Escherichia coli we develop a robust method for neutralizing technical biases (e.g. base specific RNase preferences in ribosome-protected mRNA fragments (RPF) generation), which allows for correct estimation of translation times at single codon resolution. Furthermore, we validated the method with available datasets from E. coli treated with antibiotic to inhibit isoleucyl-tRNA synthetase, and two datasets from Saccharomyces cerevisiae treated with two RNases with distinct cleavage signatures. We demonstrate that our approach accounts for RNase cleavage preferences and provides bias-corrected translation times estimates. Our approach provides a solution to the long-standing problem of extracting reliable information about peptide elongation times from highly noisy and technically biased ribosome profiling spectra.Ribosome profiling spectra bear rich information on translation control and dynamics. Yet, due to technical biases in library generation, extracting quantitative measures of discrete translation events has remained elusive. Using maximum likelihood statistics and data set from  Ribosome profiling (or Ribo-Seq) couples cell-wide profiling of the positions of translating ribosomes on messenger (mRNA) at single codon resolution  with deeCodon resolution of the RPF spectra is generally higher in eukaryotes than in bacteria. In eukaryotes, RNase I is the nuclease of choice and it cleaves precisely at ribosome boundaries . RNase IAn additional source of bias in the Ribo-Seq libraries is the local RPF sequence composition including high propensity for secondary structure formation for some RNA fragments which can interfere with the reverse transcription priming and/or with the adaptor ligation ,12. AtteIn the present work, we develop a model that accounts for the local codon context-dependent variation of peptide elongation times and RPF generation/processing biases. In total, we use 915 context-defining parameters, which are estimated by fitting the model-predicted RPF spectra to the experimental, transcriptome-wide RPF spectra using non-linear regression with maximum likelihood (ML) statistics. We also consider ribosome profiling spectra at single nucleotide resolution with homogenous fragment size to identify and neutralize RPF generation/processing biases near the 5′- and 3′-fragment ends. Our results suggest that an inner local context of five codons, including those at the A, P and E sites, accounts for the ribosomal dwell time on each A-site codon of the transcriptome. This determination of the peptide elongation times provides a basis for a detailed understanding of the dynamics of protein synthesis in living cells.Escherichia coli B strain AS19 was grown in LB medium until the culture reached an OD600 of 0.5. Cells were harvested by flash freezing and libraries from biological replicates were prepared for ribosome profiling by direct ligation of the platform-specific sequences or adapters as described , sequencing adapters were cut using cutadapt (1.8.3); minimal overlap: (1 nt) and uniquely mapped to the E. coli genome  using Bowtie (1.2.2) with parameters -l 16 -n 1 -e 50 -m 1—strata—best y. The RPF counts for each ORF were normalized per total mapped reads per million (RPM)   and calion (RPM) . The datj of the open reading frame from gene i, ORFi. The detected number of RPFs, i,j) we write as:Each RPF is assigned to a codon position ibraries ,12,14,20i,j), i in the cell population and proportional to the ORFi expression level, j in ORFi and reflects the extent of digestion/processing/ amplification biases in Ribo-Seq library preparation. We note that Here, j but is proportional to the peptide elongation cycle time: Each elongation time mentclass2pt{minimmentclass2pt{minimi and i and the expected peptide elongation cycle time j in transcript i. The major methodological task is, therefore, to elicit reliable estimates of the expected values, i, j) from the experimental sets of the sampled i,j) is A is the PCR copy number amplification factor and q is the fraction of the amplified library that has been finally sequenced, the Neyman type A distribution is close to Poisson but with variance equal to the expected value  fragment to be of Poisson type:Here, parameter rst-like  and the L for the whole transcriptome based on all We ascribe a log-likelihood function j in ORFi and that this context is composed of Lp, codons with the A site at its near middle position  among the total number Lp of context defining positions contributes with a factor Each local codon context position (c) of each one of the 64 possible codons at each position p  factors E. coli transcriptome may contain up to 1.8 × 106 distinct contexts (about 6000 ORFs and 300 codons per ORF), and the ultimate number of contexts for which 1527. In the next section, we describe how model parameters are derived from experimental data by maximizing a transcriptome-wide log likelihood function.Each factor mentclass2pt{minimmentclass2pt{minimL for the RPF spectrum takes the simple form   \documenquations ,22 to obquations  and (9) Instead of comparing experimental , each product c in the inner region of ORFi. Hence, each c in the transcriptome (Supplementary Text).Since mentclass2pt{minimEquation  varies lc at local context position p. It is defined as the standard deviation, p of the table of We also introduce the ‘sensitivity parameter’ Equation  for row Equation .j in ORFi and estimate its expected value, In order to estimate the fragment processing bias  at transcriptome position ; NLx4 table; the local nucleotide position p is counted from p= 1, via the first nucleotide at A-site position NAp = p to the third base of the last codon of the local context sequence of length NLp j in Equangth pNL .Parameters x to be the distance from the first A-site nucleotide to the 3′-end of the RPF in nucleotides, it follows that FL with nucleotide ‘b’ at 5′- and 3′-end, respectively. By applying the same ML procedure as in the codon-resolution case, we solve Equation Assuming mentclasspt{minimaTo separate the effects of bias and peptide elongation time variations on the RPF counts, we partition the context dependent factors p from 1 to 1p–1 and from 2p+1 to Lp, mainly account for the nuclease digestions/processing biases (B). The inner context dependent factors p (from 1p to 2p), mainly reflect the variation of the peptide elongation time, hence superscript (T) in As shown in Results, the outer context dependent factors relative peptide elongation time as the ribosome moves along an ORFi  translation  summarized in Equation j, so that j in ORFi  at local sequence context position (p)  near the lagging (p = 4) and leading (p = 11) ribosome edges than in A site (p = 8)  than at ribosomal A, P or E site   Figure .j of gene i. The x) from the 3′-end to the first A-site nucleotide of an RPF is constant  with the A-site codon at position Ap = 8  with the first A-site nucleotide at position NAp = 22  at positions 10, 9 and 8 for FL = 23  p equal to 10, 9 and 8 correspond to 5′- ends of the FL = 23, 24 or 25 nts fragments, respectively, implying low abundance of RPFs with G/C at their 5′-ends. Indeed, experimental RPFs with an A at their 5'- end are about 60-fold more abundant than experimental RPFs with a G at the 5′- end, in line with the previous report on strong preference of MNase to cleave before an A or a U  approach to estimate the local context factors 3 Figure , FL = 243 Figure  and FL =3 Figure , respectEquation ) moves eEquation . Irrespe1 Figure , the \do 3′-bias . The gren Figure  has a smThe strong effects of codon identities at position 11 (leading edge of the ribosome) on s Figure  can now 1 Figure , exactly1 Figure . The sam1 Figure  explainsp = 1 to 1–1p and from 2+1p to Lp , P (p = 7) or E site (p = 6) were relatively large and contributed to slow peptide elongation , P p =  or E sit), P p =  or E sit), P p =  or E sits Figure . This, w), P p =  or E sitGly3) contributed to slow peptide elongation  as exemplified in Figure r = 0.77) with similar features  when rear codons (i.e. with frequency < 0.3%) were excluded. This, we suggest, reflects the similarity of the effects of a particular A-site codon on the codon translation time in both data sets. For P and E sites, the correlation between r = 0.7). As expected, the correlation between r < 0.25), which reflects the distinct sequence preferences of the nucleases in RPF generation  than the ‘five-inner model’ for the correlation between the r-frequency profile). An intuitive explanation for this result is that in the ‘five inner model’ the five Lp = 15 local positions for modeling We wish to emphasize that to obtain the bias-corrected pausing scores Equation , we used earlier  using ne earlier , we restrevisiae  processeEquation , thus leapproach , reducesin vitro biochemistry will deepen our understanding of how the intracellular kinetic networks of mRNA translation shape the life sustaining phenotypes of living cells. In the present work, we join the ongoing and rapidly growing efforts to establish genome-wide technologies  under accession number GSE145571. Two published data sets (E. coli MG1655 and GSE 82220 (GSM2186726 and GSM2186728) for yeast. All scripts and source code for modeling and calculating the parameters used here are deposited in https://github.com/gustafGitHub/RiboTimes.The sequencing data for ata sets ,19 analygkab260_Supplemental_FileClick here for additional data file.


