Pesticides affect different organs and tissues according to their bioavailability, chemical properties and further molecular interactions. In animal models exposed to several classes of pesticides, neurotoxic effects have been described, including the reduction of acetylcholinesterase activity in tissue homogenates. However, in homogenates, the reduction in enzymatic activity may also result from lower enzymatic expression and not only from enzymatic inhibition. Thus, in this work, we aimed to investigate the neurotoxic potential of four distinct pesticides: glyphosate (herbicide), imazalil (fungicide), imidacloprid (neonicotinoid insecticide) and lambda-cyhalothrin (pyrethroid insecticide), by assessing their inhibitory effect on the activity of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase, by using direct in vitro enzymatic inhibition methods. All pesticides dose-dependently inhibited AChE activity, with an inhibition of 11 ± 2% for glyphosate, 48 ± 2% for imidacloprid, 49 ± 3% for imazalil and 50 ± 3% for lambda-cyhalothrin, at 1 mM. Only imazalil inhibited BChE. Imazalil induced dose-dependent inhibition of BChE with identical pattern as that observed for AChE; however, for lower concentrations (up to 500 μM), imazalil showed higher specificity for AChE, and for higher concentrations, the same specificity was found. Imazalil, at 1 mM, inhibited the activity of BChE by 49 ± 1%. None of the pesticides, up to 1 mM, inhibited tyrosinase activity. In conclusion, the herbicide glyphosate shows specificity for AChE but low inhibitory capacity, the insecticides imidacloprid and λ-cyhalothrin present selective AChE inhibition, while the fungicide IMZ is a broad-spectrum cholinesterase inhibitor capable of inhibiting AChE and BChE in an equal manner. Among these pesticides, the insecticides and the fungicide are the ones with higher neurotoxic potential. Channa punctatus (snakehead fish) exposed for 96 h to sub-lethal doses of CLT showed decreased activity of AChE in the brain, gills and muscle [The use of pesticides in agricultural activities is widespread worldwide for the control of undesired species of plants, insects, and fungi. With the exponential growth in pesticide use, the environmental, animal and human exposure to these chemicals increases proportionally ,2,3. Whid muscle .As seen above, although not being the structural target, AChE seems to be a target for many pesticides, and due to the primary role of AChE in neuronal physiology, i.e., in terminating neuronal signaling between synapses and transmission, its homeostasis disturbance leads to several diseases, namely those involving memory and neuromotor function, such as Alzheimer’s disease  and otheIn the case of AChE inhibition in animals, accumulation of acetylcholine affects nervous impulse transmission, with symptoms that include blood vessels vasodilation, decreased heart rate, vomiting, sweating, intestinal cramps, muscle contraction, eye pupil constriction and, ultimately, cardiac failure, paralysis and death. Pesticide-contaminated water has been reported to affect AChE in fish species by reducing their mobility and spatial awareness, reducing both the ability to feed and evade predators ,27.Carbamates and organophosphates are within the group of pesticides with higher cholinesterase inhibition; AChE is considered a molecular target for these compounds. For this reason, cholinesterases have been proposed as biomarkers to assess the environmental risk of these pesticides . VariousAChE and BChE present 65% similarity in their structure . In the Electrophorus electricus) or from Torpedo genus (electric ray) present high similarity to the human isoforms and thus being used in in vitro screening assays [Drosophila melanogaster, which present a higher number of structural differences that affect the electrostatic properties of the enzyme [N-glycosylation site [Among the known AChE isoforms found in various vertebrates, AChE isoforms obtained from electric eel , Imidacloprid , Imazalil , lamba-cyhalothrin , acetylcholinesterase from Electrophorus electricus , butyrylcholinesterase from equine serum , tyrosinase from mushroom , acetylthiocholine iodide , butyrylthiocholine iodide , 5,5′-dithiobis(2-nitrobenzoic acid) , 3,4-dihydroxy-L-phenylalanine  and kojic acid  were purchased from Merck . All other reagents and salts were of analytical grade and were also obtained from Merck .Glyphosate (PESTANALStock solution of GLY (at 40 mM) was made in distilled water, and the stock solutions of IMD, IMZ and LCT  were made in DMSO. All further dilutions were made in ultrapure distilled water. DMSO percentage at the highest tested concentration was 5%, and an enzymatic dose–response inhibition of DMSO was made in all assays. When inhibition occurred, the % of inhibition due to DMSO was subtracted from the respective sample.Pesticide-induced inhibition of cholinesterases was performed for AChE and BChE using Elman’s method . Elman´sn = 3 independent assays were made.The enzyme inhibition assays were performed in 96-well plates as described by Taghouti, et al. . For theFor BChE inhibition, an identical experimental procedure was performed (same buffers and incubation conditions), but using as enzyme substrate BCTI  and using BChE  as enzyme. In both assays, distilled water was used as negative control. The enzyme inhibition (%) was calculated according to Equation (1):Tyrosinase inhibition by selected pesticides was performed using the L-DOPA  enzymatic oxidation assay . Tyrosinn = 3 independent experiments, each one in triplicates). Statistical significance of differences between samples and the control, or between different concentrations, was assessed by the analysis of variance (ANOVA) with Tukey’s multiple comparison test (α = 0.05), using the tools of GraphPad Prism version 7 . Statistical significance between the results of the two enzymes was assessed by two-way ANOVA with Sidak´s multiple comparison test (α = 0.05), using the tools of GraphPad Prism version 7. Results from ANOVA analysis, namely degrees of freedom, F-value and p-value, are presented in The data are reported as mean ± SD (Cholinesterases (ChE) enzyme family are a group of hydrolases that break down esters of choline, such as acetylcholine, butyrylcholine or propionylcholine . AcetylcIn this study, we analyzed the anti-cholinesterase activity of glyphosate (GLY), imazalil (IMZ), imidacloprid (IMD) and lambda-cyhalothrin (LCT), using in vitro inhibition assays with the potential to be extrapolated to human exposure. Concerning human exposure to these pesticides, limited information is available unless to GLY. For example, after accidental exposure to GLY, a concentration of ~0.52 mM (89 µg/mL) was found in human serum . Considep < 0.05), as depicted. The available literature concerning AChE inhibition by GLY presents very distinct results. Samanta et al. Samanta, et al. [Anabas testudineus and Heteropneustes fossilis), a glyphosate-based herbicide increased AChE activity in the brain, muscle and spinal cord, while a study by Braz-Mota et al. Braz-Mota, et al. [Colossoma macropomum) exposed to Roundup® (containing glyphosate at 360 g/L) presented a reduced AChE activity. Similar findings were reported in other fish species (Leporinus obtusidens and Cyprinus carpio), also using Roundup®, with an observed decrease in AChE activity [50 values between glyphosate and glyphosate-based formulations [, et al.  reported, et al.  reportedactivity ,56. Neveulations , thus be50 value of 714.3 mM (~120 g/L) to human serum AChE exposed to GLY; a concentration significantly higher than the ones here tested and far above GLY concentrations found in human serum [Labeo rohita) exposed to Roundup®, an increase in BChE level was observed and correlated to a potential detoxification activity [®). Therefore, it is necessary to evaluate the toxicity of pure GLY (standard compound) using assays that allow a good correlation of its effect on cholinesterase activity and neurotoxicity.Using human erythrocytes exposed to GLY for 1 h, inhibition of AChE activity by 13% at 0.5 mM GLY was observed, but at 5 mM, the inhibition only increased to 17.6% , a low ian serum , while iactivity . Other sactivity . This reThe other studied pesticide was the fungicide imazalil. p > 0.05, comparison between concentrations). Nevertheless, it is worth mentioning that concentrations as low as 10 µM produced 22.5% inhibition of AChE activity (p < 0.05), thus demonstrating the high neurotoxic potential of this fungicide , as depicted.In this study, we also used two insecticides, IMD and LCT, to assess their inhibitory effect against AChE and BChE. Results of cholinesterase inhibition by IMD are shown in p > 0.05). At low concentrations (10 µM), IMD is potentially neurotoxic through AChE inhibition  exposed to IMD (up to 20 mg/L) for 21 days showed brain AChE inhibition, with 35% inhibition at the highest concentration [Penaeus monodon) reported elevated AChE activity, in all examined tissues, after 21 days of exposure to IMD [These results reveal that IMD has a higher affinity to AChE, which may potentially be linked to the differences between the two enzymes’ active sites. As observed to IMZ, IMD-induced inhibition reaches its