Oncogenic R248W mutation induced conformational perturbation of the p53 core domain and the structural protection by proteomimetic amyloid inhibitor ADH-6.

The involvement of p53 aggregation in cancer pathogenesis emphasizes the importance of unraveling the mechanisms underlying mutation-induced p53 destabilization. And understanding how small molecule inhibitors prevent the conversion of p53 into aggregation-primed conformations is pivotal for the development of therapeutics targeting p53-aggregation-associated cancers. A recent experimental study highlights the efficacy of the proteomimetic amyloid inhibitor ADH-6 in stabilizing R248W p53 and inhibiting its aggregation in cancer cells by interacting with the p53 core domain (p53C). However, it remains mostly unclear how R248W mutation induces destabilization of p53C and how ADH-6 stabilizes this p53C mutant and inhibits its aggregation. Herein, we conducted all-atom molecular dynamics simulations of R248W p53C in the absence and presence of ADH-6, as well as that of wild-type (WT) p53C. Our simulations reveal that the R248W mutation results in a shift of helix H2 and ß-hairpin S2-S2' towards the mutation site, leading to the destruction of their neighboring ß-sheet structure. This further facilitates the formation of a cavity in the hydrophobic core, and reduces the stability of the ß-sandwich. Importantly, two crucial aggregation-prone regions (APRs) S9 and S10 are disturbed and more exposed to solvent in R248W p53C, which is conducive to p53C aggregation. Intriguingly, ADH-6 dynamically binds to the mutation site and multiple destabilized regions in R248W p53C, partially inhibiting the shift of helix H2 and ß-hairpin S2-S2', thus preventing the disruption of the ß-sheets and the formation of the cavity. ADH-6 also reduces the solvent exposure of APRs S9 and S10, which disfavors the aggregation of R248W p53C. Moreover, ADH-6 can preserve the WT-like dynamical network of R248W p53C. Our study elucidates the mechanisms underlying the oncogenic R248W mutation induced p53C destabilization and the structural protection of p53C by ADH-6.

Influence mechanism of awns on wheat grain Pb absorption: Awns' significant contribution to grain Pb was mainly originated from their direct absorption of atmospheric Pb at the late grain-filling stage.

The spike is the organ that contributes the most to lead (Pb) accumulation in wheat grains. However, as an important photosynthetic and transpiration tissue in spike, the role of awn in wheat grain Pb absorption remains unknown. A field experiment was conducted to investigate the influence mechanism of awn on grain Pb accumulation through two comparative treatments: with and without awns (de-awned treatment). The de-awned treatment decreased wheat yield by 4.1 %; however, it significantly lowered the grain Pb accumulation rate at the late filling stage (15 days after anthesis) and led to a 22.8 % decrease in grain Pb concentration from 0.57 to 0.44 mg·kg-1. Moreover, the relative contribution of awn-to-grain Pb accumulation gradually increased with the filling process, finally reaching 26.6 % at maturity. In addition, Pb isotope source analysis indicated that the Pb in the awn and grain mainly originated from atmospheric deposition, and the de-awned treatment decreased the proportion of grain Pb from atmospheric deposition by 8.9 %. Microstructural observations further confirmed that the contribution of awns to grain Pb accumulation mainly originated from their direct absorption of atmospheric Pb. In conclusion, awns play an important role in wheat grain Pb absorption at the late grain-filling stage; planting awnless or short-awn wheat varieties may be the simplest and effective environmental management measure to reduce the health risks of Pb in wheat in regions with serious atmospheric Pb contamination.

Mechanistic insight into the destabilization of p53TD tetramer by cancer-related R337H mutation: a molecular dynamics study.

