ocean plays a more important role in affecting interannual climate variability than previously thought. The Atlantic Niño is an important mode of tropical Atlantic variability that influences the climate conditions in surrounding areas. Here, the authors use observational data and model simulations to show that positive phases of the Indian Ocean Dipole can trigger Atlantic Niño events. This climate phenomenon is called El Niño, the positive phase of El Niño-Southern Oscillation (ENSO), in the tropical Pacific2 and is referred to as the Atlantic Niño in the tropical Atlantic3.In the tropical Pacific and Atlantic Oceans, the mean sea surface temperature (SST) is warm in the west and cold in the east, under the influence of prevailing easterly trade wind forcing. The east-west SST gradient, however, is substantially weakened or even reversed in some years, which is accompanied by a significant slackening of the trade winds6. For instance, the Atlantic Niño enhances seasonal rainfall over South America and Africa8; it can also affect the African Easterly Jet9, which can subsequently influence Atlantic hurricanes11. It has been suggested that the Atlantic Niño may strengthen the Pacific Walker circulation and lead to La Niña15, the negative phase of ENSO. Through atmospheric teleconnection, the Atlantic Niño can also weaken the Indian summer monsoon18. Therefore, understanding and predicting the Atlantic Niño are of paramount importance and have large societal benefits.The Atlantic Niño is associated with prominent climate anomalies across the tropical Atlantic and has far-reaching impacts on climate in remote regions20, but some events may peak in late fall and winter22. Its development involves positive feedbacks between atmosphere and ocean24. During the Atlantic Niño, warm SST anomalies (SSTA) appear in the central and eastern tropical Atlantic basin, which lowers the sea level pressure (SLP) in the region. The associated eastward SLP gradient force induces westerly wind anomalies, which cause anomalous surface Ekman convergence, deepen the thermocline, reduce oceanic upwelling cooling and thus further increase the warm SSTA in the eastern basin. In addition, the anomalous westerlies weaken the westward South Equatorial Current and produce eastward heat advection anomalies, enhancing the warm SSTA. Surface heat flux anomalies do not contribute much to the warm SSTA associated with the Atlantic Niño25.Unlike ENSO that peaks in boreal winter, the Atlantic Niño is generally phase-locked to boreal summer26 may help trigger the Atlantic Niño. El Niño can induce wind anomalies over tropical Atlantic28, which excite oceanic waves that warm the eastern tropical Atlantic SST20. This remote ENSO influence however is overall weak29, partly due to the opposing effects of the anomalous equatorial easterlies (which generate upwelling Kelvin wave) and off-equatorial anticyclonic wind anomalies (which generate downwelling Rossby wave)30. Indeed, warm but weak SSTA is only found north of the Atlantic equator during El Niño 33. Whilst most existing studies focus on tropical Indian–Pacific and Pacific–Atlantic interactions, interaction between the tropical Indian and Atlantic Oceans is much less explored. The role played by variability of the tropical Indian Ocean in triggering the Atlantic Niño has not been investigated thus far. In the tropical Indian Ocean, the dominant interannual climate variability mode that can operate independently from ENSO is the Indian Ocean Dipole (IOD)35, which is manifested as an east-west dipole-like SSTA pattern, begins to develop in boreal summer and peaks in boreal fall.It has been recognized that inter-basin interactions among the three tropical ocean basins play a crucial role in shaping climate variability and long-term climate changeHere, we show observational evidence that positive phase of the IOD (pIOD) can cause westerly wind anomalies over the tropical Atlantic Ocean, weakening the easterly trade winds and causing warm SSTA. Consequently, the pIOD can trigger Atlantic Niño through atmospheric teleconnection. We confirm the associated physical mechanisms by performing various model experiments using standalone atmospheric and oceanic models, and fully coupled climate models.34. Similar SSTA regression patterns are obtained when the ENSO influence is removed , an index for the IOD defined as the difference of SSTA between western and eastern tropical Indian Oceanin Figs. . MeanwhiThese observational analyses suggest that the Atlantic warm SSTA peaks in 3–5 months after the pIOD peak Fig. , when th25.The generation of the Atlantic Niño by the pIOD is initiated by the westerly wind anomalies associated with atmospheric Rossby wave’s lower-level cyclones over the tropical Atlantic Ocean. The anomalous equatorial westerlies induce eastward oceanic currents Figs. , d, adve41. In addition, the equatorial westerly wind anomalies may also contribute to the enhanced coastal warm SSTA through the downwelling equatorial Kelvin waves discussed above and the subsequent poleward-propagating coastally-trapped waves along the eastern boundary  experiments forced with the tropical Indian Ocean SSTA associated with the IOD. The AGCM results show that the pIOD indeed induces prominent westerly wind anomalies over the equatorial Atlantic Ocean during its developing and peak phases Fig. , b. Expe1, plays a crucial role in the growth of the Atlantic Niño to its mature stage, we further performed pacemaker experiments using a coupled climate model, in which the Indian Ocean SSTA is nudged to the observed values whereas atmosphere and ocean are fully coupled elsewhere  Atlantic Niño (Niña) events from 1950 to 2019, 10 (10) were preceded by positive (negative) IOD events, and the composited SSTA evolution resembles that of the regression pattern on the DMI Fig. . Similar22 or late-onset Atlantic Niño21, are associated with the pIOD forcing  twentieth-century reanalysis (ERA-20C)58, and sea level data during 1958–2017 from ECMWF Ocean Reanalysis System 4 (ORAS4)59. For comparison, we also analyze satellite-derived daily sea level data during 1993–2019 obtained from Copernicus Marine Environment Monitoring Service (CMEMS), and we remove the first three harmonics of daily climatology to obtain daily anomalies. To analyze the physical causes for the SSTA, we use surface heat flux data from ERA-20C.In order to examine large-scale climate anomalies associated with tropical climate modes, we analyze SST data during 1950–2019 from the Hadley Centre Sea Ice and SST (HadISST)34, defined as differences of SSTA averaged over the western  and eastern  tropical Indian Ocean. For the Atlantic Niño, we use the SSTA averaged over the ATL3 region . In this study, we define positive IOD (pIOD) events as when the peak of 3-month smoothed DMI exceed its standard deviation, and negative IOD (nIOD) events as when the smoothed DMI falls below negative standard deviation. Similarly, Atlantic Niño events are defined as when the peak of the 3-month smoothed ATL3 index exceeds the one standard deviation threshold. Based on these criteria, we have selected 31 Atlantic Niño and 30 Atlantic Niña events during 1950–2019. Furthermore, if an observed Atlantic Niño is preceded by a pIOD within 5 months prior to its peak, the Atlantic Niño is considered associated with pIOD forcing, and similarly for observed Atlantic Niña and nIOD. We have selected 10 such Atlantic Niño events (32%), and 10 Atlantic Niña events associated with nIOD (33%) in observations . For the IOD, we use the Dipole Mode Index (DMI)To remove the influences of anthropogenic global warming, we remove the linear trend in all monthly climate variable anomalies. To exclude the ENSO-related signals  and future projections (2006–2019) from the forty-member ensemble of National Center for Atmospheric Research (NCAR) Community Earth System Model version 1 (CESM1) large ensemble (CESM1-LE)60 from Max Planck Institute (MPI) in Hamburg (HAM). Model horizontal resolution is ~2.8°, with 19 vertical levels. We performed two sets of AGCM experiments, forced by positive and negative IOD SSTA in the tropical Indian Ocean, respectively. Outside the tropical Indian Ocean, SST is set to monthly climatology. The SSTA