maximum at 500 µM, being the inhibition about to 50% of control, and from 500 µM up to 1 mM, identical inhibition was obtained , as depicted in p < 0.05) but not statistically different from IMD (p < 0.05). Nevertheless, when compared to IMZ and IMD, at 10 µM, LCT produced the lowest AChE inhibition . To the best of our knowledge, the data here presented reports for the first time on the in vitro inhibition of AChE by LCT in a system designed for extrapolation to human toxicity. Concerning other animal ecotoxicity studies, LCT was found to decrease cholinesterase activity in Rana cyanophlyctis (a frog species) at brain, liver and kidney level, using tissue homogenates [Channa punctatus (snakehead fish), brain, gills and muscle AChE activity was reduced after LCT exposure [The pyrethroid insecticide LCT produced a similar pattern of AChE and BChE inhibition  as the oogenates . In ratsogenates . In Chanexposure .Although the most common function attributed to tyrosinase is the production of the skin pigment melanin, this enzyme is also involved in the production of neuromelanin, a pigment found in neurons and associated with neurodegeneration. However, the presence of neuromelanin has also been associated with neuroprotection against heavy metal toxicity. Among the neurons affected by neuromelanin toxicity, loss of dopaminergic neurons is correlated with Parkinson’s disease onset . NeverthNevertheless, none of the pesticides tested in the present research modulated tyrosinase activity; thus, it is possible to assume that any toxicity observed by these pesticides is not due to a direct effect on the activity of tyrosinase. Regardless, it cannot be excluded the potential effect of the pesticides on the protein expression, which may induce a different outcome in vivo assays.Micromelalopha troglodyte; however, no studies were reported regarding extrapolation to human toxicity [A previous study using GLY revealed that the herbicide modulated melanin production in insect species but in a tyrosinase-independent pathway, interacting with L-DOPA oxidation . Ford ettoxicity . We repoConcerning the effect of toxicants, some in vitro assays may have limited extrapolation to in vivo . As in vivo effects may be impaired by absorption and metabolism processes, which decrease bioavailability, the effective concentrations of pesticides in target tissues are lower than the tested concentrations, making the concentration-effect correlation difficult. Thus, in vitro enzymatic inhibition assays provide a valuable tool to screen compounds for neurotoxicity dependent on key enzyme inhibition. A significant number of commonly used pesticides described to have low toxicity to animals has yet not been screened for their modulatory effect on cholinesterase-dependent neurological processes, and most studies concerning this topic are focused on ecotoxicological application. In this research, we report the comparison of AChE, BChE and tyrosinase inhibition by glyphosate, imazalil, imidacloprid and λ-cyhalothrin. It was observed that glyphosate presented the lowest neurotoxic activity, with low inhibition of AChE activity in concentrations up to 1 mM. The insecticides imidacloprid and λ-cyhalothrin present selective AChE inhibition, while the fungicide IMZ is a broad-spectrum cholinesterase inhibitor, capable of inhibiting AChE and BChE in an equal manner. None of the pesticides tested was able to modulate tyrosinase activity. While additional studies should be carried out to further understand the mechanism behind cholinesterase inhibition, we here report the use of in vitro enzyme-based methodologies to screen pesticides for their neurotoxicity.


O. niloticus and C. gariepinus fish species. The accumulation level of Cu, Zn, Fe, Cd, Pb and Al in five vital tissues was markedly elevated near the Batts drain (site2) compared to the samples from the reference site (site 1). At the same site, C. gariepinus displayed the highest accumulation capacities when compared to O. niloticus. Based on the metal pollution index, livers and kidneys, followed by gills, showed the maximum overall metal load. The degree of DNA damage (assessed by comet and diphenylamine assays) was relative to the accumulated metals in tissues with species and site specification. The values of the hazard index for human consumption showed that the studied metals were within safe values at normal consumption rates. While harmful health consequences were observed at the habitual consumption level at site 2.The present work showed the impact of long-term exposure to the Batts drain’s discharges on  Therefore, they are usually used to reflect the water quality and the impacts of deteriorating environmental conditions. Accordingly, using integrated biomarkers to monitor environmental stressors and their impacts on aquatic biota has become a valuable and reliable tool  inhabiting the same habitat based on the accumulation potency of the studied metals and the integrity of DNA in tissues; and (2) assess the human health hazards related to fish consumption from those studied sites.Water pollution is a severe problem due to the growing anthropogenic activities near different aquatic bodies , with the help of expert fishermen. Thirty-six adult, healthy and male fish (9 fish/species/site) were selected for this study. The body weights and lengths were 195.51 ± 7.46 g; 19.95 ± 0.36 cm for O. niloticus and 353.67 ± 24.4 g; 37.91 ± 1.02 cm for C. gariepinus. Fish samples were delivered to the ecology laboratory, Zoology Department, Faculty of Science, Cairo University, in large plastic containers with two portable oxygen pumps for each one. After decapitation, the dissection of the fish was done, then liver, kidney, gills, skin and muscle tissues were obtained. The Institutional Committee on Animal Care and Use (IACUC) of Cairo University, Faculty of Science, Cairo, Egypt reviewed and accredited this study with the accreditation no. CU-I-F-39-19.The concentrations of Cu, Zn, Fe, Cd, Pb and Al metals were determined in five selected tissues of both species using Inductively Coupled Argon Plasma, ICAP 6500 Duo Thermo Scientific, England. For instrumental standardization, a 1000 mg/L multi-element certified standard solution  was utilized as a stock solution. The limits of detection in ppm were < 0.002 for Cu, < 0.001 for Al, < 0.0005 for Cd, < 0.005 for Zn, Pb and Fe. The acid digestion method was conducted as fully described by Neugebauer et al. . The tisUsing the formula of Usero et al. , this in++ & Mg++ free) containing 20 mM EDTA and 10% DMSO. Then, A 10 µL aliquot of cell suspension was mixed with 80 µL of 0.5% low melting point agarose (Sigma) and diffused on a fully frosted slide pre-dipped in normal melting agarose (1%). After solidification, the slides were positioned in cold lysis buffer  for 24 h at 4°C away from any source of light. Subsequently, the slides were placed in an alkaline buffer  for 20 min. The unwinded DNA samples were electrophoresed for 20 min at 300 mA and 25 V (0.90 V⁄cm). After electrophoresis, slides were immersed in an excess amount of 0.4 M Trizma base (pH 7.5) to neutralize the alkali. Then slides were fixed in 100% cold ethanol, air-dried and finally stored at room temperature in a desiccator until they were scored. Slides were stained with ethidium bromide (2 µg⁄mL) then, images were captured at × 400 magnification by a fluorescence microscope (Optika B 353LD2 LED trinocular fluorescence microscope). DNA damage was measured as the amount of fluorescence in the comet tail (% DNA in comet tail) using TriTek CometScoreTM . In the present study, 250 cells/tissue/species/site were estimated.The alkaline (pH > 13) comet assay was conducted according to the method described by Tice et al.  and Reci2SO4 and 16 μg/mL acetaldehyde) was added to 0.5 mL supernatant of each tube. After overnight incubation, incubated the resulting blue color was measured at 600 nm against diphenylamine solution as a blank solution. The quantity of damaged DNA was expressed as a percentage of total DNA the equation of Gercel-Taylor (Both tissues (25 mg) were lysed in 0.8 mL of cold lysis buffer . The resulted homogenates were centrifugated at 10.000 r.p.m. for 10 min at 4°C to separate the intact DNA (in the pellet) from fragmented DNA (in the supernatant). The resulting pellet and supernatant were mixed with 1.5 mL Tris–EDTA buffer  containing 10% trichloroacetic acid (TCA) in separate tubes. After 10 min of incubation at room temperature, each test tube was centrifuged at 3000 r.p.m. (at 4°C) for 15 min, then the supernatant was removed. The precipitates from each tube were mixed with 0.7 mL of 5% TCA, boiled for 15 min, cooled and then centrifuged at 2000 r.p.m. for 15 min. one ml of the diphenylamine reagent  / [Absorbence of fragmented DNA (represented by supernatant) + Absorbance of intact DNA (represented by pellet)] × 100.The mean daily ingestion of a particular chemical over a lifetime was determined using the equation stated by USEPA  in eatenp < 0.05) to estimate differences between studied fish species at the same site and evaluate the difference between the same species at different sites. Data were analyzed using Statistical Processor Systems Support, SPSS software, version 