The p53 protein is a tumor suppressor crucial for cell cycle and genome integrity. In a very large proportion of human cancers, p53 is frequently inactivated by mutations located in its DNA-binding domain (DBD). Some experimental studies reported that the inherited R337H mutation located in the p53 tetramerization domain (p53TD) can also result in destabilization of the p53 protein, and consequently lead to an organism prone to cancer setup. However, the underlying R337H mutation-induced structural destabilization mechanism is not well understood. Herein, we investigate the structural stability and dynamic property of the wild type p53TD tetramer and its cancer-related R337H mutant by performing multiple microsecond molecular dynamics simulations. It is found that R337H mutation destroys the R337-D352 hydrogen bonds, weakens the F341-F341 π-π stacking interaction and the hydrophobic interaction between aliphatic hydrocarbons of R337 and M340, leading to more solvent exposure of all the hydrophobic cores, and thus disrupting the structural integrity of the tetramer. Importantly, our simulations show for the first time that R337H mutation results in unfolding of the α-helix starting from the N-terminal region (residues 335RER(H)FEM340). Consistently, community network analyses reveal that R337H mutation reduces dynamical correlation and global connectivity of p53TD tetramer, which destabilizes the structure of the p53TD tetramer. This study provides the atomistic mechanism of R337H mutation-induced destabilization of p53TD tetramer, which might be helpful for in-depth understanding of the p53 loss-of-function mechanism.

Cytopathological characteristics of angioimmunoblastic T-cell lymphoma diagnosed by fine needle aspiration.

OBJECTIVE: To analyse the cytopathological features of fine needle aspiration (FNA) in angioimmunoblastic T-cell lymphoma (AITL) diagnostics. METHODS: Fine needle aspiration lymph node biopsy samples from 12 patients with AITL were collected at a single centre between January 2014 and December 2020. The clinical, cytological and histopathological characteristics were retrospectively analysed. RESULTS: Three male and six female patients with AITL who had a median onset age of 65 years (range 51-74 years) and a mean follow-up period of 29 months (range 12-47 months) were included. The FNA cytological and morphological analysis of the reactive lymph node background revealed diffusely distributed non-homogeneous mixed lymphocytes, including mature small lymphocytes, medium-sized lymphoid cells, immune cells, and plasma cells; some mixed eosinophils, macrophages, and an occasional mixture of visible and medium-sized lymphocytes and epithelioid cells were observed. Mitotically active lymphocytes and sporadic pigmented bodies were observed occasionally. An abnormal proliferation of follicular dendritic cells observed under the microscope is important for AITL diagnosis, and these cells are often distributed in a scattered pattern of small clusters with many nuclear morphologies. Branched capillaries are another important diagnostic clue. Two patients with AITL who achieved clinical remission after treatment experienced recurrence and were diagnosed using FNA and cell block immunohistochemistry. CONCLUSIONS: Fine needle aspiration provides clues for the diagnosis of AITL in special clinical situations, and cell block immunohistochemistry is worthy of further exploration.

High mass loading NiCo-OH nanothorns coated CuO nanowire arrays for high-capacity nickel-zinc battery.

The rational design of cathode materials with core-shell heterostructures is significant to develop a Ni//Zn battery with both high gravimetric and areal energy density under high mass loading. In this work, the NiCo-OH nanothorns with a mass loading of 11.6 mg cm-2were coated on CuO nanowire arrays via a chemical bath deposition method. Thanks to the construction of 3D core-shell nanowire arrays and appropriate cobalt doping, as-fabricated Ni//Zn battery based on the NiCo-OH as cathode achieved the maximum specific capacity of 383 mAh g-1at 5 mA cm-2with high energy density of 649 Wh kg-1at 0.73 kW kg-1, indicating good energy storage performance in Ni//Zn battery.

Remarkable enhancement in the electrochemical properties of cosmetic brush-like Co3O4 nanowires via in situ surface modification with Ni2.

Cosmetic brush-like Co3O4 nanowires grown on carbon cloth were prepared by regulating the addition of trisodium citrate by hydrothermal synthesis. Then a simple liquid-phase ion exchange was carried out to improve the electrochemical performance of Co3O4 by surface modification with Ni2+. After surface modification, at a current density of 4 A g-1, the specific capacity significantly increased from 276.1 C g-1 (720.5 F g-1) to 655.9 C g-1 (1525.4 F g-1). Meanwhile, the electrocatalytic oxidation performances of methanol and urea were also improved significantly. The enhanced electrochemical properties were attributed to the modification of the surface of Co3O4 with Ni2+.

Fabrication of carbon cloth supporting MnO x and its application in Li-O2 batteries.