forcing was obtained by computing the composite of observed IOD events for both the developing and decaying years. The model was integrated for 42 years, and the first 4 years were discarded given that it takes a few years to reach the model equilibrium state. Since the SSTA forcing has a 2-year period, i.e., the developing and decaying years of the IOD, we have 19 ensemble members available for analysis.To further confirm our proposed mechanism that the IOD affects the tropical Atlantic wind anomalies through the atmospheric bridge, we perform atmospheric general circulation model (AGCM) experiments using ECHAM4.662 to examine the impact of wind anomalies induced by the IOD on the tropical Atlantic Ocean. The ocean model was forced by differences of surface wind stress anomalies during June–November between the two sets of AGCM experiments. It has 25 baroclinic modes, and a horizontal resolution of 0.5°. The model experiment started from a state of rest with a realistic background stratification and a flat ocean bottom with a depth of 4000 m. It was spun up for 5 years, and integrated forward for another 5 years, which are analyzed in this study.We also used a linear ocean modelGiven that local air-sea interaction plays a crucial role in the formation of the Atlantic Niño, we further performed pacemaker experiment using the NCAR CESM1, in which we restored the Indian Ocean SSTA to observed values (Supplementary Fig. Supplementary InformationPeer Review File


This study aims to develop a controlled release oil palm empty fruit bunch hemicellulose (EFB-H) inhibitor tablet for mild steel in 1 M HCl. As plant extracts tend to deteriorate at longer immersion time, limiting its industrial applicability, we attempted to lengthen the inhibition time by forming a controlled release inhibitor tablet. Electrochemical methods (potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS)) were employed to investigate the efficiency and mechanism of the inhibition. An optimum dosage and immersion time was determined via Response Surface Methodology (RSM). EFB-H tablet was formulated using D-optimal mixture design, and its anticorrosion action at extended immersion time was compared with EFB-H powder. PDP measurement revealed that EFB-H is a mixed type inhibitor. RSM optimization unveiled that the optimum point for a maximum inhibition efficiency (87.11%) was at 0.33 g of EFB-H and 120 h of immersion time. Tablet T3 with EFB-H to gum Arabic to hydroxypropyl methylcellulose ratio of 66:0:34 portrayed the best tensile strength (0.243 MPa), disintegration time (152 min) and dissolution behavior. EFB-H tablet exhibited a longer-lasting inhibition effect than powder, which was 360 h as compared to 120 h for powder. Overall, EFB-H tablet has been successfully developed, and its enhanced effective inhibition time has been experimentally proven. Corrosion inhibitors are substances that can minimize or eliminate metal corrosion once added to a corrosive solution. It is probably the most straightforward, inexpensive and efficacious strategy employed on a reasonably regular basis in the industrial sector . Traditi2SO4 solution. A study by Guedes et al. [−1 could inhibit approximately 90% of carbon steel corrosion in a neutral water solution. Komalasari et al. [−1 of tea leaves extract and 1.5 gL−1 of coffee leaves extract resulted in the highest inhibition efficiencies of 79% and 52%, respectively. A recent study by Lima and co-authors [−1 of aqueous soybean extract for mild steel in H2SO4 solution. El-Hashemy et al. [Calendula officinalis flower heads extract can inhibit up to 94.88% of mild steel corrosion in HCl medium. According to these reports, the capability of natural inhibitor to mitigate corrosion is undeniable.Plants-based inhibitors, particularly those that are biomass-based, have emerged as potential alternatives to conventional inhibitors. To date, numerous works have attempted to utilize plant products to mitigate the corrosion of steel in various media. Gouamid et al.  reporteds et al.  has demoi et al.  investigy et al.  reportedUpon having a proper inhibitor source, there is still uncertainty whether the inhibitor is suitable for industrial application. This is due to the biodegradability of natural inhibitors, which causes their performances to deteriorate at longer immersion time, especially after 120 h of usage ,10,11. EOil palm empty fruit bunch (OPEFB) is a fibrous material from the empty stalks of fresh fruit bunches. Conventional oil palm milling involves five primary operations: fruit sterilization, fruit loosening/stripping, fruit digestion, oil extraction and oil clarification . The oilTo characterize the functional groups and chemical constituents of EFB-H.To investigate the mechanism and kinetics of EFB-H anticorrosion action using potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS).To optimize the inhibition efficiency of EFB-H using response surface methodology-central composite design (RSM/CCD) at different dosages and immersion times.To formulate EFB-H tablet using D-optimal mixture design, determine the tablet properties and compare its anticorrosion action with EFB-H powder inhibitor at extended immersion times.The present work aims to develop a controlled release OPEFB hemicellulose (EFB-H) inhibitor tablet for mild steel in 1 M HCl. The specific objectives targeted to achieve this goal are as follows:The OPEFB used in this study was collected from Labu Palm Oil Mill located in Selangor, Malaysia. Chemical reagents such as HCl, acetone and ethanol were supplied by R&M Chemicals, Ever Gainful Enterprise Sdn. Bhd. . Mild steel specimen with a chemical composition given in The EFB-H was prepared in accordance to a typical mild acid hydrolysis method with minor adjustments ,19,20. T−1 to 650 cm−1 with a resolution of 4 cm−1.The FTIR analysis was performed using an FTIR spectrometer  equipped with an attenuated total reflectance (ATR) sampling accessory . The crystal material used in the analysis was germanium (Ge). The EFB-H sample was scanned over the range of 4000 cmThe chemical composition of EFB-H waste hydrolysate in terms of its cellulose, hemicellulose, lignin and extractives content was analyzed according to Technical Association of Pulp and Paper Industry (TAPPI) T222, TAPPI T249 and NREL TP-510-42619 standard procedures .2 of exposed surface area to the electrolyte. A platinum mesh was used as the counter electrode, while a standard Ag/AgCl electrode was used as the reference electrode. The cell was filled with 300 mL of corrosive solution, 1 M HCl solution with and without the addition of EFB-H at varying dosages. After the electrochemical cell has been set up, the system was allowed to stabilize and reach a steady open circuit potential for 30 min [−1 [RC is the corrosion rate, k is a unit conversion factor (0.13), d is the density of mild steel, EW is the equivalent weight of iron, PDPIE is the inhibition efficiency derived from PDP method and All electrochemical tests were performed using an Autolab potentiostat  equipped with Nova Software . The experimental set up of an electrochemical cell kit with a three-electrode assembly is depicted in r 30 min ,23. Thisr 30 min . After t min [−1 . Each me min [−1 . After cdlC is the double layer capacitance, maxω is the frequency at which the real impedance is at a maximum (top of semicircle) and ctR is charge transfer resistance. Finally, the inhibition efficiency can be calculated using the following formula:EISIE denotes the inhibition efficiency derived from EIS method andThe EIS measurement was conducted using the same equipment, software and experimental set up as the PDP method. After reaching a steady-state, the EIS was performed within 100 kHz to 100 mHz of frequency and an amplitude of 10 mV . The meaVarying dosages of 0.1, 0.3 and 0.5 g at a fixed immersion time at 120 h,Varying immersion times of 24, 120 and 216 h at a constant dosage.The RSM/CCD optimization was performed by Design Expert software . Prior to designing an experiment, a preliminary study was conducted using one factor at a time (OFAT) method to identify suitable ranges of EFB-H dosage and immersion time. Two sets of OFAT-based experiments were performed:All runs were conducted