25.0, IBM, Chicago, IL, USA.The results were expressed as a mean ± standard error. The student's t-test was statistically used to analyze data (p < 0.05) higher at site 2 than at site 1. C. gariepinus revealed a significantly (p < 0.05) higher accumulation of most studied metals in various tissues than O. niloticus. C. gariepinus showed higher Cu content in the liver and gills compared with O. niloticus at site 1, while the Cu content in C. gariepinus was significantly elevated in all tissues except the muscle compared to O. niloticus at site 2. The accmulated Zn in C. gariepinus was significantly increased in all studied tissues (except the muscle) compared to O. niloticus at site 1. Iron concentrations in the tissues of C. gariepinus were markedly elevated in the liver and kidney (site 1) and kidney, gills and muscle (site 2) compared to O. niloticus at the same site. The hepatic Pb content of C. gariepinus was elevated at site 1, while at site 2, Pb contents in the liver, gills and muscles were increased compared to O. niloticus. The accmulated Cd in C. gariepinus was significantly increased in all studied tissues (except the muscle at site 1 and the skin at site 2) compared to O. niloticus. The accumulated Al of C. gariepinus was significantly increased in the kidney and gills at site 1 and in all tissues at site 2. The accumulation patterns of metals showed a high ability of the liver, kidney and gills to accumulate more metals than skin and muscular tissues.The mean concentration of six accumulated metals in the liver, kidney, gills, skin and muscle tissues of both studied species from two sites was summarized in Table This index exhibited the overall accumulated metals of both selected fish species Table . The valO. niloticus and C. gariepinus which chronically inhabit site 2 was compared to that of the reference site. The smeared appearance of damaged DNA in the cells of the liver and gills was recorded and compared in terms of % of DNA in the tail as represented in Fig. p < 0.05) elevation in C. gariepinus compared to O. niloticus inhabiting the same site. In gills, the % of DNA in the tail showed insignificant differences between different species of the same site.Similarly, the level of genomic DNA fragmentation in To quantify the % of DNA damage in both fish species, the liver (the main site for detoxification and biotransformation processes) and gills  were selected. The percentages of DNA damage showed significant variability through different species and sites.p < 0.05) elevation in C. gariepinus compared to O. niloticus at site 1. While at site 2 the total DNA damage was significantly elevated in the gills of C. gariepinus at site 2.As shown in Table Hazard index (HI) values for analyzed metals in the muscle and skin (edible tissues) were presented in Table 2+, Pb2+, Zn2+, Ni2+ and Pt2+; therefore, both tissues are more susceptible to metallic damage . Freshwater fish, Labeo rohita have been used to investigate DNA fragmentation induced by different concentrations of cadmium chloride  and the high percentage of fragmented DNAin the liver and gills collected from site 2. The active biotransformation processes in the hepatic tissues and the structural location of the gills may increase the exposure rate of both tissues to genotoxic metals from their surroundings. Long-term exposure to genotoxic metals can directly affect DNAintegrity by causing physical disruption as DNAmolecules are a suitable substrate for the attachment of metal cations (Kontaş and Bostancı C. gariepinus compared to O. niloticus of the same site. This elevation was in line with the results of the accumulation of metals in fish tissues, which revealed a significant correlation between the accumulated metals and the level of DNAdamage. Fish are one of the vital sources of protein for humans. Therefore, the accumulated pollutants in fish tissues pose health threats to individuals who depend on fish in their meals (Adeogun et al. Health problems induced by chronic exposure to metal pollution cannot be assessed or expected in living organisms using chemical analysis or classical environmental monitoring techniques (Abdel-Khalek et al. f et al. , who shof et al.  mentioneh et al.  showed tThe untreated discharges of the Batts drain had a potential health concern as indicated by high metal accumulation and genotoxic damage in exposed fish. The severity of DNA fragmentation was significantly different in the studied fish species and was directly proportional to the level of metal accumulation in their tissues. The values of HI for humans showed that harmful health consequences were expected, especially for habitual fish consumers at site 2. Therefore, water treatment and constant monitoring procedures should be applied at site 2 to overcome the detrimental impacts of anthropogenic discharges.


Fixation for atlantoaxial dislocation is a challenging issue, and posterior C1 lateral mass and C2 pars–pedicle screw plate–rod construct is the standard of care for atlantoaxial instability. However, vertebral artery injury remains a potential complication. Recent literature has focused on intraoperative navigation, the O-arm, 3D printing, and recently use of robots for perfecting the trajectory and screw position to avoid disastrous injury to the vertebral artery and enhance the rigidity of fixation. These technological advances increase the costs of the surgery and are available only in select centers in the developed world.Review of the axis bone anatomy and study of the stress lines caused by weight transmission reveal that the bone below the articular surface of the superior facet is consistently dense as it lies along the line of weight transmission A new trajectory for the axis screw 3–5 mm below the midpoint of the facet joint and directed downward and medially avoids the course of the vertebral artery and holds the axis rigidly. Divergent screw constructs are biomechanically stronger. Variable screw placement (VSP) plates with long shaft screws permit manipulation of the vertebrae and realignment of the facets to the correct reduced position with fixation in the compression mode.https://youtu.be/E1msiKjM-aAThe video can be found here:  Atlantoaxial dislocation is a challenging problem. C1 lateral mass with C2 pars–pedicle plate–rod fixation is the current standard of care. Avoiding vertebral artery injury and correction of the deformity are the two main concerns which need attention. Recent literature has focused on the use of intraoperative navigation, O-arm, and 3D printing to enhance safety, but these come at an increased cost .Review of the axis vertebra anatomy consistently shows the presence of dense compact bone below the C2 facet, which has been confirmed by recent publications in the literature .1:30 The new trajectory. Starting the C2 fixation few millimeters below the midpoint of the superior facet eliminates the chances of vertebral artery injury.Directing the screw downward and medially provides for a strong hold for manipulation and rigid fixation.1:54 Cadaveric dissection and live operative clips. In this cadaveric dissection, after standard posterior midline exposure, the occiput, posterior arch of the atlas, and posterior element of axis along with both atlantoaxial joints are exposed.The new entry point for the axis screw is few millimeters below the midpoint of the facet joint, and the trajectory is downward and medially in the subfacetal region of the axis for a depth of 16–20 mm. This is decided by studying the preoperative CT scan for bone stock and volume. The tap is used to thread the drill holes for a few millimeters to allow for screw entry and direction. Overtapping must be avoided .The C1 screw is above and lateral to the midpoint of the facet joint and directed upward and medially, divergent with respect to the axis screw. The length again is decided on preoperative CT scans and bone stock. The joint space is gardened by abrading the endplate with a microburr and curette, followed by corticocancellous autografting with bone harvested from the spinous process of the axis and C3.3:57 Preop images. These preoperative images show atlantoaxial instability, both in flexion and extension with a high-riding vertebral artery.4:09 New screw and VSP plate construct and facetal realignment. The new screws with long shafts and variable screw placement plates permit realignment of the vertebra by using joysticks and by fixing the top nut at the appropriate position on the plate. The VSP plate construct permits alignment of the screws in the VSP plate in the desired position in compression mode, which is difficult in the polyaxial screw-rod construct. The extra length of the screw is cut, and now the rigid construct is seen with the screws fixed in compression mode.5:40 The biomechanics of divergent screws. Divergent screw direction is an important concept which results in better fixation. These are the special instruments made for the VSP plate-screw construct.6:01 Postoperative images and follow-up images. These postoperative images show correct realignment and screw position. These postoperative images after 6 months show solid bony fusion.6:15 Additional references. Li et al., 2015; Patkar, 2016; Menon and Raniga, 2018.