High-efficiency and low-cost electrocatalysts are generally believed to be the critical factor and have been highly researched to catalyze the oxygen reduction reaction (ORR) during the operation of Li-O2 battery (LOB). The catalysts with better ORR performance are essential for high-performance LOBs. Herein, a binder-free MnO x @carbon cloth cathode composed of Mn3O4 nanoparticles and Mn2O3 nanosheets were directly synthesized on the carbon cloth by electrodeposition and subsequently heat treatment at different temperature (from 200 °C to 400 °C). With the increase of temperature, the Mn3O4 nanospheres gradually transformed into Mn2O3 nanosheets. The MnO x obtained at 350 °C exhibited the best ORR performance. And MnO x -350 °C could operate more than 80 cycles at 340 mA g-1 with a limiting specific capacity of 1000 mAh g-1, and its first discharge specific capacity could nearly achieve 8000 mAh g-1 at 200 mA g-1.

Development of SNP, KASP, and SSR Markers by BSR-Seq Technology for Saturation of Genetic Linkage Map and Efficient Detection of Wheat Powdery Mildew Resistance Gene Pm61.

The gene Pm61 that confers powdery mildew resistance has been previously identified on chromosome arm 4AL in Chinese wheat landrace Xuxusanyuehuang (XXSYH). To facilitate the use of Pm61 in breeding practices, the bulked segregant analysis-RNA-Seq (BSR-Seq) analysis, in combination with the information on the Chinese Spring reference genome sequence, was performed in the F2:3 mapping population of XXSYH × Zhongzuo 9504. Two single nucleotide polymorphism (SNP), two Kompetitive Allele Specific PCR (KASP), and six simple sequence repeat (SSR) markers, together with previously identified polymorphic markers, saturated the genetic linkage map for Pm61, especially in the proximal side of the target gene that was short of gene-linked markers. In the newly established genetic linkage map, Pm61 was located in a 0.71 cM genetic interval and can be detected in a high throughput scale by the KASP markers Xicsk8 and Xicsk13 or by the standard PCR-based markers Xicscx497 and Xicsx538. The newly saturated genetic linkage map will be useful in molecular marker assisted-selection of Pm61 in breeding for disease resistant cultivar and in its map-based cloning.

Highly active zinc-finger nucleases by extended modular assembly.

Zinc-finger nucleases (ZFNs) are important tools for genome engineering. Despite intense interest by many academic groups, the lack of robust noncommercial methods has hindered their widespread use. The modular assembly (MA) of ZFNs from publicly available one-finger archives provides a rapid method to create proteins that can recognize a very broad spectrum of DNA sequences. However, three- and four-finger arrays often fail to produce active nucleases. Efforts to improve the specificity of the one-finger archives have not increased the success rate above 25%, suggesting that the MA method might be inherently inefficient due to its insensitivity to context-dependent effects. Here we present the first systematic study on the effect of array length on ZFN activity. ZFNs composed of six-finger MA arrays produced mutations at 15 of 21 (71%) targeted loci in human and mouse cells. A novel drop-out linker scheme was used to rapidly assess three- to six-finger combinations, demonstrating that shorter arrays could improve activity in some cases. Analysis of 268 array variants revealed that half of MA ZFNs of any array composition that exceed an ab initio B-score cutoff of 15 were active. These results suggest that, when used appropriately, MA ZFNs are able to target more DNA sequences with higher success rates than other current methods.

Coupling sensor to enzyme in the voltage sensing phosphatase.

Voltage-sensing phosphatases (VSPs) dephosphorylate phosphoinositide (PIP) signaling lipids in response to membrane depolarization. VSPs possess an S4-containing voltage sensor domain (VSD), resembling that of voltage-gated cation channels, and a lipid phosphatase domain (PD). The mechanism by which voltage turns on enzyme activity is unclear. Structural analysis and modeling suggest several sites of VSD-PD interaction that could couple voltage sensing to catalysis. Voltage clamp fluorometry reveals voltage-driven rearrangements in three sites implicated earlier in enzyme activation-the VSD-PD linker, gating loop and R loop-as well as the N-terminal domain, which has not yet been explored. N-terminus mutations perturb both rearrangements in the other segments and enzyme activity. Our results provide a model for a dynamic assembly by which S4 controls the catalytic site.

Intracellular ß-nicotinamide adenine dinucleotide inhibits the skeletal muscle ClC-1 chloride channel.