according to standard weight loss measurement method. Mild steel coupons were weighed to obtain the initial weight. Subsequently, each coupon was immersed in 300 mL of uninhibited and inhibited 1 M HCl solutions for 120 h at 25 °C. After immersion, each mild steel coupon was retrieved from the corrosive solution and rinsed using distilled water. Afterwards, the sample was air-dried to remove moisture and reweighed to acquire the final weight. Experiments were performed in triplicate, and average values were taken. The preliminary result was used to determine an appropriate range of variables with the highest possibility of finding an optimum response level. This step is important to avoid unnecessary experimental runs. The decided range was then specified in the software, and a set of experimental runs was generated . EventuaThe EFB-H tablet was formulated with the aid of D-optimal design, Design Expert software . Two polymers supplied by R&M Chemicals, Ever Gainful Enterprise Sdn. Bhd. , namely gum Arabic (GA) and hydroxypropyl methylcellulose (HPMC), were included in the formulation as binders to induce a controlled release behavior of the resulting tablet. The EFB-H powder weight was remained constant at an optimum level according to RSM optimization (0.33 g), which is 66% on the basis of 0.5 g tablet total weight. In contrast, the ranges of GA and HPMC were varied from 0% to 34%. These conditions were specified in the software, and a set of experimental design was generated. The tablets of varying formulations were prepared as described in the section that follows, and weight loss measurement was conducted at 120 h of immersion time with three replications. The formulation of selected tablets for further properties analysis are summarized in −1. Eventually, the tablet was ejected from the die and stored in a desiccator until further use. The compression process and the actual compressed tablet are illustrated in The EFB-H tablet compression was conducted using a universal testing machine  with a maximum allowable force of 10 kN . FirstlyT is the tensile strength, F is the load at fracture, D is the tablet diameter and T is the tablet thickness.The tensile strength test of the EFB-H tablet was performed using a diametrical compression test using the same instrument used for tableting . The tesThe disintegration time of the tablet was determined using a jar test apparatus according to an established pharmaceutical method with some modifications to suit corrosion inhibition application . 1 M HClE is the erosion, w0 is the weight of tablet before immersion and w1 is the weight of tablet after immersion at each interval.The dissolution profile of EFB-H tablet was analyzed using a jar test apparatus, referring to a typical method for pharmaceutical tablets with some alterations to suit corrosion inhibition application . InitialThe extended weight loss study for EFB-H tablet inhibitor was carried out according to the standard weight loss method described earlier at immersion times of 120, 240, 360, 480, 600 and 720 h.−1 indicates O–H stretching vibration in alcoholic and phenolic hydroxyl groups in cellulose, hemicellulose and lignin [−1 is due to C–H stretching of methyl and methylene groups in all components of lignocellulose, but most significant in cellulose [−1 represents the vibration of hemicellulose’s C=O uronic and acetyl groups and/or hemicellulose’s and lignin’s ester linkage of the carboxyl and p-caumaric acid [−1 corresponds to O–H stretching due to adsorbed water [−1 and 1510 cm−1 peaks is attributed to C=C stretching of benzene ring in lignin structure [−1 are attributed to C–H stretching from cellulose and hemicellulose [−1 are assigned as C–H or CH2 bending vibrations, indicating the presence of β-glycosidic linkages; a bond that joins two different types of monosaccharides. This bond is a typical structure of cellulose and hemicellulose [d lignin ,32. The ellulose ,34. The ric acid . The baned water ,37,38. Ttructure ,40,41. Tellulose . The banellulose ,44,45. Iellulose ,47.The structural compositions of raw OPEFB and EFB-H on weight bases are summarized in Potentiodynamic polarization (PDP) measurement was performed to unravel the kinetics of the electrochemical reaction of mild steel corrosion in the absence and presence of EFB-H. The Tafel polarization plot of mild steel at different dosages of inhibitor is depicted in The corrosion potential is a valuable parameter to predict the corrosion damage in a system as well as to evaluate the effectiveness of a protective film. The corrosion potential of the mild steel specimen immersed in blank 1 M HCl solution relative to Ag/AgCl reference electrode was found to be −463.51 mV, which is within the range of −450 mV to −465 mV reported in previous studies with similar experimental conditions ,53. In tThe Tafel line is helpful to determine the type of inhibitor by observing the anodic and cathodic branches change with the incorporation of an inhibitor. Based on the data, the anodic and cathodic branches have shifted to lower current densities upon the addition of EFB-H. This trend suggests that EFB-H inhibits corrosion by retarding both reactions simultaneously . DespiteWith respect to the corrosion current density, the result revealed that the values reduced appreciably upon the increase of EFB-H dosage. The corrosion current density is related to the electrochemical reaction rate and can be easily determined experimentally by extrapolating the Tafel plot . Lower cctR. The larger diameter of the capacitive loops at higher inhibitor dosage implies higher resistance to corrosion due to more molecules’ adsorption on the mild steel surface [To procure a more detailed information on the corrosion inhibition mechanism of EFB-H on mild steel, EIS experiment was also performed. In the EIS method, impedance is used to relate theoretical circuit elements to the corrosion system . The Nyq surface .To further understand the electrochemical aspect of the corrosion inhibition, the results from EIS measurement were fitted to an equivalent circuit. Ideally, the most straightforward Nyquist plot with only one perfect semicircle can be simulated by Randles equivalent circuit, which assumes homogeneous and flawless metal surface that behaves as a pure capacitor. However, in reality, the metal surface is microscopically rough and heterogeneous even with extensive cleaning and polishing. Accordingly, a constant phase element (CPE) was employed instead of a pure capacitor to accurately resemble the data . The ele2 upon the addition of OPEFB waste hydrolysate at the lowest dosage. With successive increases in the inhibitor dosage, a substantial increment of resistance was observed as anticipated. By utilizing the reduction of charge transfer resistance, the inhibition efficiency of EFB-H can be calculated. The finding is in agreement with the inhibition efficiencies obtained by weight loss and polarization techniques, which demonstrated that inhibition efficiency improved with increasing inhibitor dosages.In terms of the solution resistance, no obvious trend was observed in blank and inhibited solutions. Thus, it can be inferred that EFB-H do not alter the ionic conductivity of the solution. The charge transfer resistance resembles the metal-solution interface’s resistance that prevents the movement of ions from the metal into the aqueous solution. As can be seen in the table, the charge transfer resistance increased by 3 folds from 47.28 to 142.08 ΩcmThe deviation factor ranging from 0 to 1 indicates the deviation of the actual situation from the ideal system. A value closer to 0 implies that the CPE behaves as a resistor, while a value closer to 1 suggests that the CPE acts similar to a capacitor, as proposed in Randles equivalent circuit . Based o−2 was observed in the blank solution, and appreciable capacitance reduction was observed as EFB-H was added into the solution. The decrease in capacitance value is typically caused by the reduction of the dielectric constant (ε) and/or the thickening of the double layer. Organic compounds typically have lower dielectric constant values (ε ≈ 3–4) compared to water (ε ≈ 80), owing to their outstanding electrical insulating properties [The double