These results indicated that the ACE-inhibiting peptides from mulberry leaf protein hydrolyzed (FHMP) had the potential activities to inhibit ACE and could be used as functional food or drugs to inhibit ACE. This work provides positive support for mining the biological activity of mulberry leaves in the treatment of hypertension.Angiotensin-I converting enzyme (ACE) inhibitory peptides drew wide attention in the food industry because of their natural reliability, non-toxicity, and safety. However, the characteristics of ACE inhibitory peptides obtained from protein hydrolysate of mulberry leaf prepared by Flavourzyme were still unclear. Based on the single-factor test, the Plackett–Burman test and response surface test were used to determine the key factors affecting the ACE inhibition rate in mulberry leaf protein hydrolysate and the optimum conditions of enzymatic hydrolysis. The results showed that the optimum technical parameters were as follows: the ratio of material to liquid is 1: 25 , the Flavourzyme to substrate ratio was 3,000 U/g, the temperature of enzymatic hydrolysis was 50°C, pH was 6.3, and the time of enzymatic hydrolysis was 2.9 h. The ACE inhibitory peptides in the mulberry leaf protein hydrolysates were purified by ultrafiltration and gel filtration, aiming to obtain the highest active component. The 12 peptide sequences were identified by reverse liquid chromatography-mass spectrometry, and then, they were docked to the crystal structure of human angiotensin-I converting enzyme (1O8A), and the interaction mechanisms of 12 peptide sequences and 1O8A were analyzed. The docking results showed that among the 12 peptide sequences, ERFNVE (792.37 Da), TELVLK (351.72 Da), MELVLK (366.72 Da), and FDDKLD (376.67 Da), all had the lowest docking energy, and inhibition constant. The chemosynthetic ERFNVE (IC Improving the utilization rate of protein in the food industry has become a main choke point problem , 2. EnzyMoringa oleifera leaf protein with alkaline protease. After separation and purification, it was found that the peptides with less than 1 kDa had a significant ACE inhibitory effect, and two peptide sequences, Leu - Gly - Phe - Phe and Gly - Leu - Phe - Phe, were obtained by mass spectrometry and amino acid sequence analysis. Zhang et al.  inhibitory peptides . Hyperteg et al.  digestedMulberry leaf is rich in protein, which accounts for 27% of its dry base weight . The conN-(3-(2-furyl) acryloyl)-L-phenylalanyl-glycyl-glycine (FAPGG) were obtained from Sigma- Aldrich Co . The ACE Kit-WST was purchased from Tong Ren Chemical Co., Ltd. Cytochrome C, aprotinin, bacitracin thymopentin, and Gly - Gly - Gly was purchased from Shanghai Yuan Ye Biochemical Technology Co., Ltd. Acetonitrile was purchased from Sigma  for high-performance liquid chromatography (HPLC).Mulberry leaves were collected from the Mulberry leaf Garden of Zhejiang Academy of Agricultural Sciences; Coomassie brilliant Blue G-250 was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.; papain (≥800 U/mg), alkaline protease (≥200 U/mg), acid protease (≥50 U/mg), Flavourzyme (≥20 U/mg) and trypsin (≥250 U/mg) were purchased from Qi Yi Biotechnology (Shanghai) Co., Ltd. ACE, Captopril and MELVLK, ERFNVE, FDDKLD, and TELVLK were synthesized by Nanjing Leon Biological technology co., LTD, the purity of which was greater than 95%. Other chemical reagents used were of analytical grade.g for 10 min. The sediments were extracted in 2 L of 0.05 mol/L NaOH solution at 25°C for 2 h by shaking. The protein solution was collected by centrifugation at 2,795×g for 10 min and precipitated by adjusting to pH 3.5 using 6 mol/L HCl. The precipitates were recovered by centrifugation and were lyophilized.Fresh mulberry leaves were washed and dried in an oven at 40°C for 6 h. and crushed into homogeneous powder by a highspeed pulverizer . 100 g mulberry leaves powder was extracted with 1 L 60% ethanol at 40°C for 1 h to remove alcohol-soluble substances. The sediments were obtained by centrifugation at 2,795×g at 4°C. The supernatant was collected and lyophilized to measure the ACE inhibition rate.Mulberry leaf protein was dissolved in distilled water (10 g/100 mL) and successively hydrolyzed by Papain  5,000 U/g), Acid protease , Alkaline protease , Trypsin , and Flavourzyme  under their most suitable reaction conditions for 3 h. Enzymatic reactions were stopped by heating at 95°C for 10 min and the hydrolysate was centrifuged by 11,180×y = –4.5518x + 30.551, R2 = 0.9929).The molecular weight of mulberry leaf protein and its enzymatic hydrolysate was determined by high-performance liquid chromatography  equipped with a TSK gel 2000 SWXL  column. The mobile phase was acetonitrile/water/Trifluoroacetic acid , the detection wavelength was 220 nm, the flow rate was 0.5 mL/min, the column temperature was 30°C, and the injection volume was 10 μL. Cytochrome C (12.5 kDa), aprotinin (6.5 kDa), bacitracin (1.45 kDa), thymopentin (679 kDa), and Gly - Gly - Gly (189 kDa) were used as relative molecular weight calibration standards. The log Mw and elution time of the standard sample was used as the standard curve , enzyme to substrate ratios , enzymatic hydrolyses times ; temperatures , and pH  on the inhibitory activity of ACE were further studied. The supernatant was collected and lyophilized, prepare 5 mg/mL solution, and measure the ACE inhibition rate.Because Plackett–Burman’s design can quickly and effectively screen out the main influencing factors from many factors, it is widely used in factor-screening tests . AccordiBased on the PB test, The feed liquid ratio , pH , and enzymatic hydrolyses time , were further optimized by a central composite design with seventeen independent experiments. Three levels for each process parameter are coded as –1, 0, and +1.Refer to the method of Aondona et al. , and makThe most active fraction obtained by ultrafiltration was dissolved into ultra-pure water and loaded onto a Sephadex G-10 gel filter column (1.6 cm × 100 cm). The column was then eluted with ultra-pure water at a flow rate of 0.5 mL/min. One tube was collected every 10 min and detected at 280 nm. The elution peaks of each group were collected and lyophilized. Then prepared into 1 mg/mL solution measure the ACE inhibitory activity of the elution peak of each group was determined.18 column  coupled to a (PepMap RSLC C18) electronic spray ion.The fractions with the highest ACE inhibitory activity after gel filtration chromatography were desalted on a C18 column, and the amino acid sequences were further identified by Thermo Scientific QE mass spectrometer . The peptides were dissolved in 20 μL solution  and subjected to an LC-MS/MS system with a CThe column flow rate was 500 nL/min. The eluting conditions were as follows: mobile phase A (0.1% formic acid), mobile phase B , column temperature of 40°C, linear elution procedure . Positive ion scanning was used, and the scanning range was 100–2,500 (m/z). Tandem mass spectra were processed by PEAKS Studio version X+ .Peptide sequences were identified using the database search by PEAKS Studio X software in combination with manual de novo sequencing to process the MS/MS data and to perform peptide sequencing.DOI: 10.2210/pdb1O8A/PDB) was downloaded from the PDB database. The water molecules were removed from 1O8A, and Proton hydrogenation and energy minimization of all molecules was performed to obtain the receptor molecules needed for docking. The peptides obtained by PEAKS Studio X analysis and screening were used as the ligand, and the receptor pocket of 1O8A was used as the docking target for molecular docking. The peptide with a better docking effect was screened from the obtained results to analyze the interaction between mulberry leaf protein ACE inhibitory peptides and 1O8A.Molecular docking was performed between the identified peptides and the crystal structure of the human angiotensin-I converting enzyme (1O8A) according to the method of Samuchaya et al. . The semBased on the molecular screening of peptide sequences, selected peptides were synthesized by China Peptides Co., Ltd.  for further analysis.50 value and half of IC50. Lineweaver–Burke plots were constructed using GraphPad Prism version 8 software .According to the previous method of Shufang Ye  to exploThe ACE inhibitory activity was determined by ACE Kit-WST . Taking ultra-pure water as blank control, the inhibitory activity of ACE was calculated.P-value <0.05 was considered statistically significant.Each experimental procedure was performed three times, and data were expressed as the mean ± standard deviation. Statistical analyses were conducted by one-way analysis of variance using IBM SPSS 20 . Duncan’s test was applied to determine the significance of multiple groups. The P < 0.05), and its molecular weight was mostly <1 kDa. The molecular weight of mulberry leaf protein is mostly above 10 kDa, but the ACE inhibitory activity is very low.P < 0.05), which indicates that Trypsin treatment will destroy the protein structure of mulberry leaf protein. Therefore, the results show that different enzymes have different abilities to degrade mulberry leaf protein . Therefore, the optimum temperature of enzymatic hydrolysis is 50°C.The effect of temperature on the enzymatic reaction was significant . When thP < 0.05). However, when the enzymatic hydrolysis time reached 3.0 h, the inhibition rate of ACE continued to increase to 57.6%, which was the maximum. The inhibition rate of enzymatic hydrolysis for 4.0 and 5.0 h began to decrease, and the difference was significant (P < 0.05). It may be that the time of enzymatic hydrolysis is too long, which leads to the excessive enzymatic hydrolysis of mulberry leaf protein and a decrease in activity. Therefore, the appropriate time of enzymatic hydrolysis was selected as 3.0 h.As shown in P < 0.05). When E/S was 1,000 U/g, the ACE inhibition rate was 41.5%. With the increase of E/S, the ACE inhibition rate of mulberry leaf protein hydrolysates increased gradually, and the changing trend was larger. The inhibition rate of ACE in the enzyme hydrolysates reached 57.7% when E/S was 3,000 U/g. The analysis of significant differences showed that the ACE inhibition rate of mulberry leaf protein hydrolysates with E/S of 3,000 U/g was significantly higher than that of 4,000 and 5,000 U/g. Therefore, when the E/S is 3,000 U/g, enzyme dosage and feed liquid ratio are just in the saturated state of supply and demand balance . When pH increased to 9.0, the inhibition rate of ACE decreased significantly to 39.4%. Therefore, when the enzymatic hydrolysis of pH exceeded the optimum pH range of Flavourzyme, the catalytic activity of the enzyme decreased, which was the same as the change of ACE inhibitory activity of rice protein hydrolysates studied by Wei Kang et al.  on the ACE inhibitory activity of mulberry leaf protein hydrolysates, and the ACE inhibition rate of mulberry leaf protein hydrolysates increases significantly and then decreases with the increase of feed liquid ratio. When the feed liquid ratio was 1: 15, the ACE inhibition rate of mulberry leaf protein hydrolysates was 27.1 ± 2.37%; when the feed liquid ratio was 1: 20, the ACE inhibition rate significantly increased to 39.72 ± 0.75% (P < 0.05); when the feed liquid ratio reached 1: 25, the ACE inhibition rate of mulberry leaf protein hydrolysates reached the maximum value of 50.3 ± 1.45% (P < 0.05). However, with the continuous decrease of the feed liquid ratio, the ACE inhibition rate of mulberry leaf protein hydrolysates began to decrease. Therefore, the feed liquid ratio were selected as 1: 25 in the following study.Based on single factor test, taking the ACE inhibition rate of mulberry leaf protein hydrolysates as the main evaluation index, the Plackett–Burman factor screening test design and main effect analysis were carried out on temperature, time, feed liquid ratio, pH, and enzyme dosage. The results are shown in P < 0.01), while the effects of pH and feed liquid ratio on the response value were significant (P < 0.05). Therefore, time, pH, and feed liquid ratio were selected for the further response surface design. For the non-significant factors such as E and A, the optimal level of single factor test was selected in the follow-up experiment, that is, enzyme dosage 3,000 U/g and the temperature was 50°C.Based on single-factor results and the Plackett–Burman factor screening test, time, pH, and feed liquid ratio were selected as independent variables, ACE inhibition rate as the main response value, 3 factors, and 3 horizontal Response surface Test was designed. The results are shown in Based on the regression analysis of P < 0.0001, indicating that the model has reached a very significant level, the “R-Squared” was 0.9982 and the “adj R-Squared” was 0.9729, which indicates that there