ClC-1 is the dominant sarcolemmal chloride channel and plays an important role in regulating membrane excitability that is underscored by ClC-1 mutations in congenital myotonia. Here we show that the coenzyme ß-nicotinamide adenine dinucleotide (NAD), an important metabolic regulator, robustly inhibits ClC-1 when included in the pipette solution in whole cell patch clamp experiments and when transiently applied to inside-out patches. The oxidized (NAD(+)) form of the coenzyme was more efficacious than the reduced (NADH) form, and inhibition by both was greatly enhanced by acidification. Molecular modeling, based on the structural coordinates of the homologous ClC-5 and CmClC proteins and in silico docking, suggest that NAD(+) binds with the adenine base deep in a cleft formed by ClC-1 intracellular cystathionine ß-synthase domains, and the nicotinamide base interacts with the membrane-embedded channel domain. Consistent with predictions from the models, mutation of residues in cystathionine ß-synthase and channel domains either attenuated (G200R, T636A, H847A) or abrogated (L848A) the effect of NAD(+). In addition, the myotonic mutations G200R and Y261C abolished potentiation of NAD(+) inhibition at low pH. Our results identify a new biological role for NAD and suggest that the main physiological relevance may be the exquisite sensitivity to intracellular pH that NAD(+) inhibition imparts to ClC-1 gating. These findings are consistent with the reduction of sarcolemmal chloride conductance that occurs upon acidification of skeletal muscle and suggest a previously unexplored mechanism in the pathophysiology of myotonia.

Direct absorption of atmospheric lead by rapeseed siliques is the leading cause of seed lead pollution.

Rapeseed cultivation is a novel approach to safely utilizing lead (Pb) contaminated farmland. However, the mechanism of Pb absorption in seeds remains uncertain. A field experiment was conducted to explore this mechanism with two contrasting treatments: rapeseed exposed to atmospheric deposition and non-exposed treatment. Non-exposed treatment ultimately decreased Pb content in leaf, silique, and seed by 46.7%, 53.7%, and 53.6%, respectively. Sub-microstructure analysis further confirmed that rapeseed leaves and siliques could directly absorb atmospheric Pb. In addition, Pb isotope analysis indicates that atmospheric deposition is the primary source of silique and seed Pb. The root and silique organs had relative Pb contributions of 28.0% and 72.0%, respectively, to seed. Thus, the direct absorption of atmospheric Pb by siliques during the filling stage was found to be the leading cause of seed Pb pollution.

Elucidating the Mechanisms of R248Q Mutation-Enhanced p53 Aggregation and Its Inhibition by Resveratrol.

Aggregation of p53 mutants can result in loss-of-function, gain-of-function, and dominant-negative effects that contribute to tumor growth. Revealing the mechanisms underlying mutation-enhanced p53 aggregation and dissecting how small molecule inhibitors prevent the conversion of p53 into aggregation-primed conformations are fundamentally important for the development of novel therapeutics for p53 aggregation-associated cancers. A recent experimental study shows that resveratrol (RSV) has an inhibitory effect on the aggregation of hot-spot R248Q mutant of the p53 core domain (p53C), while pterostilbene (PT) exhibits a relatively poor inhibitory efficacy. However, the conformational properties of the R248Q mutant leading to its enhanced aggregation propensity and the inhibitory mechanism of RSV against p53C aggregation are not well understood. Herein, we performed extensive all-atom molecular dynamics simulations on R248Q p53C in the absence and presence of RSV/PT, as well as wild-type (WT) p53C. Our simulations reveal that loop L3, where the mutation resides, remains compact in WT p53C, while it becomes extended in the R248Q mutant. The extension of loop L3 weakens the interactions between loop L3 and two crucial aggregation-prone regions (APRs) of p53C, leading to impaired interactions within the APRs and their structural destabilization as well as p53C. The destabilized APRs in the R248Q mutant are more exposed than in WT p53C, which is conducive to p53C aggregation. RSV has a higher preference to bind to R248Q p53C than PT. This binding not only stabilizes loop L3 of R248Q mutant to its WT-like conformation, preventing L3-extension-caused APRs' destabilization but also reduces APRs' solvent exposure, thereby inhibiting R248Q p53C aggregation. However, PT exhibits a lower hydrogen-bonding capability and a higher self-association propensity, which would lead to a reduced p53C binding and a weakened inhibitory effect on R248Q mutant aggregation. Our study provides mechanistic insights into the R248Q mutation-enhanced aggregation propensity and RSV's potent inhibition against R248Q p53C aggregation.