Neurophysiological experiments using transcranial magnetic stimulation (TMS) have sought to probe the function of the motor division of the corpus callosum. Primary motor cortex sends projections via the corpus callosum with a net inhibitory influence on the homologous region of the opposite hemisphere. Interhemispheric inhibition (IHI) experiments probe this inhibitory pathway. A test stimulus (TS) delivered to the motor cortex in one hemisphere elicits motor evoked potentials (MEPs) in a target muscle, while a conditioning stimulus (CS) applied to the homologous region of the opposite hemisphere modulates the effect of the TS. We predicted that large CS MEPs would be associated with increased IHI since they should be a reliable index of how effectively contralateral motor cortex was stimulated and therefore of the magnitude of interhemispheric inhibition. However, we observed a strong tendency for larger CS MEPs to be associated with reduced interhemispheric inhibition which in the extreme lead to a net effect of facilitation. This surprising effect was large, systematic, and observed in nearly all participants. We outline several hypotheses for mechanisms which may underlie this phenomenon to guide future research.The online version contains supplementary material available at 10.1007/s00221-021-06183-9. A population of neurons in the primary motor cortex has axons that project across the corpus callosum to excite local inhibitory circuits in the opposite hemisphere with a net inhibitory influence on the homologous region of the brain. This pathway is the target for a developing field of brain stimulation treatments for conditions such as unilateral stroke in which the damaged hemisphere is further impaired by excessive inhibition from the healthy hemisphere  by applying a conditioning stimulus (CS) to primary motor cortex in one hemisphere prior to applying a test stimulus (TS) in the opposite hemisphere. The conditioning stimulus reduces the magnitude of the motor evoked potentials (MEPs) from the test stimulus. This reduction in the size of the MEPs is a measure of interhemispheric inhibition . Exploratory analyses of these observations suggested that there may be an association between the size of the MEP produced by the conditioning stimuli (CS MEPs) and the degree of IHI. This observation appeared to be paradoxical; although stronger conditioning stimulus intensities increased IHI, larger CS MEPs were associated with reduced IHI contrary to expectations. We, therefore, conducted a confirmatory experiment in which MEPs were measured from both hands to determine whether this paradoxical effect of facilitation was reliable.Twenty-One participants  were recruited from the Bloorview Research Institute. The one left-handed participant was not an outlier in any analysis. All participants were neurologically healthy adults who gave written informed consent. Prior to the study, all participants completed a TMS safety screening form  were placed on the belly of the first dorsal interosseous (FDI) muscle and at the metacarpal-phalangeal joint of the index finger of both the left and right hands. A common ground was placed on the left ulnar styloid process. Biofeedback was continuously available to participants via an electromyogram (EMG) display. The EMG data were collected using the BrainSight™ software  paired with the built-in, 2-channel EMG device. EMG signals being obtained from FDI muscles of both hands were baseline autocorrected prior to the start of each session. Live signals were amplified using the SENS-002-001 Model 2 amplifier incorporated into the BrainSight™ suite. This hardware applied a bandpass filter of 16–470 Hz. During magnetic stimulations, participants were instructed to flex the distal interphalangeal joint of the index finger against the thumb to produce pre-stimulation EMG activity of approximately 40 µV peak-to-peak.The Brainsight™ neuronavigation software , running on an iMac desktop computer (OS 10.13.4), was used to inform the positioning of the TMS coils. The ICBM152 MRI atlas brain .We defined an 8-by-6 point rectangular grid of neural targets over the dorsal half of peri-central cortex in both the left and right hemisphere. Targets were spaced 5 mm apart, less deformation of the rectangular grid to conform to the contour of the brain surface. Stimulation across grids with these features are reliable localizers for the motor regions that control the muscles of the hand  running Python (v2.7.1) extended with the MagPy package and connected to the stimulators by custom-built cables adapted from McNair  both FDI hand muscles pre-activated, (ii) both at rest, or (iii) pre-activated in the test hand only see Fig. . Blocks We fit linear mixed models (LMMs) in R . The residuals of models of MEP ratios deviated severely from the expected distribution indicating a violation of the assumption of normality, have non-constant variance across the range of fitted values indicating a violation of the assumption of homoscedasticity, and the relation between MEPs and MEP ratios were not constant across participants further complicating their interpretation. Together these issues present serious concerns for the accuracy of p-values derived from statistical tests on MEP ratios and the validity of the inferences that are made from them. We, therefore, report a parallel analysis in which inhibition was calculated as a linear subtraction between conditioned MEPs and the median of unconditioned MEPs.While the subtraction approach is less commonly applied in this field, it displayed few of the undesirable properties that were observed for analyses following the ratio method. The assumption of homogeneity was satisfied, although there was a minor deviation from the normality of residuals. Although LMMs are robust to minor violations of normality  for the test hemisphere and 53%MSO (11.5 SD) for the conditioning hemisphere. Mean unconditioned test MEPs were 219 (217 SD) μV, 455 (337 SD) μV, and 410 (320 SD) μV, respectively, with both hands at rest, only the test hand active, or both hands active, respectively.p = 0.0002, standardized estimate = 31.5; positive values indicate greater inhibition). The model selected for this analysis did not assess whether interhemispheric interval interacted with the configuration of the hands. The configuration of the hands affected interhemispheric inhibition . From the magnitude of the standardized estimates it is clear that this effect was considerably stronger than the effects of either interhemispheric interval or hand configuration. We refer to this effect as paradoxical facilitation because the CS did elicit inhibition, and yet the CS also produces facilitation in proportion to its effectiveness in driving its own target muscle . Paradoxical facilitation was observed less strongly when both hands where pre-activated (standardized estimate = 32.8), and when only the test hand was pre-activated (standardized estimate = 64.8) relative to both hands at rest. An examination of the random slopes of conditioning MEP with participants demonstrates that individual differences were comparatively small and paradoxical facilitation was observed in 19 of 21 participants . The configuration of the hands did not have a clear effect on inhibition ratios  with neither both hands pre-activated (standardized estimate = − 0.15) nor only the test hand pre-activated (standardized estimate = − 0.02) having notable effects on inhibition ratios relative to both hands at rest.A parallel analysis of ratio transformed data yielded qualitatively similar results, though with reduced statistical power and severe violations of common statistical assumptions (see S5). We observed a greater degree of inhibition for LIHI at 50 ms than SIHI at 10 ms . Paradoxical facilitation interacted with the configuration of the hands . Paradoxical facilitation was observed less strongly when both hands where pre-activated (standardized estimate = − 0.56), and when only the test hand was pre-activated (standardized estimate = − 0.52) relative to both hands at rest  indicated that while experimental parameters should be chosen based on the neurophysiological mechanisms under study  rather than the peripheral nervous system. Pre-contraction increased the frequency of high amplitude CS MEPs while reducing the slope of the facilitation effect. Previous studies have found that pre-activation of one hand increases IHI bidirectionally Supplementary file2 (DOCX 76 kb)Supplementary file3 (DOCX 184 kb)Supplementary file4 (DOCX 27 kb)Supplementary file5 (DOC 869 kb)Supplementary file6 (CSV 317 kb)Supplementary file7 (R 14 kb)Supplementary file8 (CSV 774 kb)Supplementary file9 (R 23 kb)Supplementary file10 (PY 11 kb)Supplementary file11 (R 15 kb)Supplementary file12 (R 7 kb)Supplementary file12 (RDATA 2595 kb)Below is the link to the electronic supplementary material.


Integrins are cellular receptors that regulate cell adhesion and many other cellular functions. Integrins can be activated via an “inside-out pathway” that is promoted by RAP1 GTPase. RAP1-GTP-Interacting Adaptor Molecular (RIAM) mediates integrin activation by linking RAP1 GTPase to talin, an integrin activator. RIAM’s function in integrin signaling is tightly regulated. In this commentary, we review recent studies of the molecular mechanisms underlying RIAM autoinhibition via both intramolecular interaction and oligomer assembly, and the phosphorylation-dependent activation of RIAM. In particular, β2 integrins are expressed exclusively on lymphocytes, whereas αIIbβ3 is strictly expressed on platelets [Extracellular matrix (ECM) surrounding cells in solid tissue provides mechanical support and communicates with cells through cell adhesion molecules (CAMs) at the cell surface ,2. Integlatelets ,5. Integlatelets . Indeed,latelets ,7.Conformational change of the ectodomains from a low-affinity binding state to a high-affinity binding state is a hallmark of integrin activation -10. Two 4β1), α5β1 and α6β1 of β1 integrins and LFA-1 (αLβ2) of β2 integrins [RIAM is abundantly expressed in leukocytes and is essential for the signaling of leukocyte-specific integrins. In particular, T cells highly express two types of β integrins that include VLA-4  in the PM via the PH domain [RIAM is a RAP1 effector and mediates the “inside-out” integrin activation by activating talin and recruiting it to the PM ,30. The H domain . RIAM alH domain .The structural properties and the resulting activity states of these signaling proteins are often controlled by various types of post-translational modifications (PTMs). Among all PTMs, phosphorylation is the most commonly observed event in the integrin signaling pathway. The earliest studies of phosphorylation events in the integrin signaling pathway can be traced back to the 1980s -36. It hLβ2 integrin) and triggers downstream signaling that activates Phosphoinositide-3 kinase (PI3-K) and Syk [FAK was known for its function of regulating signaling downstream of integrin activation. Integrin-mediated cell adhesion to ECM relieves FAK from autoinhibition, allowing phosphorylation of its activation loop ,41. Acti and Syk . In addi and Syk -48. Rece and Syk ,50.in vitro RAP1:RIAM interaction and the PM co-localization of RAP1 and RIAM. Bioinformatic analyses suggest that Tyr45 is a substrate of FAK kinase, which was further confirmed by phosphorylation analyses using cells treated with FAK-specific inhibitors [RIAM adapts an autoinhibitory conformation in which the IN segment binds to the RA domain. The IN:RA interface involves multiple side chain interactions that include Tyr45, Glu60, and Asp63 in the IN segment, and is further stabilized by the CC segment that resides on top of the IN segment . Both Glhibitors . It is nhibitors . We havehibitors .2 in the PM [2-binding site. This autoinhibitory interaction of the PH domain was first revealed by structural analyses and further validated by biochemical and functional characterization. Five crystal structures that contain the RA-PH module of RIAM have been reported [Besides the RA domain, the RAP1-dependent RIAM translocation to the PM also requires the PH domain to engage PIPreported . These ireported ,50,53. Treported ,55. Thesreported . Interesreported . RIAM di2-binding site in the dimer configuration of RIAM RA-PH results in the suppression of the PM translocation of RIAM. The two β strains of the neighboring PH domain that interact with the helical extension accommodate a positively charged patch composed of Lys327, Lys328, Lys331, Arg332, and Arg333 that engages with PIP2 in the PM in response to RAP1 activation [2-binding site needs to be unmasked for RIAM translocation to the PM. Indeed, RIAM-A435Y, the helical extension mutation that disrupts dimer assembly and unmasks the PIP2-binding site, exhibited enhanced PM translocation [IIbβ3 integrin. Similar to the RA domain autoinhibition, this dimerization-induced inhibition of PH: PIP2 interaction is also regulated by phosphorylation. RIAM dimerization is significantly reduced upon phosphorylation as indicated by the co-IP assay. Moreover, RIAM is highly phosphorylated during T cell activation, and the phosphorylation of RIAM is coupled with its PM translocation, consistent with the model that phosphorylation of RIAM disrupts RIAM dimerization, in turn promotes its association with the PM. Further analyses revealed that RA-PH module is the substrate of Src family kinases. An LCK inhibitor, but not FAK inhibitors, suppressed the phosphorylation of the RA-PH module of RIAM upon T cell activation [2-binding site in RIAM PH domain, thus promoting the PM translocation of RIAM and subsequent integrin activation [Masking of the PIPlocation . Consequtivation . The LCKtivation .2-binding site in the PH domain is regulated by homo-oligomerization and phosphorylation. Nevertheless, bioinformatic analyses of crystal structures of PH domains using ProtCID revealed that Lpd may also possess a similar oligomer assembly that masks the PIP2 binding site [In vitro kinase assays using synthetic peptides derived from Lpd suggested six tyrosines in the PH domain that were phosphorylated by both Abl and Src [2-binding site in Lpd may be regulated by a similar, phosphorylation-dependent mechanism. Moreover, Lpd’s function in cell motility is regulated by phosphorylation mediated by Abl and Src. Lpd interacts with both the EVH1 domain of Ena/VASP and Abi of the SCAR/WAVE complex via the C-terminal polyproline motifs. Although the binding specificity to EVH1 or Abi of these polyproline motifs remains unclear, the interaction of Lpd with Ena/VASP or with SCAR/WAVE complex is controlled by Lpd phosphorylation. Nonetheless, phosphorylation of Lpd by Src only promotes its association with the SCAR/WAVE complex, while phosphorylation of Lpd by Abl promotes the interaction of Lpd and Ena/VASP and its association with the SCAR/WAVE complex [in vitro kinase assays suggest similar phosphorylation patterns of Lpd in the C-terminal region by Abl and Src kinases [To our knowledge, this is the first example of such a regulatory mechanism of a PH domain, in which the exposure of PIPing site . The RA- and Src ,58. Stru and Src , support complex ,58. The  kinases ,58. In cTogether, these recent studies demonstrate that both the RA and PH domains of RIAM are autoinhibited for its adaptor function in mediating integrin activity, and phosphorylation of RIAM mediated by FAK and Src kinases regulates RIAM activity . Other aIn addition to PTMs, the function of the adaptors can also be modified prior to translation by RNA splicing, which often results in the loss of distinct functional modules in various isoforms. For instance, RIAM has a splicing isoform that lacks the PH domain and the C-terminal polyproline motifs , and Lpd