is a high correlation between the test value and the fitting value, and. the model can explain at 97.3% of the variation in the data. The “lack of fit” was not significant (P > 0.05) indicating that the equation of the model fits the data well and can better reflect the relationship between the respective variables and the response value. Therefore, the regression equation can be used to fit and predict the test results. The differences in the interactive term AC were extremely significant, according to the P values of the factors in The statistical analysis for the model  showed tBased on the results of the analysis of variance, the interaction of substrate and liquid ratio, pH and time was analyzed by Design-ExpertV8.0.6 software, and the results are shown in It can be seen from Under the above optimal process conditions, three verification tests were carried out, and the results showed that the inhibition rate of ACE was 60.3, 60.9, and 60.3%, and the average clearance rate was 60.5%, which was close to the theoretical value of the model. It shows that the model can be used to predict the preparation process of ACE inhibitory peptides from mulberry leaf protein hydrolysates.As shown in At the concentration of 1.0 mg/mL, the ACE inhibition rate of FHMP-V was 62.63 ± 0.48% and higher than those of the other five components. Previous studies have shown that some plant protein peptides can inhibit ACE activity, such as sesame peptide samples  and MoriGel filtration chromatography is usually used to separate and enrich peptides . AccordiSince the ACE inhibitory activity of FHMP-V-3 was higher than that of other fractions, it was inferred that it might contain more active ACE inhibitory peptides. Peptides were identified using LC-MS/MS analysis and de novo sequencing on Peak Studio 8.0. Twelve peptides were simultaneously identified in the FHMP-V-3 fraction by LC-MS/MS combined with de Novo sequencing, all sequences are listed in To further explore the interaction between polypeptide sequences and ACE, 12 polypeptide sequences were used for molecular docking with the crystal structure of the human angiotensin-converting enzyme (1O8A).Low molecular weight angiotensin-converting enzyme inhibitory peptides have been shown to have better antihypertensive activity , 44 and The results of molecular docking show that the ACE inhibition rate of these peptides is realized by forming hydrogen bonds with the residues inside and outside the active center, closing the active center, or distorting the catalytic configuration. Among the 12 peptide sequences, there are eight peptides with binding energy less than –16 with residues around the active site of 1O8A, as shown in TELVLK had four hydrogen bonding forces at Asp121, Ser219, Arg522, and Asn66 ; ERFNVE 50 values  seed studied by Nurul Dhania et al. , S2 , and S1′ (Glu162) and Znterature . The resterature ; ERFNVE terature ; MELVLK terature ; FDDKLD terature . More imterature , which iterature . In the Furthermore, the ACE inhibitory activities of peptides within MW 500–800 Da are higher than that of peptides above 800 Da . In the In this study, the preparation process of ACE inhibitory peptides by Flavourzyme was optimized by a single factor experiment, Plackett–Burman experiment, and response surface experiment, and 12 peptide sequences were obtained from FHMP-V-3 by further separation, screening, and structure identification. Among these 12 peptide sequences, 4 peptide sequences ERFNVE, TELVLK, MELVLK, and FDDKLD have lower binding energy and more interaction binding bonds force to 1O8A. The peptides with hydrophobic amino acids account for a large proportion and contribute more to its ACE inhibitory. This is the first report on four novel ACE inhibitor peptides, produced from the hydrolysates of HMP using Flavourzyme, which have potential applications in several industries.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.YC, YZ, and WW conceived and advised on all aspects of the study. YC, YZ, QQ, FL, and XL performed experiments, analyzed data, and wrote the manuscript. YJ and WW supervised all aspects of the study. QC, NW, KB, and SS completed language changes. XW put forward the advice. All authors discussed and commented on the manuscript.


Inhibitory neurons take on many forms and functions. How this diversity contributes to memory function is not completely known. Previous formal studies indicate inhibition differentiated by local and global connectivity in associative memory networks functions to rescale the level of retrieval of excitatory assemblies. However, such studies lack biological details such as a distinction between types of neurons (excitatory and inhibitory), unrealistic connection schemas, and nonsparse assemblies. In this study, we present a rate-based cortical model where neurons are distinguished , connected more realistically, and where memory items correspond to sparse excitatory assemblies. We use this model to study how local-global inhibition balance can alter memory retrieval in associative memory structures, including naturalistic and artificial structures. Experimental studies have reported inhibitory neurons and their subtypes uniquely respond to specific stimuli and can form sophisticated, joint excitatory-inhibitory assemblies. Our model suggests such joint assemblies, as well as a distribution and rebalancing of overall inhibition between two inhibitory subpopulations, one connected to excitatory assemblies locally and the other connected globally, can quadruple the range of retrieval across related memories. We identify a possible functional role for local-global inhibitory balance to, in the context of choice or preference of relationships, permit and maintain a broader range of memory items when local inhibition is dominant and conversely consolidate and strengthen a smaller range of memory items when global inhibition is dominant. This model, while still theoretical, therefore highlights a potentially biologically-plausible and behaviorally-useful function of inhibitory diversity in memory. Broadly, there are two types of neurons: excitatory and inhibitory. Inhibitory neurons are amazingly diverse compared with excitatory neurons. Why? Using a computational model with realistically-sized groups of excitatory neurons (representing memories) associated together in a network of memories, we highlight a potentially biologically-plausible and behaviorally-useful function of inhibitory neuron diversity in memory. Two findings in particular standout: (1) inhibitory diversity can quadruple the range of memory retrieval; and (2) balancing the strength of different inhibitory neurons’ influence on excitatory neurons can dramatically change how the network of memories become activated, balancing and extracting both geometric and topological information about the network.The mechanisms by which our brains flexibly perform the complex tasks of learning and memory are not completely understood. Hebbian learning , the relThe majority of experimental and computational work focusing on assemblies as representations of memory items has focused on the role of excitatory neurons. However, emerging evidence suggests inhibitory neurons play a nontrivial role in cortical networks. Throughout the brain, inhibitory neurons have classically been thought to coarsely keep excitation in check with a broad, nonspecific blanket of inhibition . But morPrevious modeling work in a formal model with binary neurons  has showIn order to embed memories in the network, we generate binary patterns as vectors of length ion rule  designed10.1523/ENEURO.0023-22.2022.f1-1Extended Data Figure 1-1Figure 1-1, EPS file.An example of spiking rates of all units in the stimulated pattern, its first neighbor (pattern adjacent to the stimulated pattern), the neighbor’s neighbor (second neighbor), the neighbor’s neighbor’s neighbor (third neighbor), and all other patterns. This is from a simulation using Two populations of inhibitory neurons are also modelled, one with global connectivity . At eac=ψ(IiG)ViL˙=ψ of the underlying neurons  showed different resolutions of clustering. For most graphs, there was a trend of more and small clusters at We also observed how excitation spreads across the associative memory structure across time, after activation of vertices of interest, in the Tutte and multiroom graphs. For the Tutte graph we chose the central vertex, which branches off into three separate “rooms,” and for the multiroom graph we chose a location within one of the rooms that also led through a “doorway” to a neighboring room. We chose these vertices since they represent points of behavioral interest and ecological importance in animals—they are points at which an animal may make significant choice between which room to enter, explore, or exploit. In the Tutte graph, for ee rooms A. This i c=0.525 C quicklyactivity B, arrowsThe multiroom graph showed a similar trend in broadening and maintaining a larger range of retrieval with increases in nd c=0.7 . InteresThe clustering and geometric indices, Clustering indices (Geometric indices (Previous modeling studies have conflated excitatory and inhibitory neuron identities and learning rules  or ignorIt is important to emphasize these results are generated in the context of a memory structure which relies on the correlation of semantically close units, implying that memory retrieval in such a structure is functionally optimized when nearby units are correlated. Biological evidence for such correlations was first prominently shown in monkey anterior ventral temporal cortex by In our study, we selectively stimulate single memory patterns and see memory retrieval of the pattern and surrounding associating patterns in ∼100–200 ms of simulation time. Is this biologically realistic? Single neurons in human medial temporal lobe which learn to selectively encode associative episodic memories within just a few trials can be recruited in subsequent activations within ∼500–700 ms ; maximalRecent experimental evidence in mice  shows thIn Inhibitory neurons also contribute to the initiation, maintenance, and modulation of rhythmic oscillations in local electrical activity . One exaTheoretically, in the absence of noise and with a sufficiently large network, an associative memory structure with patterns , N/log(Ne errors , and few results  thereforConceivably, it is possible to functionally enter into the range of spatial correlation patterns , howeverThe effects generated by these modifications, such as stable extension in the range of retrieval, appears limited because of increases in noise in strongly local-inhibition dominant states. This is likely because of a finite field effect and may indicate a necessary minimum size of local excitatory-inhibitory assemblies for such states. For example, stability in the case of  in mice , most re in mice . Among o in mice  and in a in mice . It therAlternatively, it is possible this mechanism requires a hybrid sparse-dense coding schema, as has long been suggested operates in hippocampus , cerebelExtension of the range of retrieval was not simply the only apparent function of the inhibitory mechanism in sophisticated associative memory structures, the mechanism also permitted multiscale segmentation of the associative memory structure. Local-inhibition dominant states typically activated coarser topological segments of the graphs whereas global-inhibition dominant states consolidated activity in more densely associated clusters, highlighting finer topological features. These results were similar to those found in a more abstract model of binary neurons , except  studies , which wAn intriguing aspect of this inhibitory mechanism is its ability to dramatically affect not just the range of retrieval but also which parts of the memory structure become dominant given an initial stimulation. For example, it appears in global-inhibition dominant states, global inhibition drives a “winner-takes-all” dynamic  whereby Probing such circuits and behaviors may provide insights on the potential influence such inhibitory mechanisms have on neuropathologies, especially those associated with cognitive defects . For insWhile this study has made some advances over prior models  in termsIn our model, making a seemingly subtle change to the network structure by introducing some of the complexities and diversities of inhibitory neurons had a profound impact on retrieval. We have shown how this phenomenon mainly persists in a sparse, associative memory structure which obeys Dale’s Law and has more biologically-plausible connections than prior models. We have also shown and discussed some of the potential functional roles of this mechanism in graph-based cognitive tasks and discussed how this mechanism may contribute to a type of sparse-dense coding schema.