layer capacitance is a capacitance value associated with the double layer formed within the metal/electrolyte interface. Physically, the double layer capacitance exists in a corrosion system due to the charge distribution of ions and polar molecules such as water in the interface. According to the result, the highest capacitance of 145.36 µF cmoperties ,69. Sincoperties . Furtheroperties ,71. On tAn inhibitor’s inhibition efficiency is immensely dependent on the inhibitor dosage and immersion time . Hence, IE is inhibition efficiency, A is EFB-H dosage and B is immersion time.After determining the appropriate range of variables, RSM/CCD was performed to achieve optimum parameters for maximum inhibition efficiency. A total of 13 runs were generated in the design matrix, as depicted in The final part addresses optimizing the parameters for maximum inhibition efficiency of EFB-H using the generated model. For this purpose, the response (inhibition efficiency) was set to “maximum” to achieve the highest possible value. Based on the specified condition, the maximum efficiency was predicted to be 85.95% at an inhibitor dosage of 0.33 g and an immersion time of 120 h. A validation experiment at the optimum point revealed that the inhibition efficiency was 87.11 ± 1.91%, which proved that the model is accurate and reliable for prediction. Such a conclusion was made on the basis of a small error between the actual and the predicted value, which was only 1.35%.As discussed earlier, the effectiveness of EFB-H reduced after 120 h of immersion time, which is possibly caused by the desorption of inhibitor molecules and the hydrolysis degradation of inhibitor molecules in acid solution. In an attempt to prolong the effective duration of the inhibitor, a well-established concept in the pharmaceutical field, a sustained release tablet is applied herein as a proof of concept for enhancements in corrosion inhibition application. In addition, corrosion inhibitor tablets’ application is industrially advantageous in the sense of ease of handling, eliminating the need for regular dosing and ensuring a safer working environment . In thisIE is the inhibition efficiency, C is the composition of GA and D is the composition of HPMC, respectively. When dealing with formulations of a two-component mixture or more, in this case, GA and HPMC, it is worth to know if the components are working synergistically (better together) or antagonistically . Based on the last coefficient of Equation (8) (−0.02), it seemed that the polymers exhibited antagonistic effect [The expression representing the effect of GA and HPMC composition towards the inhibition efficiency EFB-H tablet for mild steel in 1 M HCl at 25 °C is given as follows:c effect . The response trace plot of various GA and HPMC combinations is depicted in In the previous section, the effect of different tablet formulations on the corrosion inhibition efficiency was investigated, and two best tablet formulations were determined (T2 and T3). Those two tablets, as well as a control tablet (T1) were further investigated in terms of their tensile strength, disintegration time and dissolution profile. The tensile strength is an essential mechanical property to determine the tablets’ integrity. It is greatly affected by the excipients used in the formulation and their particle size distribution . The tenIn terms of the tablets’ disintegration time in 1 M HCl media, T3 showed a longer disintegrating period than T1 and T2. This finding indicates that HPMC better facilitates the slower release of EFB-H to the corrosive solution. HPMC is excellent in slowing down the disintegration due to its high hydrophilicity, enabling the polymer to swell and become gelatinised upon contact with water . The forThe dissolution profile is also an essential property of a corrosion inhibitor tablet as it is crucial to have a sufficient and continuous supply of inhibitor over time to avoid localised corrosion. The dissolution profiles of T1, T2 and T3 are illustrated in In general, based on the tensile strength, disintegration time and dissolution profile, T3 portrayed the best performance, followed by T1 and T2. The addition of polymer in the EFB-H tablet had modified the tablet properties substantially. Despite the promising results, the tensile strength of T3 is still meagre (0.243 MPa) compared to other HPMC bound tablets reported elsewhere (0.16 to 1.75 MPa) ,86. HencTo prove the EFB-H tablet’s effectiveness at an extended time, a prolonged weight loss study (120 to 720 h) for both powder and tablet forms of EFB-H was performed. T3 formulation was selected for this examination due to its excellent properties, as discussed in the previous section. The trends of the inhibition efficiency of T3 and EFB-H powder at extended immersion times are illustrated in As expected, EFB-H powder showed a reduction in inhibition efficiency after 120 h of immersion. Contrarily, the tablet showed nearly persistent performances over 360 h despite having slightly lower efficacies relative to the powder at 120 h of immersion time. The longer inhibitive time of the tablet is explicable to the sustained release of EFB-H active compounds. The gradual release prevents desorption and degradation of the inhibitor molecules, which are the leading causes of efficacy reduction at longer immersion time ,77,87. BOn the basis of this finding, it can be deduced that the sustained release tablet has a longer-lasting inhibition effect than the powder. Therefore, the implementation of a well-acknowledged pharmaceutical technology in corrosion engineering is proven to be successful. While preliminary, this finding leads to a number of possibilities and offers a revolutionary step in the industrialisation of nature-based corrosion inhibitors, which is a huge and unbridged gap between research and commercialisation .In general, this project aimed to develop a controlled release EFB-H inhibitor tablet to inhibit mild steel acid corrosion. This study contemplated the conventional fundamental study of a corrosion inhibitor as well as its engineering application. Based on the findings, it can be summarized that the presence of heteroatoms and reactive functional groups in EFB-H has been validated through FTIR analysis, and 89.51% of hemicellulose was quantified via compositional analysis. In addition, the PDP measurement has shown that EFB-H inhibited mild steel corrosion by a mixed mechanism of anodic and cathodic reactions retardation. A reduction of current density and corrosion rate was observed as the dosage was increased, reflecting that more inhibitor molecules are adsorbed at higher dosages. From EIS analysis, it has been revealed that EFB-H increased the charge transfer resistance from metal to the solution and reduced the double layer capacitance within the metal/electrolyte interface. Furthermore, it was discovered that the corrosion inhibition system could be integrated into a modified Randles circuit with a CPE. RSM/CCD optimization has unveiled that the optimum point for a maximum inhibition efficiency (87.11%) was at 0.33 g of EFB-H dosage and 120 h of immersion time. Regarding tablet development, T3 with EFB-H:GA:HPMC ratio of 66:0:34 portrayed the best tensile strength (0.243 MPa), disintegration time (152 min), dissolution behavior and adequate corrosion inhibition performance (84.30%). In comparison with EFB-H in powder form, the EFB-H tablet exhibited a longer-lasting inhibition effect, which was 360 h compared to 120 h for powder. Overall, these results suggest that the EFB-H tablet has been successfully developed, and its enhanced effective inhibition time has been experimentally proven. The innovative approach of developing a controlled release inhibitor tablet may have unraveled the emerging issues when dealing with natural inhibitors. This pioneering discovery provides a colossal foundation to utilize nature-based inhibitors for commercial applications. The precise inhibition mechanism of EFB-H remains to be elucidated. Hence, a study that assesses the inhibition mechanism through adsorption isotherm study, comprehensive chemical characterization, quantum chemical calculation and molecular dynamic simulation will need to be carried out in the future.