Continuous monitoring of ice cover belongs to the key tasks of modern climate research, providing up-to-date information on climate change in cold regions. While a strong advance in ice monitoring worldwide has been provided by the recent development of remote sensing methods, quantification of seasonal ice cover is impossible without on-site autonomous measurements of the mass and heat budget. In the present study, we propose an autonomous monitoring system for continuous in situ measuring of vertical temperature distribution in the near-ice air, the ice strata and the under-ice water layer for several months with simultaneous records of solar radiation incoming at the lake surface and passing through the snow and ice covers as well as snow and ice thicknesses. The use of modern miniature analog and digital sensors made it possible to make a compact, energy efficient measurement system with high precision and spatial resolution and characterized by easy deployment and transportation. In particular, the high resolution of the ice thickness probe of 0.05 mm allows to resolve the fine-scale processes occurring in low-flow environments, such as freshwater lakes. Several systems were tested in numerous studies in Lake Baikal and demonstrated a high reliability in deriving the ice heat balance components during ice-covered periods. Recently, the interest in the ice season has been remarkably increasing among limnologists ,2, causein situ autonomous continuous monitoring of vertical temperature distribution in the near-ice air, the ice strata and the under-ice water layer for several months with simultaneous registration of solar radiation incoming at the lake surface and passing through the snow and ice covers, under-ice current velocities and snow and ice thicknesses.Seasonal formation of ice is an essential feature of the hydrological regime in temperate and (sub-)polar climatic zones. In this regard, freshwater lakes are a special class of hydrological objects both in terms of thermal and hydrodynamic processes that control the formation and melting of ice and from the point of view of the impact of the ice regime on the global freshwater budget. Seasonally freezing lake systems—Lake Baikal, Laurentian Great Lakes, great European Lakes Ladoga and Onego and lake systems of Fennoscandia and Northern Canada—accumulate the bulk of the world’s surface freshwater. Their seasonal ice regime defines the climatic balance of precipitation and evaporation as well as the ecosystem state and the water quality of the lakes themselves ,4. A steThe first autonomous stations aimed at monitoring of ice properties were the so-called ice mass balance (IMB) buoys designed for marine conditions, among which there were the Cold Regions Research and Engineering Laboratory (CRREL) IMB buoy  and the Modernization of the SAMS IMB buoy resulted in the Snow Ice Mass Balance Array (SIMBA) buoy, which is currently widely used, in particular because of its cost-cutting design. The buoy does not have any built-in rangefinders, but possesses a dense spatial distribution of temperature sensors in its thermistor string, spaced every 2 cm. Additionally, small heaters are mounted underneath every temperature sensor, emitting heat impulses once per day for 60–120 s. The different heating rates of the sensors in the various environments help to distinguish the air/snow, air/ice and ice/water interfaces. Despite this approach, retrieval of snow depth and ice thickness from the SIMBA temperature profiles remains a non-trivial task requiring manual data processing, while several automated algorithms of snow and ice depth determination were recently proposed ,18. MostThe Polar Research Institute of China and Taiyuan University of Technology have developed their own ice-tethered buoys. This type of buoy is similar to SIMBA, and has a 4.5 m long thermistor string consisting of 150 sensors spaced every 3 cm. The buoys also include acoustic rangefinders in air and water . The buoDespite the advances in automated ice monitoring, all of these buoys provide relatively rough estimates of heat and mass balance. The temperature sensors used have a resolution of 0.0625 °C and initial accuracy of ±0.5 °C , which are insufficient to resolve the fine-scale processes occurring at the ice–water interface and beneath it where small vertical temperature gradients are crucial for the melting rate at the ice base. Moreover, the massive body of the SIMB-3 buoy (12 cm in diameter) has a significant impact on the surrounding temperatures. The underwater rangefinder used in the SIMB-3 also has a resolution of only 1 cm.The developers of the “Multilayer Sea Ice Temperature Sensor”  addresseThe prototype ice monitoring system of Aslamov et al.  has beenThe autonomous system for lake ice monitoring (ASLIM) is designed to record the vertical temperature distribution in the air, within the ice cover and sub-ice water layer together with short-wave solar radiation entering and passing through the snow and ice as well as the thickness of snow and ice. The 32-bit microcontroller PIC33EP256MU806 , a link between all elements of the system, determines the logic of the entire device.Ultra-low noise (4 https://hlserver.lin.irk.ru/shs/icemon/ (accessed on 16 December 2021)). It is also possible to download raw data in tabular format for further processing and analysis after authorization. An example of a webpage with previewed data is shown in The information collected by the ASLIM is transmitted via a GSM network to a remote Internet server with configurable periodicity, typically every hour. The server hosts a database and a dynamic webpage for online visualization of information from the network of ice measuring systems , through which the logger body is immersed. Two cuts from the hole at right angles are made with the help of an ice saw. In one cut the measurement system is inserted, and positioned so that the second light sensor from the top and the corresponding temperature sensor are aligned with the upper ice surface a,b. The To measure the ambient temperature, two types of sensors were used in the instrument.MAX31725 chips were chosen as the main temperature sensors. The chip is a miniature temperature sensor based on a P-N junction with integrated on-chip ADC, a digital core and It system , minimizt system .Additionally, fast-response analog temperature sensors were used in the range of the possible location of the ice–water interface to obtain higher resolution in temperature and space. Glass sealed semiconductor thermistors were used as sensing elements  with 1 mThe device has an adjustable burst mode for recording the readings of all low-inertia analog temperature sensors with a 1 Hz frequency. The data on high frequency and fine-scale temperature fluctuations can provide information on under-ice convection and the characteristics of turbulent mixing in the ice–water boundary layer as well as evaluate the dissipation rate of the turbulent kinetic energy (TKE) .The highly integrated optical sensors, MAX44006 and MAX44008 , were used to measure downward planar irradiance. These chips combine five types of high-sensitivity (down to 0.001 Lux) photodiodes, which can measure intensity in the red, green, blue and infrared spectrum  and also intensity of ambient light . A built-in temperature sensor is used to compensate for the influence of temperature changes on the illumination and infrared sensor readings. Six on-chip ADCs enable simultaneous measurement of signals in all channels. The sensor outputs via digital interface values calibrated in W/mollector . The othThe presence of light sensors in the air, ice and under-ice water allows for estimating the attenuation of solar radiation in the ice and the water column, whereas the use of information on the thickness of the snow enables studying its effect on the amount of downward solar radiation passing through the snow.R, from the ice surface. The essence of the hydroacoustic method is that a modulated acoustic signal is emitted into the water, then reflected from the ice bottom and recorded by the receiver. The distance to the ice–water interface L and, hence, the ice thickness, H, can be calculated from the information on the time elapsed between transmission and reception and the sound speed in water  S and hydrostatic pressure P. This dependence has been established experimentally and is represented by various empirical equations, in particular, the widely known Chen–Millero formula for freshwater [To accurately determine the distance to the ice bottom, it is necessary to know the sound speed profile in the water. The propagation of acoustic waves in a medium is determined by its elastic properties. Liquids and gases do not have shear elasticity. On the other hand, when two adjacent layers come together or move apart, return forces occur in them, preventing shear and tensile deformations. As the vibrations of the particles of the medium occur in the direction of the returning elastic forces, the propagation of the initial displacement front is possible only in the form of a longitudinal wave. The following expression determines the velocity of longitudinal waves, eshwater . In our eshwater , in whiceshwater ,31, whic10−6 K−1 , its relA lead zirconium titanate-based piezoelectric ceramic transducer in the form of a 20 mm diameter disc with a 6 mm thickness and a resonance frequency of 360 kHz was used as a transmitting and receiving element. To emit a powerful prolonged phase-manipulated signal, it is necessary to drive the piezoelectric transducer with a high voltage. For this purpose, a DC-DC step-up converter from 12 V to 400 V based on the LTC3721  chip was used in the transmitter circuit. The PWM signals from the converter are applied to the transformer primary winding, shortening it to the ground with transistors connected in a push-pull circuit. The feedback is organized on the TL431 chip via an optocoupler. The transmitter is designed with a full bridge circuit using N-channel transistors and high-power drivers, IRS21867 . Each of the drivers receives two control signals from the main microcontroller to drive the upper and lower transistor, respectively. The power part of the MOSFET gate drivers is supplied by the galvanically isolated supply voltages generated on the additional secondary windings of the transformer. The transistors of the two drivers operate in anti-phase, alternately connecting the piezoelectric transducer to +400 V and to “ground”, thus the peak-to-peak range of the output signal is 800 V.The receiver is based on a high-speed, low-noise two-stage OPA2350 amplifier. A digital potentiometer chip, MCP4551 , is used to adjust the gain in the feedback of the second amplifier stage. To protect the amplifier from possible high voltage peaks on the piezoceramic, two oppositely directed diodes are placed at its input, so the limiting threshold of the input signal is about ±0.6 V. The amplified signal is fed to a high-speed 8-bit ADC ADS830  operating at 16 MHz. A 64KB IDT7208  high-speed asynchronous dual-port FIFO buffer is used to record the digitized data. Thus, the instrument directly records the waveform of the reflected signal, allowing its further post-processing.To increase the measurement accuracy of the time taken by an acoustic pulse to travel to the ice bottom and back, an M-sequence with a duration of 20 bits was applied as an emitting sound pulse. Such binary sequences have low autocorrelation sidelobe levels, so they improve the signal-to-noise ratio in radar applications . The conAn ultrasonic range finder similar to the underwater one was developed to measure snow thickness. The sensor is positioned in the air at a fixed