The mismatch between bony endplates (BEPs) and grafted bone (GB) triggers several complications biomechanically. However, no published study has identified whether this factor increases the risk of screw loosening by deteriorating the local stress levels. This study aimed to illustrate the biomechanical effects of the mismatch between BEP and GB and the related risk of screw loosening. In this study, radiographic and demographic data of 56 patients treated by single segment oblique lumbar interbody fusion (OLIF) with anterior lateral single rod (ALSR) fixation were collected retrospectively, and the match sufficiency between BEP and GB was measured and presented as the grafted bony occupancy rate (GBOR). Data in patients with and without screw loosening were compared; regression analyses identified independent risk factors. OLIF with different GBORs was simulated in a previously constructed and validated lumbosacral model, and biomechanical indicators related to screw loosening were computed in surgical models. The radiographic review and numerical simulations showed that the coronal plane’s GBOR was significantly lower in screw loosening patients both in the cranial and caudal vertebral bodies; the decrease in the coronal plane’s GBOR has been proven to be an independent risk factor for screw loosening. In addition, numerical mechanical simulations showed that the poor match between BEP and GB will lead to stress concentration on both screws and bone-screw interfaces. Therefore, we can conclude that the mismatch between the BEP and GB will increase the risk of screw loosening by deteriorating local stress levels, and the increase in the GBOR by modifying the OLIF cage’s design may be an effective method to optimize the patient’s prognosis. The anterior lateral single rod (ALSR) fixation system can reconstruct instant postoperative stability in a single incision for oblique lumbar interbody fusion (OLIF) patients. As a hardware-related complication, screw loosening has been widely reported, negatively affecting patients’ rehabilitation and deteriorating long-term prognosis . The detClinical studies have shown that the mismatch between BEP and GB triggers complications, including nonunion and cage subsidence , and theStudies proved that screw loosening was related to these complications, and insufficient anterior support was also reported as a risk factor for stress concentration in the bone-screw interfaces and resulting screw loosening . ConsideIn this study, we verified whether the mismatch between BEP and GB triggers a higher incidence of screw loosening and investigated the biomechanical effects of this phenomenon. Imaging data of OLIF patients fixed by ALSR have been retrospectively reviewed, and the biomechanical changes in ALSR and bone-screw interfaces have been computed in an anteriorly constructed numerical lumbo-sacral model. This study could provide theoretical guidance for understanding the screw loosening mechanism and optimizing the design of OLIF cages.The ethics committees of West China Hospital reviewed and approved the protocol of this study (2020-554). Informed consent was waived for this retrospective study. We retrospectively reviewed patients who underwent single segment OLIF with ALSR screw fixation from May 2017 to August 2019. The age, sex, and BMI of these patients were recorded. A senior spine surgeon performed all operations. Screw types and sizes were identical in these patients. All screws were placed in a single attempt and penetrated the contralateral cortex.Patients who underwent single segment OLIF with ALSR screw fixation for lumbar degenerative diseases, including spinal stenosis, grade 1 and grade 2 degenerative spondylolisthesis, and lumbar disc herniation, were included in this study. The exclusion criteria were as follows: 1) Patients with a history of lumbar surgery; 2) Patients with primary or metastatic spinal tumors, lumbar tuberculosis, rheumatic immune diseases, and secondary osteoporosis caused by medication or other metabolic diseases; 3) Patients with grade 3 and grade 4 degenerative spondylolisthesis or spondylolysis; 4) Patients who underwent lumbar revision surgery within the clinical follow-up period of 12 months for complications other than screw loosening; 5) Patients who underwent intraoperative screw replacement.All patients underwent lumbar computational tomography (CT) three times in the imaging center of our hospital, including 1 week before, 1 week after, and 1 year after OLIF surgery (tube voltage: 120 kV) . The CT The screw loosening status of the cranial and caudal vertebral bodies was identified separately. In the postoperative 1-year CT imaging data, vertebral bodies with ≥1 mm width radiolucent zones around the screw were defined as screw loosening . The BMDRadiographic and demographic indicators are presented as the mean ± standard deviation for continuous variables and number (percentage) for categorical variables. We conducted statistical analyses in SPSS software. The intraclass correlation efficiency (ICC) was computed to identify the repeatability of continuous variables (ICC ≥ 0.8 represents excellent reliability) . The kapt test was used for continuous variables, and the chi-square test was used for the categorical variables. We performed binary logistic regression to identify independent risk factors for screw loosening. Univariate analyses of each potential risk factor were performed, and the variables that achieved a significance level of p < 0.1 were entered into multivariate analyses. Variables with p < 0.05 were considered independent risk factors in the multivariate analyses . Bone structures of the FE model include cortical, cancellous, and BEPs. The cortical thickness was set as 0.8 mm, and the thickness and morphology parameters  of BEPs were defined separately based on anatomic studies . NonbonyThe L4-L5 segment was selected to simulate oblique lumbar interbody fusion (OLIF) with ALSR screw fixation, and we performed surgical simulation according to a literature review and our surgical experience . In thisThree different grades of contact sufficiency between the BEP and GB  were simulated by changing the GBOR in the coronal plane. The ideal contact was defined completely match between BEP and GB, GBOR in acceptable and poor contact models were defined as 80 and 60%, respectively. Based on the review of radiographic data, the mismatch on the superior side was mainly in the central region, while that on the inferior side was in the peripheral region. By combining different contact sufficiency grades between the GB and superior and inferior BEPs, five different OLIF models were constructed , 5.During the simulation of ALSR screw fixation, two titanium alloy (TI) screws were inserted into the L4-L5 vertebral bodies and penetrated the contralateral cortex. The axes of the screws in the transverse plane were parallel to the OLIF cage, whereas those in the coronal plane were parallel to the BEPs . Screw tFinite element analyses in this study were performed in the “Ansys workbench 2020 r2 academic”. Hybrid elements  with different sizes were set in different components of the FE model. Mesh refinement was set in structures with low thickness and large deformation  . The degin vitro studies could be reduced  in the L4-L5 segment and adjusting ligament stiffness, the differences between computed ROMs and measured values from widely cited  reduced . As a re reduced . The com process .p = 0.002). The cranial side’s GBOR was significantly lower than that of the caudal side in coronal and sagittal planes (p = 0.009), and there were no significant differences in HU between cranial and caudal vertebral bodies (p = 0.519).A total of 56 patients  with an average age of 56.57 ± 11.96 years treated by single segment OLIF with ALSR screw fixation were recorded. The interobserver and intraobserver results during the judgment of screw loosening were substantial, with Kappa values of 0.778 and 0.759, respectively. The reliability of continuous variable measurement was excellent, with ICCs of 0.894 and 0.862, respectively . The ovep = 0.033) and had significantly lower coronal plane GBOR and HU than those without screw loosening. The p value of HU was 0.003, and that of GBOR was 0.013. Based on the computational results of univariate logistic regression analyses, these three indicators were also entered into the multivariate analysis to identify independent risk factors. The results showed that reducing HU and coronal plane GBOR were independent risk factors for screw loosening on the cranial side  could not be excluded effectively in a small sample size study  after spinal fixation, the variation tendency was evident at the 1-year clinical follow-up. As a radiographic review with limited sample sizes and a short follow-up period, we still should admit that the results of this study cannot be generalized to long-term clinical outcomes. In addition, in FEA, we did not identify the mechanical effects of the sagittal plane’s GBOR. Although this factor is not an independent risk factor for screw loosening, it should be considered in our subsequent studies to evaluate the interaction of coronal and sagittal plane GBOR and their mechanical effects on the risk of screw loosening. Additionally, although this was the common method for the same type studies , the mulBased on the radiographic review and numerical surgical simulations, we can conclude that the mismatch between the BEP and GB will lead to stress concentration on the ALSR and bone-screw interfaces and increase the resulting risk of screw loosening. Cage design modification is of great significance for reducing screw loosening risk biomechanically.