The Met allele of the brain-derived neurotrophic factor (BDNF) gene confers reduced cortical BDNF expression and associated neurobehavioral changes. BDNF signaling influences the survival, development, and synaptic function of cortical networks. Here, we compared gamma-aminobutyric acid (GABA)ergic network activity in the human primary motor cortex (M1) between the Met  and non-Met  genotype groups. Short- and long-interval intracortical inhibition, short-latency afferent inhibition (SAI), and long-latency afferent inhibition were measured using transcranial magnetic stimulation (TMS) as indices of GABAergic activity. Furthermore, the considerable inter-individual variability in inhibitory network activity typically measured by TMS may be affected not only by GABA but also by other pathways, including glutamatergic and cholinergic activities; therefore, we used 3-T magnetic resonance spectroscopy (MRS) to measure the dynamics of glutamate plus glutamine (Glx) and choline concentrations in the left M1, left somatosensory cortex, and right cerebellum. All inhibitory TMS conditions produced significantly smaller motor-evoked potentials than single-pulses. SAI was significantly stronger in the Met group than in the Val/Val group. Only the M1 Glx concentration was significantly lower in the Met group, while the BDNF genotype did not affect choline concentration in any region. Further, a positive correlation was observed between SAI and Glx concentrations only in M1. Our findings provide evidence that the BDNF genotype regulates both the inhibitory and excitatory circuits in human M1. In addition, lower Glx concentration in the M1 of Met carriers may alter specific inhibitory network on M1, thereby influencing the cortical signal processing required for neurobehavioral functions. The gamma-aminobutyric acid (GABA)-mediated inhibitory network of the primary motor cortex M1) is critical for motor coordination. Paired-pulse paradigms delivered non-invasively via transcranial magnetic stimulation (TMS) can evaluate various forms of the local GABAergic activity in the human M1. These stimulus paradigms include short-interval intracortical inhibition (SICI) evoked by inter-stimulus intervals (ISIs) of 1–5 ms, long-interval intracortical inhibition (LICI) evoked by ISIs of 100–150 ms  is criti, and comIf the inhibitory network defined using TMS measurements depends on the BDNF genotype, it may ultimately be associated with differences in neurotransmitter such as GABA within M1 rather than altered excitatory neurotransmission. However, recent studies that used magnetic resonance spectroscopy (MRS), which is a reliable means of directly measuring the concentrations of neurometabolites, reported no relation between GABA concentration in sensorimotor cortex and either SICI or LICI ,16,17,18The influence of the BDNF Met allele on the inhibitory and excitatory circuits in the human M1 has not been investigated with TMS and MRS. Using both TMS and MRS would provide with unique information from different perspectives. In addition, to elucidating BDNF-related changes in inhibitory and excitatory circuits on M1 may help understand how the BDNF genotype influences behavioral changes. In the present study, we investigated three hypotheses: (1) BDNF genotype affects TMS-induced inhibitory networks as evidenced by changes in SICI, LICI, SAI, and/or LAI; (2) BDNF genotype influences cortical excitatory neurometabolite concentrations; (3) there is a relationship between TMS-induced inhibitory networks and M1 excitatory neurometabolites. To test this hypothesis, we also analyzed neurometabolite concentrations in the primary somatosensory cortex (S1) and cerebellum in addition to M1 because motor-evoked potentials (MEPs) reduction induced by somatosensory inputs may affect the ways in which S1 and cerebellum receive these signals. Collectively, these findings may suggest that the BDNF Met genotype disrupts M1 function through the inhibitory and excitatory networks.A total of 58 right-handed healthy participants  without a history of neurological or psychiatric disorders were recruited for this study. Participants participated in the TMS experiment , MRS experiment . Seventeen participants were overlapping in both experiments . A total of 13 participants who participated in the magnetic resonance (MR) experiment did not participate in the TMS experiment. TMS was performed in accordance with the current TMS safety guidelines  by an exAll participants underwent blood sample collection and genomic DNA extraction, and the samples were analyzed based on the SNP database (BDNF-rs6265) of the National Center for Biotechnology Information. Polymerase chain reaction was used for BDNF genotyping as previously described . ParticiThe EMG was recorded using disposable Ag/AgCl electrodes placed over the right first dorsal interosseous muscle in a belly−tendon montage. Recordings were sampled at 4000 Hz using an A/D converter , amplified 100× , and band-pass filtered between 20 and 1000 Hz.TMS pulses were delivered through a figure-of-eight coil (9.5 cm diameter) connected to two Magstim 200 stimulators . The coil was oriented at an angle of 45° to induce a posterior-to-anterior current over the left M1 at the location producing the largest and most consistent MEPs. The individual position and orientation of the coil were digitally registered according to the structural magnetic resonance images of a template using Visor2 TMS Neuronavigation System . TMS intensity was adjusted to evoke an MEP amplitude of approximately 1 mV peak-to-peak in the first dorsal interosseous muscle. The resting motor threshold (RMT) was determined as the minimum stimulus intensity that elicited MEPs > 50 µV in a minimum 5 of the 10 trials.The M1 inhibitory network was evaluated using paired-pulse paradigms and single TMS pulses paired with peripheral electrical stimulation. The test stimulus intensity was set to evoke approximately 1.0 mV MEP amplitudes from single-pulse TMS. For SICI, the conditioning stimulus intensity was set at 80% RMT with an ISI of 2 ms . To asse2, acquisition matrix = 256 × 256 mm2, number of slices = 160, and slice gap = non-gap.Magnetic resonance data were acquired using a 3 T Vantage Galan MRI scanner  with a 32-channel head SPEEDER coil. Anatomical images were acquired using a T1-weighted 3D magnetization-prepared rapid gradient echo sequence with the following parameters: repetition time (TR) = 5.8 ms, echo time (TE) = 2.7 ms, flip angle = 9°, inversion time (TI) = 900 ms, slice thickness = 1.2 mm, field of view = 23 × 23 cm2, acquisition matrix = 64 × 64 mm2, number of slices = 34, and slice gap = 1 