distance from the ice surface and measures the transit time of the acoustic signal to and from the upper boundary of the snow. During precipitation, the reflection boundary shifts towards the sensor, thereby reducing the time between transmission and reception. To improve measurement accuracy, temperature and humidity sensors were integrated into it to make corrections to the sound velocity in the air depending on these parameters. The carrier frequency of the emitted signal is 40 kHz. The developed sensor has a distance resolution of about 1 mm and an accuracy of 1 cm. A photo of the sensor is shown in The first test run of the ASLIM was conducted in 2014 in the southern part of Lake Baikal a. Lake BStation 1 was installed in the area of the Bolshiye Koty settlement, at the site with increased under-ice currents with the following coordinates: 51°51.850ce water b,d, incoce water e, ice thce water c and dirce water f.The variations in air temperatures and incident solar radiation on both stations were rather similar a,e. Afteed there d due to ed there b.After a large snowfall on March 16, a stable snow cover was formed at Station 1, leading to a decrease in the fraction of penetrating solar radiation to 5% and an increase in the temperature of the ice surface due to its thermal insulation from the air b,e. DespIn contrast to Station 1, the ice at Station 2 grew until mid-March, with only a slight slowdown during the February warming c. Therefz at the top of the ice; The heat balance at the ice–water interface (IWI) can be expressed as follows :(2)Qcw=QTemperature at the IWI is fixed at the melting point of 0 °C that corresponds to the following thermodynamic characteristics: molecular heat diffusion coefficient for fresh water is  K−1 m−3 .Equation  can be asolution . CalculaAs the ice thickness grows, the lower ice boundary gradually approaches the sensors originally located in the water, which is equivalent to the motion of sensors toward the ice–water interface. Based on the changes in ice thickness with high resolution c, it is Evidently, before freezing into the ice, the sensors moved from a turbulent water mass with large temperature fluctuations and passed through the laminar layer, with an almost linear change in temperature. Based on the obtained temperature profiles, we can estimate the thickness of the laminar layer, which ranged from 2 to 3 mm. Based on the temperature gradient in the laminar layer, we can estimate the heat flux from the water according to Equation  conditions on the formation of heat flux at the ice–water interface . A mathe−2) .The next year, we carried out a special field experiment to closely estimate the effects of under-ice current intensity on the ice–water heat flux under the same meteorological conditions , combiniThese studies have revealed that in different years and different areas of Lake Baikal the heat flux from water to ice varies within a wide range (from 2 to 50 W m 1 W m−2 , and the 1 W m−2 ,42.In 2018, one of the measurement systems was used to continue the previous studies ,43 on thOur monitoring platform implements modern advances in the environmental sensing to obtain a reliable autonomous solution suitable for application in a wide range of ice-covered conditions. Originally designed for freshwater lakes, the configuration of the sensing platform reflects specific features of lake ice cover: high transparency of the freshwater ice and, as a result, the critical importance of short-wave solar radiation for the total heat budget of the ice and underlying water , relativIn our device we used a sensor spacing of 5 cm, which is higher than that of SIMBA buoys, where temperature sensors are distributed every 2 cm. Results of our studies together with the mathematical modeling have revealed that the 5 cm step between the sensors is sufficient to verify ice thermodynamic models. Using the typical values of the ice thermal conductivity (2 W mAnother a distinctive feature of ASLIM is the high resolution of the ice thickness meter of 0.05 mm, which allows for resolving the fine-scale processes occurring in low-flow environments, and measuring the ice growth every 2–4 min at the initial stage of its formation. For comparison, the resolution of rangefinders on SIMB-3 and TUT buoys are 1 cm , and typAn important difference between our system and commercially available buoys is the presence of several light sensors in the air, in the ice and in the water which allows for estimating the attenuation of solar radiation in the ice and snow covers and in the water column. In this regard, the experimental development of a chain of 48 side-ward viewing multispectral irradiance sensors at 5 cm vertical spacing  is of paCurrently, four units of the ASLIM have been assembled and more are under development. The monitoring systems can be used both for long-term observations of the ice cover dynamics in different climatic conditions and for development of and verifying ice models, with their subsequent integration into the climate modeling and weather prediction systems. Another direction of ASLIM use consists in investigation of the spatial heterogeneities in the ice cover characteristics in Lake Baikal, extendable to other large ice-covered lakes and marginal seas. Additionally, it would be interesting to investigate with several systems the areas of appearance of giant ring structures on the Baikal ice cover ,47. AparOne potential extension of the monitoring platform consists in parallel monitoring of weather parameters: wind, air humidity and long-wave radiation balance between ice surface and the atmosphere. It will allow estimation of the total heat and mass budget of lake ice cover including the ice–atmosphere boundary layer ,49 and cWe have developed an autonomous system providing in situ continuous monitoring of vertical temperature distribution in the air–ice–water system for several months with simultaneous records of solar radiation incoming at the lake surface and passing through the snow and ice as well as of snow and ice thicknesses. The use of modern electronic components and miniature analog and digital sensors enabled us to create a spatially distributed set of addressable sensors operating on a common four-wire bus. This approach allows for flexibly expanding the number of sensors in the measurement system and freely planning their spatial arrangement. The system was tested in several studies on Lake Baikal and demonstrated high reliability in deriving the ice heat balance components during the ice-covered period.


There are a lack of disease‐modifying treatments for peanut allergy, which is lifelong in most instances. Oral immunotherapy has remained at the forefront of prospective treatments, though its efficacy is consistently undermined by the risk of adverse reactions and meager sustained effects.This review discusses the current state of oral immunotherapy, its strengths and limitations, and the future of therapeutics for the treatment of peanut allergy.The persistence of peanut allergy is currently attributed to reservoirs of peanut‐specific memory B cells and Th2 cells, though the cellular and molecular interplay that facilitates the replenishment of peanut‐specific IgE remains elusive. Uncovering these events will prove critical for identification of novel targets as we forge ahead to a new age of peanut allergy treatment with biotherapeutics. A growing body of literature suggests that some patients exhibit lasting clinical benefits following cessation of OIT. This phenomenon has been termed sustained unresponsiveness (SU) and refers to the continuance of a desensitized state (assessed by OFC) following discontinuation of the maintenance dose. SU has been almost exclusively assessed at 1–2 months post‐cessation, where half or more of desensitized patients pass an in‐clinic supervised OFC. However, beyond this arbitrary 1–2 months timeframe the prevalence of SU declines, with the POISED study reporting only 13% SU (vs. 4% on placebo) at 1 year post‐OIT.3,Recently, evidence has emerged that critically appraises the safety of OIT. In the process of allergen up‐dosing and maintenance, numerous adverse reactions can occur. Reactions involve mild to severe gastrointestinal, respiratory, and/or dermatological symptoms, with the most severe unintended effect being anaphylaxis. The safety profile of PN‐OIT has been systematically reviewed in the PACE study,OIT has been a dominant theme in the field of food allergy research. However, the same core approach has been researched and implemented for over 20 years4Anisakis spp., drastically decline following 10 months without fish consumption in Anisakis spp.‐allergic humans.+ plasma cells (PCs). Similarly, IgE‐secreting cells in peripheral blood of food‐allergic individuals were discovered to have an immature transcriptional profile with downregulated expression of plasma cell survival genes.In IgE‐mediated disease, IgE levels have been shown to decline in periods of non‐allergen exposure. In humans affected by seasonal allergic rhinitis, there is documented evidence of this decline, where IgE titers are cyclical coinciding with allergen exposure.+ MBCs has been described in human peripheral blood mononuclear cells of allergic subjects. This does not, however, discount the existence of IgE+ MBCs/PCs at secondary lymphoid or non‐lymphoid tissue sites. In gastrointestinal biopsies from subjects with PN allergy, Hoh et al.+ PCs (IgE+ CD138+) in the stomach and duodenum, but did not detect IgE+ B cells . The longevity of gastric and duodenal IgE+ PCs was not described, though identification of non‐IgE isotypes within IgE clonal lineages at the same tissue sites suggests local IgE class switch recombination.+ MBCs and IL4‐transcribing CD4+ T cells from Th2‐immunized mice have been shown to drive IgE responses in recipient mice, demonstrating that IgG1+ MBCs are sufficient for the perpetuation of IgE responses.+ T cells to undergo PC differentiation. A specific subset of Th2‐polarized CD4+ T cells, termed “Th2A” cells, has been proposed to drive allergic responses.+ T cell subset uniquely expands and contracts in pollen‐allergic individuals, concordant with on‐ and off‐season allergen peaks. Moreover, in patients achieving clinical desensitization following a 20‐week PN‐OIT regimen, the Th2A cell subset declined, but remained at detectable levels.+ T cells capable of subverting SU. An overview of immunological memory to food allergens is provided in Figure In this regard, memory B cells (MBCs) have been a focal point of recent investigations on the maintenance of food allergy. Through the application of advanced flow cytometry strategiess IgE+ CD38+ in th5+ MBCs and Th2A as direct targets for biotherapeutics. In recent years, the field of oncology has seen the rise of approaches, such as CAR‐T cells among others, aimed towards the destruction of malignant cells. Such approaches could theoretically be adopted for food allergy, with an aim to specifically kill pathogenic allergen‐specific cell repertoires  Elucidate the molecular requirements for recall responses to allergens. Knowledge of the critical molecular interplay is necessary to identify novel therapeutic targets, above and beyond IL‐4/IL‐4Rα. (2) Characterize the tissue localization of allergen‐specific memory lymphocytes. Most human food allergy research carried out to date has utilized blood samples. While informative, these data are limited in their biological applicability due to exclusion of a potentially key reservoir of tissue‐resident lymphocytes. Moreover, this may inform important considerations as to the route of therapeutic delivery, as systemically administered therapeutics  may not provide adequate efficacy at tissue sites. (3) Evaluate the plasticity of allergen‐specific B and T cells. Whether pathogenic allergen‐specific lymphocytes retain a malleable phenotype will inform as to the requirement of lifelong versus transient therapeutic regimens.To overcome the plateau in food allergy treatment and advance to the “future of therapeutics” proposed above, we foresee at least three immediate 6Herein, we have summarized the current state of knowledge on OIT, described some of the key cells involved in the perpetuation of allergy, and highlighted the advent of a new era of research and treatment in allergic disease. The way forward will center around targeted biotherapeutics. The practical implementation of these new therapies, however, may require marriage with the strategies, such as OIT, that have been developing for over decades. Strict allergen avoidance may be necessary while biotherapeutics offer comfort in raising the threshold of reactivity in the case of accidental exposure, or while waiting for the gradual tapering of existing IgE and IgE‐secreting cells. OIT may be instrumental in administering significant doses of allergen in the safest way possible in order to incite the immune system to reprogram pathogenic cell types or expand cells that will eventually maintain lifelong tolerance in a previously food‐allergic patient. Optimizing the safety and efficacy of OIT is sensical, given that its clinical benefits are appealing for some patients. However, OIT at its current stage does not appear to be a cure for food allergy. The lack of disease‐modifying therapies beckons towards gathering further fundamental insights into the cellular and molecular mechanisms that drive and perpetuate food allergy, and the subsequent implementation of targeted therapies with curative potential.The authors declare no conflicts of interest.Kelly Bruton, Paul Spill, Derek K. Chu, Susan Waserman and Manel Jordana. conceived, wrote, and edited the manuscript.All authors consent to the publication of this review.