The Value of Immunocytochemical Staining for the HPV E7 Protein in the Diagnosis of Cervical Lesions.

Purpose: To investigate the value of immunocytochemical (ICC) staining for human papillomavirus (HPV) E7 protein (E7-ICC staining) as a new-generation immunological method in the cytological diagnosis of cervical lesions. Methods: The exfoliated cervical cell samples of 690 women were subjected to a liquid-based cytology test (LCT), high-risk HPV (HR-HPV) test, E7-ICC staining, and cervical biopsy for pathological diagnosis. Results: E7-ICC staining as a preliminary screening scheme for cervical precancerous lesions was comparable to the HR-HPV test in sensitivity and to the LCT in specificity. E7-ICC staining was advantageous in facilitating the secondary triage of HR-HPV-positive patients; therefore, this method can be used as an auxiliary scheme to routine LCT for diagnostic grading to improve the accuracy of cervical cytology. Conclusion: E7-ICC staining as a primary or auxiliary cytological screening scheme can effectively reduce the colposcopy referral rate.

Application of Big Data Technology and Visual Neural Network in Emotional Expression Analysis of Oil Painting Theme Creation in Public Environment.

With the progress of science and technology and the arrival of the big data era, people increasingly rely on computers to deal with daily life and related affairs. In recent years, machine learning has become more and more popular and has achieved good results in some fields, which also makes machine learning widely used. Among them, visual neural network technology can more intelligently analyze the emotional expression of oil painting, which is one of the current research hotspots, involving machine vision, pattern recognition, image processing, artificial intelligence, and other fields. However, in the art field, oil painting is still very different from other images. At present, there is no deep learning algorithm to identify the application of emotional expression analysis in oil painting theme creation. This paper will start with the neural network algorithm and combine the big data recognition technology to analyze the emotional expression of the oil painting subject in the public environment and establish the emotional expression analysis model of oil painting creation based on big data and neural network. The experiment shows that the graphics synthesized by this model have high resolution and good definition, but the speed is slow in the process of experimental operation. It takes about one hour to complete a round of image optimization.

Hernioscopy Revealing Rare Abdominal Cocoon Syndrome in an Elderly Patient: A Novel Technique for Abdominal Pathology.

BACKGROUND Incarcerated inguinal hernias (IGHs) combined with abdominal cocoons (ACs) are uncommon in adults. Abdominal cavity exploration using laparoscopy via the hernial sac (hernioscopy) has rarely been reported. Here, an elderly man with unilateral IGH complicated by a contralateral inguinal hernia and AC was found using hernioscopy. We present the surgical decision-making points in an elderly patient with IGH, enrich the diversity of AC, and propose a relatively novel hernioscopy approach. CASE REPORT A 90-year-old man presented with chronic constipation and reducible right inguinal masses. A lump in the right groin was strangulated for 2 days, accompanied with progressive abdominal pain, distension, and vomiting. The levels of inflammatory markers were elevated. Abdominal computed tomography revealed a dilated small bowel and a large mass in the right groin. Subsequently, the patient's condition quickly deteriorated. Therefore, he underwent surgical repair of bilateral hernias. Additionally, in our hospital, a total of 46 patients underwent hernioscopy because of IGH. No intraoperative or postoperative complications were observed. According to our clinical practice, hernioscopy via the bilateral hernial sacs was performed in this elderly patient. We found that almost the entire small bowel and colon were encapsulated in a fibrous and cocoon-like membrane, which postoperative pathological results revealed as AC. CONCLUSIONS This is the first report to reveal that AC complicated with IGH could occur in a 90-year-old man. Hernioscopy is a relatively novel and safe surgical approach to abdominal pathology associated with incarcerated or strangulated inguinal hernias. It is rarely used in adults with IGH.

A Novel In Vitro Platform Development in the Lab for Modeling Blast Injury to Microglia.