mm. Participants were instructed to repeatedly tap their right index finger at a frequency of 1.0 Hz. Resting and finger tapping conditions were alternated for 30 s each in three blocks (1 block = 60 s). Three recording spans at the beginning of data acquisition were removed from the analysis owing to the unstable signal. Maximum activation of the left M1 was determined by analyzing the BOLD signal using the M-power V5.0 MRS application prior to MRS recording. The S1 hand area was identified based on the location of the left M1 hand area and central sulcus. The position of the right cerebellum was set at the top of the right cerebellum corresponding to the upper voxel. Functional (f)MRI was performed to confirm the location of the M1 hand area using the following echo planar imaging sequence: TR = 2000 ms, TE = 25 ms, flip angle = 85°, slice thickness = 3 mm, field of view = 24 × 24 cm3, voxels of interest over the right cerebellum covering 10 × 20 × 20 mm3, and chemical shift selective saturation to suppress the water signal. The exclusion criteria for data were as follows: full width at half maximum (FWHM) > 15 Hz for M1 and S1 voxels and 30 Hz for cerebellum voxel.MRS data were acquired using a point resolved spectroscopy sequence (PRESS) with the following parameter settings: TR = 2000 ms, TE = 32 ms, 128 averages for each region, voxels of interest over left M1 and left S1 covering 15 × 15 × 15 mmFor TMS experiments, participants sat in a comfortable reclining chair with a mounted headrest. A total of 40 single-pulse MEPs and inhibitory TMS conditions were measured in a randomized order with an inter-trial interval of 4–6 s. Forty MEPs per condition were collected over two TMS sessions (1 session = 20 MEPs per condition) separated by approximately 10 min. LabChart8 software (AD Instruments) was used for MEP analysis. Trials were rejected when the root mean square background EMG activity exceeded 30 µV from −100 ms before the TMS trigger to the end of the MEP waveform. The peak-to-peak amplitudes of single-pulse MEPs and paired MEPs in each inhibitory TMS condition were averaged for each participant within session 1, within session 2, and across both sessions. The MEP amplitudes for the inhibitory TMS conditions were normalized to single-pulse MEPs.MRS data were analyzed using the LCModel software (Version 6.3-1 M) , which cStatistical analysis was performed using PASW software version 25 . Data normality was assessed prior to analysis using Kolmogorov-Smirnov tests, with log transformation applied when a violation was identified . For cla1 mV), RMS, and electrical stimulation intensity were compared among BDNF genotypes by one-way ANOVA with post hoc Bonferroni tests. Glx/tCr and Cho/tCr values in each brain region were compared between the Val/Val group and Met group  by independent samples t-test. The age difference between the two groups was analyzed by an independent samples t-test. In addition, Pearson’s correlation analysis was used to compare the Glx/tCr and inhibitory TMS conditions. A significant threshold of p < 0.05 was used.Single-pulse MEP amplitudes and MEP amplitudes recorded under inhibitory conditions were compared by paired t-tests. Individual variability within sessions is expressed by the coefficient of variance and that between sessions by Pearson’s correlation. Inhibitory parameters , age, TMS intensity required to elicit a 1 mV MEP (TS1 mV (mean ± SD) was 61.5% ± 8.1%, RMT was 50.7% ± 7.5%, and electrical stimulation intensity was 10.1 ± 2.2 mA. Some MEP trials (mean ± SD per participant across two sessions) were excluded owing to the presence of movement artifacts in the EMG recordings . No participant dropped out or presented with side effects. For 45 of the 58 participants, TSn = 58) as Val/Val, Val/Met, or Met/Met genotypes . Moreover, independent t-test revealed no significant difference in age between Met  and Val/Val genotype groups participating in the MR experiment (p = 0.643). The genetic analysis classified all participants , session 2 , and across both sessions . The average single-pulse MEPs and inhibitory metrics from TMS are shown in r-values > 0.60, all p-values < 0.001).The coefficient of variance values was high for all inhibitory TMS conditions . Moderate to strong positive correlations were observed for all inhibitory TMS conditions between sessions 1 and 2  = 5.358, p = 0.008, partial η2 = 0.203] and SAI_2 [F = 6.721, p = 0.003, partial η2 = 0.242). Post hoc tests demonstrated that SAI_1 was significantly stronger in the Met/Met group compared to the Val/Val group (p = 0.010), while there is a significant trend that SAI_1 was stronger in the Val/Met group compared to the Val/Val group (p = 0.053). Furthermore, SAI_2 was significantly stronger in the Val/Met and Met/Met groups compared to Val/Val group . However, there was no significant difference between the Val/Met and the Met/Met groups . One-way ANOVA revealed no significant effects of BDNF genotype on single-pulse MEP amplitude  = 1.501, p = 0.685, partial η2 = 0.004) or on the other inhibitory TMS conditions .The effects of BDNF genotype on single-pulse MEP amplitudes and MEP ratio averages are shown in t(28) = 2.456, p = 0.021, d = 0.899), while there were no significant differences in S1 and cerebellum . Regional Cho levels also did not differ significantly between the Val/Val and Met groups .The representative voxel positions and spectra for the left M1 are presented in r = 0.485, p = 0.049; r = 0.457, p = 0.065). However, there were no significant correlations between SAI_1 or SAI_2 and Glx in the others . Since BDNF genotypes only influence the SAI-related inhibitory circuit, correlation analysis was performed between SAI and Glx in each region. Positive correlation was observed between SAI_1 and the M1 Glx level  SAI is stronger in Met allele carriers compared to the Val/Val group; (2) M1 Glx concentration is lower in the Met group than the Val/Val group, while S1 and cerebellar concentrations are not dependent on BDNF genotype; (3) there is a positive correlation between SAI and Glx in M1. Several studies have reported large inter-individual variations in SICI, with coefficient of variance values of 0.532–0.793 . The curA positive modulator lorazepam both influenced SAI [The present study revealed that Met carriers demonstrate stronger SAI than Val/Val carriers. The common Met polymorphism is associated with an approximately 18%–30% reduction in BDNF secretion , which rnced SAI ,38. TherMRS studies have found no relationship between GABA concentration and TMS measurements of GABAergic activity  ,17,18,30One previous human study reported lower Glx concentrations