In vivo, male C57/BL6 mice were subjected to myocardial ischemia 30 min/reperfusion 4 h. Rg2 (50 mg/kg) or vehicle was intravenously infused 5 min before reperfusion. Cardiac function and the signaling pathway involved in necroptosis were investigated. Compared with H/R group, Rg2 significantly inhibited H/R-induced cardiomyocyte death. Rg2 treatment effectively inhibited the phosphorylation of RIP1, RIP3, and MLKL in H/R cardiomyocytes, and inhibited RIP1/RIP3 complex (necrosome) formation. In mice, Rg2 treatment manifested significantly lower ischemia/reperfusion (I/R)-induced myocardial necroptosis, as evidenced by decrease in phosphorylation of RIP1, RIP3, and MLKL, inhibited lactate dehydrogenase (LDH) release and Evans blue dye (EBD) penetration. Mechanically, an increased level of tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-6, and MCP-1 were found in MI/R hearts, and Rg2 treatment significantly inhibit the expression of these factors. We found that TNFα-induced phosphorylation of RIP1, RIP3, and MLKL was negatively correlated with transforming growth factor-activated kinase 1 (TAK1) phosphorylation, and inhibition of TAK1 phosphorylation led to necroptosis enhancement. More importantly, Rg2 treatment significantly increased TAK1 phosphorylation, enhanced TAK1 binding to RIP1 while inhibiting RIP1/RIP3 complex, ultimately reducing MI/R-induced necroptosis. These findings highlight a new mechanism of Rg2-induced cardioprotection: reducing the formation of RIP1/RIP3 necrosome by regulating TAK1 phosphorylation to block necroptosis induced by MI/R.Necroptosis contribute to the pathogenesis of myocardial ischemia/reperfusion (MI/R) injury. Ginsenoside Rg2 has been reported to have cardioprotective effects against MI/R injury; however, the underlying mechanism remains unclear. This work aimed to investigate the effect of ginsenoside Rg2 on necroptosis induced by MI/R and to explore the mechanism. In this study, hypoxia/reoxygenation (H/R) injury model was established in H9c2 cells.  As terminally differentiated cells, cardiomyocytes have highly limited ability to regenerate. Excessive death of cardiomyocytes induced by injury stress and their pathological effects leads to a variety of cardiovascular diseases, such as myocardial infarction (MI), malignant arrhythmia, heart failure (HF), and sudden cardiac death . IschemiIn the past, most of the studies on cardiomyocyte death focused on apoptosis, which is usually considered as programmed cell death. Necrosis is initial considered to be an unregulated process. In 1988, studies pointed out that tumor necrosis factor α (TNFα) can trigger a kind of cell death with necrotic morphological features . With thGinsenosides are the main components of ginseng, which exert a variety of pharmacological effects, such as vasodilation, anti-tumor, anti-diabetes, anti-inflammation, anti-oxidation, and so on. Ginsenoside Rg2 is one of the compounds in the protopanaxatriol group . Studiesin vivo.Transforming growth factor-activated kinase 1 , as a node regulator of apoptosis and necrosis, plays an important role in regulating the formation of RIP1–FADD–Caspase8 and RIP1–RIP3 necrotic complex . StudiesHere, we demonstrated that Rg2 treatment promotes the phosphorylation of TAK1 and enhances its binding to RIP1, thereby inhibiting the RIP1/RIP3 complex formation, and ultimately preventing MI/R-induced necroptosis.ad libitum in conventional facility on conditions of 12:12-h light/dark cycle.All animal experiments were approved by the Animal Ethical Experimentation Committee of the Fourth Military Medical University. Male C57BL/6 mice at age of 12 weeks weighting 22–25 g were used. All animals were breed with regular pellet diets Evans blue was purchased from Sigma-Aldrich . The antibody against RIP1 (3493), RIP3 (95702), MLKL (37705), phosphor-MLKL (91689), phosphor-RIP1 (31122), phosphor-TAK1 (9339S), TAK1 (5206S), and β-tubulin (15115) were obtained from Cell Signaling Technology , phosphor-RIP3 (AF7443) were obtained from Affinity. TNF-α was purchased from Novoprotein and caveolin 3 (CaV3) (ab2912) was obtained from Abcam . The level of serum lactate dehydrogenase (LDH) was detected by the kit purchased from Genesource.in vitro, the cells were added DMEM without sugar and serum upon reaching 70–80% confluence, and then were placed in an anoxic box, and replaced the air in the anoxic chamber with 95% N2, 5% CO2 mixed gas until the oxygen concentration was less than 1%, and cultured in the incubator at 37°C for 9 h  with 10% fetal bovine serum and 1% Penicillin–Streptomycin Solution. To establish the hypoxia-reoxygenation cell model  for 9 h . After t4/well. After incubating in 5% CO2 incubator at 37°C for 1 h, CCK8 assay solution (10 μl) contained water-soluble tetrazolium salt (WST8) was then added to each well and the cells were incubated for 1 h. The optical density of each well was measured using a microplate reader at 450 nm.Cell viability was detected using a Cell Counting Kit-8 (CCK8) assay. H9c2 cells were cultured in 96-well plates at about 2 × 10In vitro, the supernatant culture medium 40 μl after H/R added with 200 μl working reagent was ready to detect relative LDH release. In vivo, blood samples were collected from control and MI/R (30 min ischemia/4 h reperfusion) mice subjected to vehicle or Rg2 and centrifuged for 10 min at 3,000 r.p.m. to obtain serum. The serum 2 μl plus 38 μl double distilled water and then the working reagent of 200 μl was added to detect.Lactate dehydrogenase determination kit (rate method) produced by Shanghai Kean Science and Technology Company uses continuous monitoring method to determine the activity of LDH, liquid A and liquid B are mixed into working reagent according to the ratio of 1:5. via caudal tail injection 5 min before reperfusion  dissolved in saline (10 mg/ml) 12 h before MI/R operation. The heart was excised to separate the ventricular myocardium, then embedded it in optimal cutting temperature (OCT) compound, and immediately froze it in liquid nitrogen, finally cut into 5 μm cryosections. The Cav-3 antibody was performed to label viable cardiomyocytes while EBD-labeled necroptosis as previously described . The imaThe heart was obtained in control, MI/R (30 min ischemia/2 h reperfusion) group and MI/R + Rg2 group, fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. The tissue was cut into 5 μm slices, and incubated with TNF-α antibody overnight at 4°C, then the second antibody was incubated at 37°C for 1 h, finally observed under microscope.Ejection fraction (EF) and fractional shortening (FS) were measured as previously described . To evalSerum levels of TNFα were measured in control and MI/R (30 min ischemia/2 h reperfusion) mice subjected to vehicle or Rg2 according to instructions (Beyotime.PT512).Total protein was extracted quantitatively, and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (30 g/lane), then transferred to PVDF membrane. The membranes were blocked with 5% slim milk with TBST and incubated with the primary antibody at 4°C overnight, then washed with TBST and incubated with second antibody for 1 h at room temperature. β-tubulin was used as the loading control.t-test and one-way ANOVA with Turkey’s test correction were applied to analyze significance between two groups, statistical significance was considered at p < 0.05.Quantitative data were analyzed by Prism 8.0, and were expressed as mean ± SEM. double-tailed unpaired Student’s By observing the effect of Rg2 concentration on cell survival rate by CCK8 assay, the results showed that Rg2 at a concentration of 1–10 μM has no effect on survival rate of H9c2, although an excessively high concentration of Rg2 (20–100 μM) will inhibit cell survival . Next, tTo determine whether there is a relationship between Rg2 and H/R-induced necroptosis in cardiomyocytes, RIP1, RIP3, and MLKL phosphorylation was determined. Compared with the control group, H/R significantly increased the RIP1, RIP3, and MLKL phosphorylation  combinedin vivo, mice were subjected to 30 min ischemia followed by 4 h or 4 weeks reperfusion in vivo with vehicle or Rg2 treatment. The results demonstrated that Rg2 markedly reduced the I/R-induced myocardial infarct size to the area at risk, although the groups had comparable areas at risk  myocardium. Myocardial TNFα level was markedly increased by MI/R injury, which was effectively mitigated by Rg2, as evidenced by enhanced TNFα IHC staining , which iIn order to further clarify how Rg2 regulates TNFα-induced necroptosis, H9c2 cardiomyocytes were stimulated by TNFα (10 ng/ml) to induce necroptosis. The results showed that 2 h after TNFα exposure, the RIP1 and RIP3 phosphorylation levels increased significantly  associatin vitro experiments, we further clarify whether Rg2 can regulate myocardial necroptosis through phosphorylation of TAK1. Indeed, TAK1 phosphorylation was markedly decreased in H/R injury H9c2 cells. However, compared with the H/R group, Rg2 effectively enhanced TAK1 phosphorylation  can directly interact with TAK1 to inhibit its hyperphosphorylation and thus inhibit c-JNK/P38 signaling pathway, thus effectively alleviating the progression of NAFLD .Transforming growth factor activated kinase 1 is essential for regulating many important biological processes, such as immune cell activation, inflammation, cell differentiation, and cardiac hypertrophy. Previous studies have reported the role of TAK1 in regulating cardiomyocyte apoptosis and necroptosis . TAK1 red by DOX . TAK1 phd by DOX . Previoud by DOX . And thed by DOX . At presof NAFLD . Meanwhiof NAFLD . Howeverin vivo and in vitro experiments, the study demonstrates that TAK1 phosphorylation plays an important role in regulating myocardial necroptosis induced by TNFα. Rg2 enhances TAK1 phosphorylation to inhibit necroptosis. This research is important in furthering our understanding of Ginsenoside-induced cardioprotection.In conclusion, based on The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Animal Ethical Experimentation Committee of the Fourth Military Medical University. Written informed consent was obtained from the owners for the participation of their animals in this study.YL, HY, and HH performed the material preparation, data collection, and analysis. LY, HM, and HZ wrote the first draft of the manuscript. All authors commented on previous versions of the manuscript, contributed to the study conception and design, and read and approved the final manuscript.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.