Inhibitory signaling in the brain organizes the neural circuits that orchestrate how living creatures interact with the world around them and how they build representations of objects and ideas. Without tight control at multiple points of cellular engagement, the brain’s inhibitory systems would run down and the ability to extract meaningful information from excitatory events would be lost leaving behind a system vulnerable to seizures and to cognitive decline. In this review, we will cover many of the salient features that have emerged regarding the dynamic regulation of inhibitory signaling seen through the lens of cell biology with an emphasis on the major building blocks, the ligand-gated ion channel receptors that are the first transduction point when the neurotransmitter GABA is released into the synapse. Epilepsy association will be used to indicate importance of key proteins and their pathways to brain function and to introduce novel areas for therapeutic intervention. Epilepsy is a chronic neurological disorder characterized by repeated, unprovoked seizures. Seizure prevalence in those with epilepsy can range from one seizure every few months, to hundreds of seizures a day, the latter of which is usually seen in cases of genetic epilepsies . Yet, evNeurological disorders characterized as epilepsy can emerge throughout the lifespan, with cases of genetic epilepsy syndromes emerging in the first few years of life, childhood-restricted epilepsies, and new onset epilepsy in adulthood . FrequenThough numerous classes of medications have come into use for epilepsy in the past several decades, nearly all of them come with a heavy load of side effects that may exacerbate symptoms co-morbid with epilepsy such as mood disorders and learning and memory difficulties . AdditioARs)  . Furtherepilepsy .Replication of these studies, along with more recent immunohistochemical datasets from surgically resected tissue of patients with intractable epilepsy, have produced inconsistent findings that often do not validate earlier reports . AdditioARs and their efficacy as the brain’s major inhibitory sensor. We will explore regulation of (1) GABA receptor trafficking, stabilization, and localization, (2) GABAAR subunit composition (with associated changes in channel properties), (3) inhibitory synapse formation and elimination, and identification of (4) vulnerable GABAergic neuronal populations. Additionally, we will provide examples where a particular nexus of control is known, or proposed, to be dysregulated with epilepsy, either in animal models or in tissue samples collected from human epilepsy patients. Finally, we will discuss how the proliferation of single cell technologies and genetic disease models may allow for a more refined understanding of inhibitory brain dynamics that impact seizure disorders and how this may inform the development of more targeted therapies.Over the past 20 years there has been an expanded exploration into the basic mechanisms of inhibitory plasticity. A complex and dynamic signaling landscape has emerged that contains unique cell populations which deliver GABA into the synapse and a tightly controlled feedback system that regulates its receptors in discrete cellular compartments of specialized neuronal populations. In this review, we will examine various mechanisms that have been shown to alter inhibitory signaling, with a special focus on GABAARs to exert an inhibitory effect in neurotransmission, they must be located within the plasma membrane, either at the synapse (synaptic) or away from the synapse (extrasynaptic). Given the need for fine-tuned control of inhibitory signaling, neurons possess numerous mechanisms that they use to dynamically regulate GABAARs at the cell surface , and the phosphorylation status of a particular subunit, determining whether they are recycled back to the cell surface membrane, or targeted to the proteasome for degradation , and Golgi glycoprotein 1 (Glg1). However, only the presence of CLPTM1 altered GABAergic currents, leading to decreased amplitude of GABAAR mediated currents, but having no effect on the frequency of currents. Importantly, by preventing the progression of GABAARs out of the ER, and to the plasma membrane, CLPTM1 modulates both tonic and phasic inhibition.Trafficking of GABAmembrane . The γ2 ilepsies . In facteticulum . Interessynapses . A tandeARs to the plasma membrane comes from proteins that play a role in promoting re-insertion of receptors that have undergone endocytosis . This important study also highlighted the specificity of the HAP1- GABAAR subunit interaction which occurs with β1, β2, or β3 subunits. Given that all GABAARs will have one of these β subunit subtypes, HAP1 could have an important role in the receptor recycling of most GABAARs in the nervous system  , leading to de-phosphorylation of key residues on the subunit. Due to this dephosphorylation event, the β3 is no longer protected from association with endocytic machinery, mainly the adaptor protein AP2 . Ultimat(IPSC’s) .AR endocytosis plays a critical role is in activity dependent trafficking, both under physiologic and pathologic conditions  , and even inhibitory synaptogenesis (One important setting in which GABAnditions . Perhaps(NMDARs) . Part ofogenesis .ARs at the cell surface. This receptor-dependent potentiation of inhibitory synaptic plasticity relied on NMDAR-mediated calcium influx, and the activation of Calcium/Calmodulin-dependent protein kinase II (CAMKII). The investigators also identified a necessary role for N-Ethylmaleimide sensitive factor (NSF), GABA Type A receptor-associated protein (GABARAP), and Glutamate receptor interacting protein (GRIP) in the insertion of new synaptic GABAARs in response to NMDAR activation , Chiu and colleagues identified a form of inhibitory potentiation at dendrites that occurs in response to calcium influx through NMDARs , was required, rather than simply CAMKIIa activation from any calcium source. A role for CAMKII in iLTP was also demonstrated in a 2014 report that focused on mechanisms leading to increased clustering and the stabilization of GABAARs at synapses, again in the setting of iLTP. The investigators found that increased clustering of GABAARs at synapses relies on CAMKII-mediated phosphorylation of Ser383 in the β3 subunit of GABAARs, leading to recruitment of gephyrin from extrasynaptic sites to synaptic. Here, gephyrin mediates not only the increased recruitment of GABAARs but their stabilization at synapses, playing an essential role in the long-term potentiation of inhibition   .in vivo studies in the lithium chloride-pilocarpine SE model report that phosphatase inhibitors prevent the activity-dependent trafficking of γ2 subunit-containing GABAARs away from the synapse, suggesting that indeed, calcineurin-dependent dephosphorylation of γ2 promotes dispersal of γ2-containing GABAARs with increased neuronal activity , glycogen synthase kinase 3 Beta (GSK3B), and cyclin-dependent kinase 5 (CDK5). Many of the exogenous factors that disrupt gephyrin clustering likely act through these pathways, including estradiol/estrogen and brain-derived neurotrophic factor (BDNF) . Given tsynapses . In a reassembly .ARs at the synapse, as gephyrin-independent clustering of γ2-containing GABAARs has been identified. In a search for potential auxiliary subunits associated with GABAARs, like those previously seen and defined at excitatory synapses, lipoma HMGIC fusion partner-like 3 and 4 [referred to as the GABAAR regulatory LFRPL (GARLH family proteins that contain LH3- and LH4-like protein)] were identified as putative auxiliary GABAAR subunits that act as alternative scaffolding molecules at GABAergic synapses , serves to stabilize GABAARs at synapses by interacting with the γ2 subunit, in association with the GARLH protein LH4-like (see above) that helps to stabilize γ2-containing GABAARs at the synapse  leads to a decrease in gephyrin clustering , a protein involved in stabilization of extrasynaptic GABAantile 9 . PCDH19 currents .ARs in brain inhibition is influenced not only by their presence at the plasma membrane, as opposed to the abundant intracellular pool of GABAAR subunits, but critically whether they are located synaptically or extrasynaptically. Most importantly, there is a ready exchange between synaptic and extrasynaptic sites for receptors via lateral diffusion  could be found at the synapse . However, certain receptor subunit combinations were found more often at the synapse than others and intra-hippocampal kainic acid injection (IHKA) SE mouse models.In addition to subunit composition, specific interacting partners of GABApartment . Also imraplegia . In theiARs, it resulted in greater localization of these receptors to the postsynaptic membrane. The functional response to altering protrudin levels likely stems from its role in exocytosis, potentially through its physical interactions with the GABAAR and GABARAP . Using drugs that selectively block tonic inhibition, these same investigators demonstrated that protrudin overexpression increased the amplitude of sIPSC’s in both tonic and fast synaptic signaling. Additionally, they found that while protrudin did not impact overall levels of β2 or β3-containing GABA GABARAP . As discsynapses . In contmobility .ARs, some important progress has been made. In a study focusing on α5-subunit containing GABAARs that are normally located extrasynaptically, Torben and colleagues describe an anchoring protein, radixin, that typically serves to keep α5-containing GABAARs at the extrasynaptic membrane by preventing lateral diffusion. However, dephosphorylation of radixin, via Ras homologue family member A (RhoA)/Rho-associated protein kinase (ROCK)-mediated signaling disrupts