in Met carriers but in the human hippocampus . A mouseThere was no relationship between SAI and Glx concentration in S1 or cerebellum. SAI evoked at ISI = 20 ms is generated in M1 via S1 ,45. StudBDNF acts as a vital trophic protein for neuronal survival , and alsA receptors. However, pharmacological studies have indicated that drugs affecting distinct GABAA receptor subtypes differentially influence SICI and SAI [B receptor [A-mediated process similar to SAI [A receptor subtypes may differ between LAI and SAI. Further, because LAI requires a longer ISI than SAI, LAI may be induced by different cortical regions.The BDNF genotype did not affect SICI, LICI, or LAI, possibly due to the involvement of distinct pathways and inhibitory networks. Similar to SAI, SICI is induced by GABAergic activity mediated by GABA and SAI . Althougreceptor . Recent r to SAI . HoweverWe did not directly measure GABA concentrations, owing to the application of the PRESS MRS sequence, because GABA measurement generally acquired using a MEGA-PRESS sequence . TherefoOur data show that the SAI-related inhibitory network is stronger in Met allele carriers than in Val/Val carriers. On the other hand, M1 Glx concentration is a lower in Met carriers (with no difference in S1 and the cerebellum) than Val/Val carriers. These results suggest that BDNF genotype controls specific inhibitory neurotransmission and excitatory neurometabolite in the M1 only. Additionally, a lower Glx concentration in the M1 predicts stronger SAI. In other words, the Glx concentration in the M1 of Met carriers may antagonize a specific inhibitory network on the M1 through the unique relationship. These findings suggest that Met polymorphism decreases M1 plasticity, and poor motor learning, consistent with previous studies.


Integrated omics analysis  identified caspase-9 as a kidney disease risk gene. Genome-wide association studies (GWAS) have identified hundreds of genetic risk regions for kidney dysfunction [estimated glomerular filtration rate (eGFR)]; however, the causal genes, cell types, and pathways are poorly understood. Integration of GWAS and human kidney expression of quantitative trait analysis using Bayesian colocations, transcriptome-wide association studies, and summary-based Mendelian randomization studies prioritized caspase-9 (CASP9) as a kidney disease risk gene. Human kidney single-cell epigenetic and immunostaining studies indicated kidney tubule cells as a disease-causing cell type. Mice with genetic deletion or pharmacological inhibition of CASP9 showed lower apoptosis while having improved mitophagy, resulting in dampened activation of cytosolic nucleotide sensing pathways (cGAS-STING), reduction of inflammation, and protection from acute kidney disease or renal fibrosis. In summary, here, we prioritized CASP9 as an eGFR GWAS target gene and demonstrated the causal role of CASP9 in kidney disease development via improving mitophagy and lowering inflammation and apoptosis. More than 800 million people in the world suffer from kidney disease. Kidney disease is one of the strongest risk factors for cardiovascular death, which can be as high as 15 to 20% annually, once end-stage renal failure is reached  and the gene expression of nearby genes, termed expression quantitative trait loci (eQTL) studies  models and the unilateral ureteral obstruction (UUO) kidney fibrosis models  GWAS locus and prioritized P = 7.0 × 10−24) , glomeruli (P = 9.3 × 10−8), and whole-kidney samples (P = 9.0 × 10−6)  (fig. S2 and table S1). On the contrary, we did not observe an association between the genetic variant (rs12736181) and expression of DNAJC16 and CLEA2B, genes located next to CASP9 .In this study, we decided to focus on the chromosome 1 eGFR GWAS locus, which showed a strong and reproducible association with kidney function (× 10−24)  (5). The × 10−6) . MultitiCASP9 expression) share causal variants at this locus, we conducted Bayesian colocalization analysis using coloc (CASP9 expression in kidney tubules were shared (PP4 = 0.95 and PP3 = 0.05). CASP9 expression in the kidney were strongly genotype dependent   . TranscrR levels .CASP9 expression, it has been shown that the SNP with the best eQTL effect size or P value is not always the causal SNP but, rather, SNPs located in the regulatory region such as enhancer are likely to be causal  (r2 = 1).While multiple variants showed association with eGFR and CASP9, we deleted open chromatin regions containing these SNPs and examined CASP9 expression –induced kidney fibrosis, the uninephrectomy and streptozotocin (STZ)–induced diabetes (UNx-STZ), renal aging, UUO, podocyte-specific expression of APOL1 risk variant–induced glomerulosclerosis (APOL1) and the cisplatin-induced toxic AKI (Cis) models . Wes models . CASP9 as models . Furthere models .Lotus tetragonolobus lectin) demonstrated CASP9 expression in renal proximal tubules  mice, with about 50% change in Casp9 expression (Casp9 HZ mice at 6 and 24 months of age (table S2).Kidney disease risk allele was associated with a roughly 30% higher pression  (37). AtCasp9 HZ when compared to WT mice after cisplatin injection (Havcr1 or Kim1) expression, a typical renal tubular injury marker, was markedly lower in kidneys of cisplatin-treated Casp9 HZ mice . Although apoptosis per se is a noninflammatory cell death mechanism, expression of cytokines  and Icam1 was also markedly lower in kidneys of cisplatin-injected Casp9 HZ mice compared to cisplatin-injected WT mice  and Icam1 expression were lower in inhibitor-treated mice after cisplatin injury , which interferes with lysosomal function halting autophagosome-lysosomal fusion, enabling the quantification of the rate of autophagy. The LC3-II expression was higher in Casp9 HZ renal tubule cells exposed to cisplatin, and its level further increased following BafA1 treatment , phosphorylated p65 (pp65), and phosphorylated interferon regulatory factor 3 (pIRF3) were lower in cisplatin-treated Casp9 HZ renal tubule cells  were improved in FA-treated CASP9 HZ mice  were preserved in the FA-treated Casp9 HZ mice , renal fibrosis, and profibrotic genes  was lower in the FA-treated Casp9 HZ mice  was lower in UUO kidneys from Casp9 HZ mice (Casp9 HZ and WT mice were compared (fig. S11A). The higher LC3-II levels in UUO kidney of Casp9 HZ mice were consistent with improved autophagy  in UUO kidney of Casp9 HZ mice , downstream inflammation, and fibrosis development.Here, we demonstrate that CASP9 expression levels, where higher CASP9 expression levels were associated with lower eGFR levels. SMR and TWAS analysis indicated that CASP9 expression mediated the effect of the genotype on