Traumatic brain injury (TBI), which is mainly caused by impact, often results in chronic neurological abnormalities. Since the pathological changes in vivo during primary biomechanical injury are quite complicated, the in-depth understanding of the pathophysiology and mechanism of TBI depends on the establishment of an effective experimental in vitro model. Usually, a bomb explosive blast was employed to establish the in vitro model, while the process is complex and unsuitable in the lab. Based on water-hammer, we have developed a device system to provide a single dynamic compression stress on living cells. A series of amplitude (∼5.3, ∼9.8, ∼13.5 MPa) were generated to explore the effects of dynamic compression loading on primary microglia within 48 h. Apoptosis experiments indicated that primary microglia had strong tolerance to blast waves. In addition, the generation of intercellular reactive oxygen species and secretory nitric oxide was getting strongly enhanced and recovered within 48 h. In addition, there is a notable release of pro-inflammatory cytokine by microglia. Our work provides a reproducible and peaceable method of loading single dynamic compression forces to cells in vitro. Microglia showed an acute inflammatory response to dynamic loadings, while no significant cell death was observed. This insight delivers a new technological approach that could open new areas to a better understanding of the mechanism of cell blast injuries.

The risk factors of early hemorrhage after emergency intravenous thrombolysis in patients with acute ischemic stroke.

BACKGROUND: At present, the treatment of acute ischaemic stroke (AIS) by aticepase (rt-PA) in emergency veins has become the main treatment mode in hospital, but the research on early hemorrhage complications in patients with emergency thrombolysis is rarely reported. This research aims to study the earlier warning index of early hemorrhage complications in patients with emergency thrombolysis. METHODS: A retrospective analysis was performed on the clinical data of rt-PA intravenous thrombolysis-treated AIS patients in the advanced stroke center of the emergency department of a tertiary grade hospital from January 2018 to May 2020. Patients were divided into a hemorrhage group and non-hemorrhage group according to the hemorrhage situation within 24 hours after thrombolytic therapy. The differences between the 2 groups in terms of pre-thrombolysis risk factors were analyzed. Logistic regression analysis was used to analyze the independent risk factors associated with post-thrombolysis hemorrhage. RESULTS: After intravenous thrombolysis, the hemorrhage group had 91 cases and the non-hemorrhage group had 146 cases. Logistic regression analysis showed that atrial fibrillation, systolic blood pressure before thrombolysis, platelet count, and antiplatelet drugs were independent risk factors for hemorrhage after intravenous thrombolysis (P<0.05). CONCLUSIONS: Patients with AIS have a higher incidence of hemorrhage after intravenous thrombolysis. Atrial fibrillation, systolic blood pressure before thrombolysis, platelet count, and antiplatelet drugs were independent risk factors for hemorrhage after intravenous thrombolysis. These independent risk factors can provide a basis for clinical nurses to evaluate hemorrhage risk in AIS patients after intravenous thrombolysis.

Proton-dependent inhibition, inverted voltage activation, and slow gating of CLC-0 Chloride Channel.

CLC-0, a prototype Cl- channel in the CLC family, employs two gating mechanisms that control its ion-permeation pore: fast gating and slow gating. The negatively-charged sidechain of a pore glutamate residue, E166, is known to be the fast gate, and the swinging of this sidechain opens or closes the pore of CLC-0 on the millisecond time scale. The other gating mechanism, slow gating, operates with much slower kinetics in the range of seconds to tens or even hundreds of seconds, and it is thought to involve still-unknown conformational rearrangements. Here, we find that low intracellular pH (pHi) facilitates the closure of the CLC-0's slow gate, thus generating current inhibition. The rate of low pHi-induced current inhibition increases with intracellular H+ concentration ([H+]i)-the time constants of current inhibition by low pHi = 4.5, 5.5 and 6 are roughly 0.1, 1 and 10 sec, respectively, at room temperature. In comparison, the time constant of the slow gate closure at pHi = 7.4 at room temperature is hundreds of seconds. The inhibition by low pHi is significantly less prominent in mutants favoring the slow-gate open state (such as C212S and Y512A), further supporting the fact that intracellular H+ enhances the slow-gate closure in CLC-0. A fast inhibition by low pHi causes an apparent inverted voltage-dependent activation in the wild-type CLC-0, a behavior similar to those in some channel mutants such as V490W in which only membrane hyperpolarization can open the channel. Interestingly, when V490W mutation is constructed in the background of C212S or Y512A mutation, the inverted voltage-dependent activation disappears. We propose that the slow kinetics of CLC-0's slow-gate closure may be due to low [H+]i rather than due to the proposed large conformational change of the channel protein. Our results also suggest that the inverted voltage-dependent opening observed in some mutant channels may result from fast closure of the slow gate by the mutations.