the interaction between α5 and radixin, allowing α5-containing receptors to diffuse to the synapse as needed . The role of Shisa7 at inhibitory synapses was discovered incidentally when authors were trying to study the role of Shisa6–9 at excitatory synapses , and the ultimate properties and localization of their resulting ligand-gated channels. However, even after the synthesis of new GABAAR subunits, there are several additional layers of regulation at play that influence their ultimate functional contribution, and thus whether alterations in the transcript levels of unique GABAAR subunits will ultimately impact inhibitory transmission of the nervous system is still an active area of investigation.While the most dynamic regulation of inhibitory signaling in the brain may occur via a change in the trafficking, stabilization, and/or localization of GABAARs can play a critical role in the localization, and thus the resulting function of those receptors. Synaptic receptors are capable of mediating fast phasic synaptic transmission, whereas generation of additional receptors that localize to extrasynaptic spaces, especially α5γ and α4δ-containing GABAARs, will alter tonic inhibition. On a more refined level, changes to the subunit makeup of GABAARs can influence what endogenous and exogenous modulators exert effects on the receptors, and even impact the dynamics of the resulting IPSC’s produced with opening of the chloride channels. For detailed review of the major effects of subunit composition on GABAAR function please see the earlier reviews of Rabow et al. and Fisher and Macdonald  were some of the earliest transcriptomic datasets to be examined in epilepsy, both in resected patient tissue and animal models. Since the identification of these changes nearly 3 decades ago, significant work has illuminated the intracellular pathways that associate with epileptogenesis. To date, a range of molecular pathways have been implicated in epilepsy, from the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathways to tropomyosin receptor kinase B (TrkB) and its neurotrophin brain derived neurotrophic factor (BDNF), REV-Erb alpha (Rev-Erbα), mammalian target of rapamycin (mTOR), among others  and subiculum in the long-term after SE, once spontaneous seizures arise in these models  model of SE-induced TLE in mice, and rats, differ in which GABAe models . Additioe models . In somee models . Alterate models , 2015. Re models . HoweverARs, along with clear evidence demonstrating alterations in mRNA levels and protein expression of numerous receptor subunits in animal and human TLE cases, an important area of past and current research exists for the development of new epilepsy therapeutics based on identifying the intracellular pathways that contribute to the genome expression of GABRs. Work from our own lab, in collaboration with our colleagues in the Brooks-Kayal lab, has helped to identify the intracellular signaling that leads to the transcriptional alterations in α1 and α4 gene expression in the context of epilepsy  through sequences upstream of the InR can be upregulated via the binding of the c-AMP response element binding protein (CREB) in response to protein kinase C signaling that solidifies its role in learning and memory, as well as in synaptic plasticity  can convert the “On” response to “Off”. Interestingly, this can occur in response to c-AMP signaling or in the case of BDNF, through a specific activation of the Janus Kinase (JAK)/signal transducer and activator of transcription (STAT) pathway that is dependent on TrkB (GABRA4p and GABAR subunit α4 transcript and protein levels through induction of early growth response factor 3 (EGR3) via MAPK signaling  within the intracellular domain of the α2 subunit of GABAARs that promotes interactions between the receptors and their binding partners, collybistin and gephyrin, that helps cluster GABAARs to produce effective inhibitory synapses  significantly decreased GABAergic neurotransmission at DIV8, whereas shRNA-KD of ST3 at DIV2 did not, suggesting that NLGN2 is essential at that time but ST3 is not. Later in development, elimination of either ST3 or NLGN2 at DIV10 significantly decreased mIPSCs at DIV18, representative of more mature neurons, and the effects of KO were additive. MAM Domain Containing Glycosylphosphatidylinositol Anchor proteins (MDGAs), on the other hand, inhibit the role of NLGN2 in forming inhibitory synapses, as KD of these molecules in the adult brain led to increased inhibitory synapse formation and upregulation decreased the number of inhibitory synapses  serving as a key transcription factor that is responsive to local neuron activity levels and plays a role in determining the number of functional inhibitory synapses that are formed . AnotherWhen it comes to the elimination of inhibitory synapses, microglia are a key cell type involved in the process. During development, microglia play an essential role in synaptic pruning, disruptions of which have been implicated in various neuropsychiatric disorders, including autism and schizophrenia. In addition to this developmental function, microglia continue to play a role in refinement of synaptic connections into adulthood . In orde+ interneurons in the hippocampus are particularly vulnerable to insults , and calretinin (CR), serve an important role as a buffering system for transient dysregulation in intracellular calcium levels. Thus, in the context of excessive excitatory signaling, calcium binding proteins that can buffer more calcium may provide added resilience against such insults when compared to the neuronal subtypes that primarily contain calcium binding proteins with lower buffering capacity. Of the 3 primary calcium binding proteins that are known to define distinct sets of inhibitory neurons in the hippocampus, PV has the lowest buffering capacity, while CR has the highest, which may contribute to the almost invariant vulnerability of PVs of TLE . And yets of TLE . While Ctermined . In addie points . In conte insult . Taken t+ neurons that may be particularly susceptible to degeneration in TLE. As for transcriptomic alterations, subsets of many broad classes of inhibitory neurons, defined by the authors using known subtype-specific expression markers derived from literature, including PV+ neurons expressing SULF1, PV+ neurons expressing nitric oxide synthase 1 (NOS1), vasoactive intestinal peptide positive neurons expressing cerebellin 1 precursor, SST+ neurons expressing tachykinin precursor 1, and inhibitor of DNA binding 2 positive neurons expressing lysosomal associated membrane protein family member 5 and NOS1, displayed dramatic shifts between control and TLE cases, providing more detailed information than was previously possible regarding GABAergic neurons and their signaling in the setting of human chronic TLE.With the advent and large-scale application of single cell technologies, future research will be poised to identify selectively vulnerable classes of inhibitory neurons, whose protection may offer a new therapeutic avenue in epilepsy . AdditioIn parallel with technological advances in single cell RNA-sequencing, recent advances in spatial transcriptomics are allowing researchers to capture spatially resolved single cell or even subcellular level transcriptomic and proteomic data. These spatially resolved techniques are a particularly valuable resource in epilepsy, as they will allow for an in-depth investigation of whole circuits that are disrupted and detail how alterations in one brain region may relate to those in another on an epigenetic, transcriptomic, and proteomic level. Spatial approaches that give context to transcriptomic findings may indeed provide important insights into the progression of epilepsy from the hyperactivity of neuronal subsets to a circuit level disorder in which epigenetic or genomic changes may drive epileptogenesis, providing new strategies for therapeutic intervention.ARs, the primary receptors responsible for synaptic and extrasynaptic neurotransmission, as well as the formation, maintenance, and elimination of inhibitory synapses in both health and disease. In part, this progress has been informed by the genomic revolution, which in the past two decades has allowed for identification of specific gene mutations in an increased number of individuals with genetic epilepsies, the exploration of which have propelled our understanding of GABAergic signaling forward. In the same vein, the single cell and spatial transcriptomic revolutions of the past five years have already begun to illuminate advancing complexity in the identity and function of cell types throughout diverse organs, including the brain, and offer a rich opportunity for enhancing our understanding of GABAergic neurons in the context of neural circuits that are dysregulated in a plethora of brain disorders, including epilepsy. The promise of multiomics to bring together the regulation of the genome with the functional proteomic landscape that is carved into the brain’s connectome will open new opportunities to rethink the development of curative therapies to restore the capacity of the brain for rebalance using its dynamic and complex inhibitory signaling properties.Significant progress has been made in the past three decades to elucidate the various cellular mechanisms that allow for fine grained control over inhibitory signaling within the CNS. Great strides have been made in our understanding of factors regulating the formation, properties, stability, and localization of GABAAT generated the original text for the document which was edited in collaboration with SR. AT generated the figures for general information to synthesize key concepts. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.