kidney function. Integration of Bayesian colocalization signal with single-nuclei open chromatin information prioritized likely causal variant and indicated tissue-specific (kidney), and cell type–specific (tubules) regulation of CASP9 expression. CRISPR-Cas9 gene editing confirmed the functional role of the open chromatin region containing likely causal variants.While GWAS identifies risk regions in the genome, orthogonal datasets are needed for the functional interpretation of GWAS risk variants, such as kidney eQTL and single-nuclear epigenetic information. Bayesian colocalization analysis revealed that the GWAS risk variants had a shared effect on both eGFR and kidney tubule Casp9 mRNA expression levels correlated with fibrosis severity. Higher Casp9 expression and activity and more severe fibrosis were observed in the FA and UUO models compared to UNx-STZ and aging mice. The FA and UUO models are widely recognized as kidney fibrosis models  data following the FUSION pipeline  was calculated for each SNP-gene pair and tissue tested. The significance cutoff of m > 0.9 was used to discover high-confidence eQTLs.The eQTL summary results of 44 other human tissues were downloaded from GTEx (Human kidneys were homogenized and single-nuclear suspension for snATAC sequencing (snATACseq) was prepared according to the manufacturer’s protocol (10X Genomics) as previously described (Human embryonic kidney (HEK) 293 cells stable expressing Cas9 was a gift of L. Wang and L. Song (University of Pennsylvania). Guide RNAs were designed by CRISPOR and cloned into pLKO.sgRNA plasmid. After confirming insert sequences, plasmids were transfected into Cas9-expressing HEK293 using Lipofectamine 3000  at 70 to 80% confluent according to the manufacturer’s instruction. Cultured cells were collected 48 hours after transfection, and RNA was extracted using TRIzol as previously described  reached genome-wide significance in eGFRcrea GWAS . RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit , and qRT-PCR was run in the ViiA 7 System (Life Technologies) instrument using SYBR Green Master Mix  and gene-specific primers. For quantitative analysis, samples were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the ΔΔTissue lysates were homogenized in SDS lysis buffer  and transferred onto polyvinylidene difluoride membranes. After blocking in 5% milk in tris-buffered saline and 0.1% Tween 20, membranes were incubated overnight with the following antibodies at 4°C: CASP9 , cleaved CASP3 , RIP3 , BAX , BCL2 , GSDMD , NLRP3 , ACSL4 , LC-3 , cGAS , STING , TBK1 , pTBK1 , IRF3 , pIRF3 , p65 , pp65 , and GAPDH . Membranes were incubated with horseradish peroxidase–conjugated secondary anti-rabbit or anti-mouse antibody  for 1 hour at room temperature. Signal was detected by enhanced chemiluminescence . Western blots were quantified using the Fiji software , according to the manufacturer’s instructions. Electrolytes  and hemoglobin were measured with the iSTAT Portable Clinical Analyzer using i-STAT CHEM8+ cartridge . Kidney tissue was stained with hematoxylin and eosin to investigate renal injury. The degree of tubulointerstitial damage was scored semiquantitatively on a 0 to 5+ scale, according to the percentage of the area affected by hydropic degeneration, hyaline casts, cytoplasmic vacuolization, loss of the brush border, and tubular lumen dilation . Sirius red staining was performed according to the manufacturer’s protocol  to determine the degree of fibrosis. For each analysis, five to eight fields were randomly selected and quantified using the Fiji software  for 8 hours in the presence and absence of BafA1 .Primary kidney tubule cells were isolated from 3- to 4-week-old mice. Kidneys were minced and incubated for 30 min at 37°C with collagenase I . Digested kidney cells were filtered through the 100-, 70-, and 40-μm mesh to isolate single cells. Cell suspensions were cultured in RPMI 1640  supplement with 10% fetal bovine serum , EGF , insulin-transferrin-selenium (ITS) , and 1% penicillin-streptomycin  at 5% COCasp9 HZ mice were seeded on 96-well plates . After reaching 80% confluency, cells were treated with cisplatin (20 μM) for 8 hours. Live cells were monitored according to the manufacturer’s protocol . Fluorescent signal at 400/505 nm (excitation/emission) was detected in a microplate reader .Renal tubule cells from WT and Ct method and shown as fold change. Primers are listed in table S4.Cytosolic, mitochondrial, and nuclear fractions from kidney tubules were extracted as described previously, by lysing cells with digitonin buffer  (EGFP-LC3 (#11546), ptfLC3 (#21074), and Cox8-EGFP-mCherry (#78520) were purchased from Addgene. Plasmids were transfected with Lipofectamine 3000 according to the manufacturer’s protocol . After transfection, tubule cells were incubated for 24 hours. The fluorescent signal was detected by a confocal microscope . The number of puncta was counted in 10 cells, and mean puncta number were plotted.2O2 for 10 min. Sections were incubated in 5% goat serum (blocking buffer) at room temperature for 1 hour. In addition, avidin/biotin blocking was performed using the avidin/biotin blocking kit . Sections were incubated overnight at 4°C with primary antibody for cleaved CASP3 . After washing, sections were incubated with goat anti-rabbit secondary antibody for 1 hour at room temperature. ABC ready-to-use reagent  and ImmPACT DAB Peroxidase (HRP) Substrate  were used for visualization.Kidneys were fixed in 10% neutral formalin and 5-μm-thick sections were prepared. Sections were deparaffinized, followed by antigen retrieval in citrate buffer at 95°C for 10 min. Sections were allowed to cool slowly, washed in distilled water, and incubated in 3% HFor immunofluorescence studies, sections were incubated with CASP9  antibody, washed with PBS, and incubated with donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 594  and fluorescein isothiocyanate-labeled LTL , coverslip-mounted, and examined under a fluorescence microscope (OLYMPUS DP73).Kidney tissue was homogenized in lysis buffer according to the manufacturer’s protocol . Protein concentrations were measured using the Pierce BCA Protein Assay Kit . Fifty micrograms of total protein was incubated at 37°C for 1 hour with reaction buffer  and CASP3 or CASP9 substrate . The enzyme-catalyzed release of AFC was quantified in a fluorimeter (excitation of 400 nm and emission of 505 nm).t test was used for comparisons between two groups. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used to compare multiple groups. P < 0.05 was considered significant.Data are presented as means ± SEM. Unpaired two-tailed Student’s