R = 0.931, p < .001, vs. POF: R = 0.931, p < .001). RF strongly correlated with the ISQ value using linear approximation . For the cadaveric vertebrae study, the correlation coefficients between RF and the peak torque/POF were significant regardless of approximation method  . Thus, the results of this study revealed a constant correlation between RFA and conventional methods as a measurement validation, predicting favorable support for intraoperative PSF. RFA has the potential to be a new index for evaluating the implant fixation force.There is no evaluation method currently available to assess intraoperative pedicle screw fixation (PSF) strength. In this study, we established a laser‐based resonance frequency analysis (RFA) system with high‐speed, noncontact, quantitative measurements of PSF. Clinical investigations in the future can assess surgical failure risk of implants. We investigated the characteristics of the laser RFA and compared them with the conventional methods. We inserted a pedicle screw in the vertebral pedicle of human cadaver or model bone, followed by screw pull‐out, peak torque, implant stability quotient (ISQ) value obtained by the magnetic dental RFA system, and fixation force of laser RFA. We compared the outcomes using best‐fit linear or logarithmic approximations. For the model bone study, the resonance frequency (RF) versus peak torque/pull‐out force (POF) demonstrated strong correlations using logarithmic approximation (vs. peak torque:  This method is referred to as resonance frequency analysis (RFA). The most notable assessment, which is used in the engineering field, is the percussion method wherein the sound of a tunnel wall is determined by the sound of hitting the wall with a hammer.To date, the medical application of RFA has only been with dental implants.There are multiple studies in which noninvasive measurements of orthopedic implant stability in orthopedic surgery have been performed in the laboratory.The magnetic RFA  already in use in dentistry requires a smart peg to be attached before measurement, thus making it difficult to use deep within the body, other than in the oral cavity where it can easily access the implant.The basic principle involves the irradiation of an evaluation sample with a pulse laser to induce vibration on the surface through the laser ablation process and the subsequent detection of the induced vibration using a laser Doppler vibrometer. The scheme of laser RFA is similar to that of a hammering inspection, and it can provide high‐speed, noncontact, and quantitative measurements. The advantages of an impact scheme using laser pulse irradiation have been previously reported and shown to be massless and remote laser sensing is comparable to physical excitation measurements.In this study, we sought to investigate the characteristics of laser RFA using biomechanical test materials  and human cadaveric vertebrae and compare the technique with mechanical test forces (insertion torque and POF) and the ISQ value.22.1Five types of solid rigid polyurethane forms and one type of cellular polyurethane form   were prepared as test materials to represent diverse human vertebrae Table . Five teThese biomechanical test materials were homogeneous and consistent with human cancellous bone.2.2For the experimental use of fresh, nonfrozen human cadavers, written informed consent was obtained from each donor as per the ethical guidelines of Keio University School of Medicine. The cadavers were stored at <4°C shortly after death and dissected within seven days of death. The discs and ligaments were removed from the spine to obtain the vertebrae Figure . All exp2.3We prepared single‐threaded, non‐cannulated, titanium alloy , monoaxial pedicle screws  with lengths of 45 mm, an outer threaded diameter of Ø 5.5 mm, an inner thread diameter start point of Ø 3.8 mm (an endpoint of Ø 4.6 mm), and a screw pitch of 2.5 mm. Using an awl, we made a small hole in the target area where a model bone or vertebra screw was to be inserted at the center of the 60 mm × 60 mm material and the entry point of the vertebral pedicle.Before insertion, a 1.5 mm drill bit was used to drill a pilot hole in the material. A pedicle screw was inserted at its 40‐mm length, and the root of the screw was left 5 mm Figure . Paik et2.4A digital torque gage HTGA‐5N  was used to measure the insertion torque (peak torque)2.5Magnetic RFA was performed using a specific device  after the pedicle screw insertion was complete and without contacting the screw, per previous reports.2.6e2 values from the peak values of the Gaussian fitting for the bean profile  were performed. We described two types of best‐fit approximations: linear and logarithmic. Correlation coefficient values < 0.3, 0.3–0.7, and >0.7 were defined as weak, moderate, and strong relationship, respectively. Statistical analyses were performed using SPSS statistics software version 26 . The significance level of all tests was set at 3The primary purpose of this study was to investigate the characteristics of Laser RFA; therefore, results not obtained with the Laser RFA are described in Figure R = 0.931, p < .001; POF: R = 931, p < .001) compared to the linear approximation  .We used linear and logarithmic analyses to compare RF and conventional methods  Figure . The RF ) Figure . Further) Figure . The resR = 811, p < .001; logarithmic: R = 0.716, p < .01)    Figure . Neverth) Figure . Finally) Figure . RF and 4This study is the first to investigate the strength of pedicle screw installation using the Laser RFA method. In the study of the biomechanical test materials, the RF is strongly correlated with the ISQ value Figure , and it Amerini et al.In the future use of laser RFA, it will be necessary to analyze the effects of these two forces  on the screw during fixation by combining clinical data/screw mechanics testing and finite element analysis. Nakashima et al.In the dentistry field, the ISQ value has negatively correlated with the displacement of an implant after the application of a lateral load.RF, such as the ISQ value, was indicated to be a type of index that had different characteristics from those of the mechanical fixation force measures. As described above, peak torque and POF may represent different phenomena from RF and ISQ. However, as a logarithmic fitting was used in this study, it is possible to say that RFA and ISQ are sensitive in the region of low peak torque and POF, while RF is not sensitive in the region of high peak torque and POF. In future, there should be investigations to increase the sensitivity of the RFA, particularly in the region of high peak torque and POF.In the study using cadaveric vertebrae, the comparison with the ISQ value showed the same high correlation with linear approximation as the studies using the biomechanical  test materials Figure . HoweverNakashima et al. investigated the correlation between the three test forces of peak torque, POF and ISQ values, and imaging parameters (bone density and bone morphometry with microcomputed tomography (CT)/multidetector CT).In addition to insertion torque and POF, there are other methods of measuring implant fixation force, such as the toggle test,Laser RFA has several advantages over previous pedicle screw fixation evaluation systems. First, the laser system is a noncontact system and requires short steps to perform RFA. Laser RFA requires only 10 s from laser irradiation to frequency spectrum analysis. However, the hammer method is more complex as it needs 16 impacts to obtain sufficient data for analysis.In dentistry, the ISQ values correlate with the percentage of the implant in contact with the bone .Two important factors for ensuring final stability have been reported, primarily in dentistry using the ISQ value: initial fixation at the time of implant placement and postoperative osseointegration.A future RFA system for a series of pedicle screw insertion procedures is shown in Figure This study has the following limitations: First, we used cadaveric vertebrae with conditions that differed from those of living bone and were different from clinical conditions. In contrast to the cadaveric bone, in actual surgery, the vertebrae are connected to each other by ligaments and intervertebral discs, and paravertebral muscles are present around them. Therefore, the results may differ from this study. Further, in vivo studies are warranted. Second, the RF was evaluated under dry conditions. In particular, the effect of blood exposure to the implant needs to be considered, but the current assumption is that the effect of blood and other light‐mass materials on vibration is expected to be small. For future high‐precision diagnosis, it is necessary to examine the effects of the wet environment caused by biological fluids and blood. Third, the current laser system involves reflection by fixed mirrors making the irradiation range nonflexible.Because the current system does not allow the direction of laser irradiation to be changed freely, the pedicle screw needs to be placed in a fixture to make it easier to irradiate with the laser. Therefore, the position of the human body needs to be altered vertically and horizontally to evaluate pedicle screw stability. The free movement of the human body in the operating room is not possible rendering assessment difficult. The use of an optical fiber system to enable the operator to irradiate an arbitrary point can help overcome this problem owing to its flexibility. Fourth, although there exists a cyclic test value as well as torque and POF for implant fixation force, this test was not performed in this study. This test may be close to the multidirectional force reflected by the RF and needs to be investigated in the future.Fifth, we did not examine the difference in direction and location of laser irradiation. Since the RF obtained by laser RFA is the characteristic vibration of the implant, it can be induced regardless of the direction of excitation. Therefore, it is thought that the RF fluctuations because of the direction and location of irradiation do not occur. In this study, laser irradiation was performed from the side of the screw by raising the root of the screw by 5 mm, but if the entire screw was inserted and irradiated from the longitudinal direction, no change in RF was expected. Further research is warranted to investigate the differences in irradiation location and direction.The experimental subjects were examined in a uniform and reproducible manner. We believe that this study provides a new perspective on the definition of implant fixation force and presents useful information for implant placement strength research and design developments and improved clinical outcomes. In conclusion, laser RFA has the potential to be a new evaluation index for implant fixation force with excellent reproducibility and usability, replacing the conventional test forces.The authors declare that there are no conflict of interests.Daisuke Nakashima was in charge of all procedure, analysis, and manuscript write‐up. Katsuhiro Mikami was in charge of laser system construction, data gathering, data analysis, and manuscript write‐up. Shunsuke Kikuchi handled cadaver dissection and bone model preparation. Masaharu Nishikino, Toshiyuki Kitamura, and Noboru Hasegawa performed data analysis. Morio Matsumoto, Masaharu Nishikino, and Takeo Nagura took responsibility for conception.Supporting information.Click here for additional data file.


This work reports the development of an enzymatic biosensor using reduced graphene oxide (RGO) as a key nanomaterial for the detection of contaminants of emerging concern (CECs). RGO was obtained from the electrochemical reduction of graphene oxide (GO), an intermediate previously synthesized in the laboratory by a wet chemistry top-down approach. The extensive characterization of this material was carried out to evaluate its proper inclusion in the biosensor arrangement. The results demonstrated the presence of GO or RGO and their correct integration on the sensor surface. The detection of CECs was carried out by modifying the graphene platform with a laccase enzyme, turning the sensor into a more selective and sensitive device. Laccase was linked covalently to RGO using the remaining carboxylic groups of the reduction step and the carbodiimide reaction. After the calibration and characterization of the biosensor versus catechol, a standard laccase substrate, EDTA and benzoic acid were detected satisfactorily as inhibiting agents of the enzyme catalysis obtaining inhibition constants for EDTA and benzoic acid of 25 and 17 mmol·L A chemical sensor is defined by IUPAC as a device that transforms chemical information into an useful analytical signal . The useAmong others, graphene and its derivates have gained popularity in electroanalysis due to their interesting inherent properties, such as biocompatibility, low-cost raw materials, and their easy tuning ,4. In thsp2 configuration. Some examples of bottom-up approaches are the chemical vapor deposition (CVD) or the epitaxial growth 3-/4- 5 mmol·L−1 (diluted in phosphate buffer 50 mmol·L−1 at pH 7.4) as analyte. The goal was to find a volume that covers all the electrode surface and did not saturate the response. As can be observed in The optimization of the sensor assembly was carefully addressed. First, the optimal amount of GO solution to be deposited onto the electrode was studied. In this case, drop-casting was chosen as the modification method, due to its rapid and easy application. The effect of four different volumes  of a 1 mg·mLRMS) of 73.5 nm; meanwhile, the surface modified with GO has a RRMS of 123.3 nm. Those values, along with the AFM images, verify the presence of graphene flakes. Moreover, the 3D AFM image , limit of quantification (LOQ), repeatability, intermediate precision, selectivity, and lifetime.−1. The correlation coefficient for this range was 0.992, indicating a satisfactory linearity.The linear range is generally defined as the area in the calibration curve where the signals are directly proportional to the concentration. This parameter is typically evaluated using the correlation coefficient. As can be observed in the inset of −1 and a LOQ of 7.7 µmol·L−1.Regarding the LOD and LOQ, there are multiple ways to calculate them, such as the use of the calibration curve for the target analyte (see Equation (1), where m is the slope of the calibration curve and σ the standard deviation of the response at a 95% confidence level), the measure of signal/noise ratio or by the use of the blank standard deviation ,58. In t−1 catechol solution were performed, obtaining a relative standard deviation (RSD) value equal to 8%. Moreover, a second repeatability test was done to check the reliability of the biosensor during the day. For this purpose, 3 calibration experiments were repeated in 3 different time instants of the day .The repeatability is defined for the European Medicines Agency (EMEA) as the “term that expresses the precision under the same operating conditions over a short interval of time” . In thisTo visualize the selectivity of the biosensor, a Principal Component Analysis (PCA) (executed in Matlab R2020a) was carried out to show the ability of the biosensor to distinguish between the different substances considered. In particular, six substances from different families were analyzed with a Lac-RGO-modified electrode. As can be seen in max). This typical behavior is clearly observable in M is the Michaelis–Menten’s constant, defined as the concentration of substrate needed to reach half of the maximum rate ) with the reaction rate (V). In this model, the enzyme rate depicts a saturation curve arriving to a steady state situation. At this point, the reaction rate achieves its maximum ) . HoweverM and Vmax, obtained from Equation (4), were 2.62 ± 0.50 µmol·L−1 and 1.90 × 10−4 ± 0.13 × 10−4 A, respectively.In this work, the h estimated value from the catechol calibration curve was 0.99 ± 0.09, demonstrating that the Lac-RGO biosensor followed the already explained kinetics. The rate parameters K−1 for the benzoic acid and 600 µg·L−1 for the EDTA  [As commented in the introduction section, there are common household products that have been used for a long period of time, that are now under scrutiny due to their possible effects on the environment and health. Examples are the emerging pollutants found in cosmetics, processed food, or pharmaceutical compounds. The inhibition study of the Lac-RGO biosensor was carried out using benzoic acid and EDTA, two examples of CECs. Benzoic acid is a colorless crystalline compound that is widely used in pharmaceutical products either as an active product ingredient  or as an excipient (as a lubricant in tablets). Both in the food and cosmetics industry, it is used as a preservative ,74. On tg-water) ,78.M and Vmax to K’M and V’max.Any substance that in a particular context can decrease the kinetics of an enzyme will be considered as an enzymatic inhibitor. As mentioned before, the Michaelis–Menten constants (Equation (2)) are unique for a specific enzyme-substrate system, so the presence of inhibitors will lead to renaming the KM < K’M and Vmax = V’max. The second situation is named non-competitive inhibition; in this case, the inhibitor is not a direct competitor but is bound in an allosteric site that deactivates the enzyme. The changes in the constants are noted as KM = K’M and Vmax > V’max. Finally, the last scenario is the uncompetitive inhibition, where the inhibitor is bound, producing a bigger affinity between the substrate and the enzyme, leading to fewer active sites in the system, in that case KM > K’M and Vmax = V’max [There are three modelled scenarios . The fir = V’max .max and zone 2 in the increase in KM with the inhibitor concentration. In M and V’max , and the constant trend for the maximum velocity . This state is supported by M’ and Vmax’ values, and their trends, are plotted. Experimentally, the changes can be elucidated comparing the Lineweaver-Burk linearization for different concentrations of inhibitor. In this sense, I) for each inhibitor. −1) to Laccase than EDTA (17 mmol·L−1). To calculate KI values, expression (5), corresponding to competitive inhibition, was used.Moreover, the inhibition constant (K−1 of catechol. A bigger effect for benzoic acid, as expected from the previous comments, was fulfilled. In detail, a maximum ≈ 60% of inhibition was reached for the benzoic acid system in front of the ≈25% for EDTA. Voltammograms presented in On a further visualization of the inhibition effect, The reported work has shown the capabilities of graphene as a nanomaterial to improve and facilitate the development of electrochemical biosensors, specifically those employing redox enzymes. The advantages observed can be attributed to two factors: the first one is the amelioration of the electrochemical transduction properties, thanks to inherent electrical characteristics of graphene. The second, the conjunction with the chemical properties of the material, as it serves as a linkage platform for immobilization of biomolecules such as enzymes.All this makes this combination an unbeatable choice for electrochemical biosensing. The case specified in here, the biosensing of catechol, has demonstrated an improvement of sensitivity of ca. 30%, which in turn improves the direct determination of phenolic compounds or opens the door to consider inhibition. In this inhibitory biosensing application, two different chemicals, EDTA and benzoate, have been verified as inhibitors of the enzyme reaction, which may constitute the basis for a future analytical device or system to monitor CECs in water.


CFTR gene cause cystic fibrosis, the most common lethal autosomal genetic disease in the Caucasian population. In recent years, major efforts have led to the development of CFTR modulators, small molecules targeting the underlying genetic defect of CF and ultimately rescuing the function of the mutant channel. Recent evidence has highlighted that this class of drugs could also impact on the phosphorylation of the R domain of the channel by protein kinase A (PKA), a key regulatory mechanism that is altered in various CFTR mutants. Therefore, the aim of this review is to summarize the current knowledge on the regulation of the CFTR by PKA-mediated phosphorylation and to provide insights into the different factors that modulate this essential CFTR modification. Finally, the discussion will focus on the impact of CF mutations on PKA-mediated CFTR regulation, as well as on how small molecule CFTR regulators and PKA interact to rescue dysfunctional channels.Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel expressed on the apical membrane of epithelial cells, where it plays a pivotal role in chloride transport and overall tissue homeostasis. CFTR constitutes a unique member of the ATP-binding cassette transporter superfamily, due to its distinctive cytosolic regulatory (R) domain carrying multiple phosphorylation sites that allow the tight regulation of channel activity and gating. Mutations in the  CFTR gene in 1989 (The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride and bicarbonate channel expressed at the apical surface of secretory epithelia, including the airways, sweat glands, gastrointestinal tract, and other tissues . The CFT in 1989  has prom in 1989 .CFTR gene, expressed on the long arm of chromosome 7  is approved both as a single therapy for G551D mutants and as a combination with either the corrector VX-809 -5-chloromethyl-isoxazoles, like UCCF-152, were found to stimulate potent and rapid phosphorylation of the R domain of WT, temperature-rescued F508del-CFTR and G551D-CFTR, while also increasing iodide currents, leading to their classification as CFTR activators . The speOverall, our current knowledge of how CFTR modulators interfere or promote PKA-dependent phosphorylation of the channel remains scarce, and future efforts are needed to allow a better understanding of their impact on this essential molecular modification of the channel.In conclusion, PKA-dependent phosphorylation plays a key role in multiple steps during the life cycle of CFTR. While interactions resulting in phosphorylation at the CFTR R domain regulate channel opening and activity, other phosphorylation events at the C and N terminal ends of CFTR modulate channel stability and trafficking at the PM. Importantly, CFTR mutations leading to CF impair different steps of CFTR biogenesis that are regulated by these phosphorylation events. Targeting PKA-mediated phosphorylation thus represents a promising strategy to rescue the activity of different CF-causing CFTR variants. Nonetheless, how correctors and potentiators, including their highly effective combinations, like the recently approved Trikafta, impact on the PKA-mediated phosphorylation of CFTR still needs to be thoroughly investigated. Future studies in this direction might help to maximize therapeutic efficacy, ultimately normalizing the life expectancy of CF patients.ADS designed the overall layout and wrote the bulk of the manuscript. GP was involved in the writing of the manuscript and referencing. EH reviewed the manuscript and produced the figure. AG was involved in the editing and critical revision of the manuscript. AG and EH acquired the funding. All authors contributed to the article and approved the submitted version.AG and EH are founders and board members of Kither Biotech, a company focused on the development of PI3K inhibitors for airway diseases not in conflict with statements in this article. The other co-authors declare no conflict of interest.


All the parameters of the ADME study revealed that geraniol is an acceptable drug candidate. A docking study showed that the binding energy of geraniol  was lower than that of acetylcholine  with AChE, which exhibited around a 12.58-fold higher binding affinity of geraniol. Furthermore, molecular dynamics simulation revealed that the RMSD of AChE alone or in complex with geraniol fluctuated within acceptable limits throughout the simulation. The mean RMSF value of the complex ensures that the overall conformation of the protein remains conserved. The average values of Rg, MolSA, SASA, and PSA of the complex were 3.16 Å, 204.78, 9.13, and 51.58 Å2, respectively. We found that the total SSE of AChE in the complex was 38.84% (α-helix: 26.57% and β-sheets: 12.27%) and remained consistent throughout the simulation. These findings suggest that geraniol remained inside the binding cavity of AChE in a stable conformation. Further in vivo investigation is required to fully characterize the pharmacokinetic properties, optimization of dose administration, and efficacy of this plant-based natural compound.Acetylcholinesterase (AChE) inhibition is a key element in enhancing cholinergic transmission and subsequently relieving major symptoms of several neurological and neuromuscular disorders. Here, the inhibitory potential of geraniol and its mechanism of inhibition against AChE were elucidated in vitro and validated via an in silico study. Our in vitro enzyme inhibition kinetics results show that at increasing concentrations of geraniol and substrate, Vmax did not change significantly, but Km increased, which indicates that geraniol is a competitive inhibitor against AChE with an IC The cholinergic network affects various cellular functions through neurotransmission via acetylcholine (ACh), a neurotransmitter released from presynaptic neurons into the synaptic cleft, which binds to acetylcholine receptors (AChR) on the post-synaptic membrane, relaying the signal from the nerve or hydrolyzed by acetylcholinesterase (AChE) . A signiMoreover, cholinesterase enzymes such as AChE further decreased the concentration of ACh by hydrolyzing it . TherefoThe approved medications (cholinesterase inhibitors) for neurodegenerative disorders have several side effects such as nausea, vomiting, loss of appetite, headache, constipation, confusion, and dizziness ,12. SubsMost natural products are promising for the effective treatment of various ailments, and our team previously reported their antioxidant, anti-inflammatory, antidiabetic, hypolipidemic, and neuroprotective properties by inhibiting key regulatory enzyme activity ,20,21,2210H18O and is well known for its antioxidative , where “V” is denoted as the velocity (change in absorbance), which represents the enzyme activity, and “[S]” is denoted for substate concentration A. This lxon plot B and obtllalobos , who fouanalysis .Y-axis (Y-intercept = 1/Vmax), which showed the varying Vmax, but intersected at the same locations on the X-axis (X-intercept = −1/Km) of the plot, showing similar Km (2 = 0.22) with an increase in the concentrations of tacrine, and Vmax decreased significantly (R2 = 0.81), which indicated that tacrine is a non-competitive inhibitor of AChE and binds to the non-catalytic site of the enzyme. Our results are in agreement with a previous report where tacrine  was reported as a reversible non-competitive inhibitor against AChE enzyme [Moreover, we also investigated the AChE enzyme inhibition kinetics of tacrine through a Lineweaver–Burk plot and found that all the trendlines of different concentrations of tacrine at various concentrations of the substrate intersected at different locations on the milar Km A. We furmilar Km B and obtE enzyme ,59, whicE enzyme  and contE enzyme , who rep2) than tacrine (38.91 Å2) and that both can easily cross the BBB.Physicochemical properties, drug-likeness, and pharmacokinetics of geraniol and tacrine were evaluated using the SwissADME tool . Both liIn the present study, based on our in vitro results, it is well established that tacrine behaves as a non-competitive inhibitor and that geraniol showed competitive inhibition against AChE and binds to the catalytic site residues, which are mainly used for binding of the substrate (acetylcholine). Therefore, an analysis of molecular docking between AChE–geraniol and AChE–acetylcholine was performed, which revealed that both ligands were bound to the central active site cavity of AChE . AChE inThe binding pose of geraniol at the active site of AChE was further compared with the binding mode of a control ligand, that is acetylcholine . It was −1 and 1.02 × 103 M−1, respectively, for the AChE–acetylcholine interaction,—5.6 kcal mol−1 and 1.28 × 104 M−1, respectively, for the AChE–geraniol interaction at CAS cavity, and −6.8 kcal mol−1 and 9.72 × 104 M−1, respectively, for the AChE–geraniol interaction at the PAS cavity. We found that the binding affinity of geraniol for AChE was around 12.58-fold higher than that of the control ligand, acetylcholine, in the CAS cavity.Interestingly, the amino acid residues of AChE commonly engaged in the interaction with geraniol as well as acetylcholine in the CAS cavity including Trp84, Gly118, Glu199, Ser200, Phe330, His440, Gly441, and Tyr442, which show that geraniol interacted with all the specific residues of acetylcholine, except Tyr130 and Ile439 and that geraniol interacted with four more residues: Tyr121, Phe290, Phe331, and Ile444. Our molecular docking results confirmed that geraniol is a competitive inhibitor of the AChE enzyme. Moreover, the docking energy and the corresponding binding affinity were estimated to be –4.1 kcal mol−1, indicating that most of the terpenoids have high affinity for catalytic site residues, such as Asp74, Trp86, Gly120, Gly121, Ser125, Ser203, Phe295, Phe297, Tyr337, Phe338, His447, and Gly448. However, the report did not explain the residues that specifically interacted with geraniol. Moreover, we also analyzed the binding of non-hydrolysable structural analogues  in the active site of AChE at the catalytic site and at the peripheral anionic site, as previously reported by Colletier et al. [Our results are in agreement with a previous report , where gr et al. , and theThe dynamic nature of the interaction and the stability of the AChE and geraniol complex was assessed by molecular dynamics simulation under physiological conditions. The initial frame of the AChE–geraniol complex was subjected to molecular dynamics for 100 ns, and the results are presented in N- and C-terminal zones. The mean RMSF value of AChE–geraniol complex was determined to be 1.28 Å, which is much lower than the acceptable limit. It is evident that the RMSF of AChE did not deviate significantly in the presence of geraniol, assuring that the overall conformation of the protein remained conserved.The root-mean-square fluctuation (RMSF) of a protein provides insight into the local conformational changes in the side chains of a protein during simulation. 2, respectively (The dependency of radius of gyration (Rg) and surface area, such as molecular surface area (MolSA), solvent accessible surface area (SASA), and polar surface area (PSA) of a ligand on simulation time, provide information about the behavior of the ligand inside binding pocket of the enzyme. The Rg may also indicate whether the complex remains folded during MD simulation. The variation in Rg of geraniol bound to AChE as a function of simulation time is presented in ectively D. These The formation of a stable protein and ligand complex was established by determining the total number of contacts formed between them during the simulation A. It is The interaction between a ligand and protein often leads to changes in the protein’s secondary structural elements (SSE). Thus, a check on the variation in SSE during simulation is critical to provide an overview the establishment of a stable complex between geraniol and AChE. The variation in the total SSE (α-helix + β-sheet) of AChE bound with geraniol during simulation is presented in An analysis of the interaction between AChE and geraniol during the molecular dynamics simulation is presented in 50: 98.06 ± 3.9 µM) in the catalytic active site. Geraniol also exhibited the finest drug-likeness, pharmacokinetics, and physiological properties that can cross the BBB, as well as high absorption through the GI tract, suggesting that it can be a potential drug candidate for neurological disorders. The in vitro results were validated through in silico study and found that geraniol bound to most of the residues of the catalytic active site with a 12.40-fold higher binding affinity and lower binding energy  than the substrate binding energy . Moreover, molecular dynamics simulation study concluded that complex (AChE–geraniol) RMSD fluctuated within acceptable limits, and the overall conformation of the protein remained conserved and remained consistent throughout the simulation. These findings suggest that geraniol remained inside the binding cavity of AChE in a stable conformation. Further in vivo investigation is required to fully characterize the pharmacokinetic properties, optimization of dose administration, and efficacy of this plant-based natural compound.Neuromuscular and neurodegenerative disorders are generally treated by inhibiting acetylcholinesterase (AChE) enzyme to increase the level of acetylcholine in synapses. Through our in silico and in vitro findings, it has been concluded that geraniol inhibits AChE in a concentration-dependent manner and that it bound more effectively (IC