N spikes of M input neurons are propagated, and these “first spike patterns” can be readout by downstream neurons with homogeneous weights and no desensitization: as a result, the exact order between the first spikes does not matter. This paper also introduces a third option—“Ranked-NoM” (R-NoM), which combines features from both ROC and NoM coding schemes: only the first N input spikes are propagated, but their order is readout by downstream neurons thanks to inhomogeneous weights and linear desensitization. The unifying mathematical framework allows the three codes to be compared in terms of discriminability, which measures to what extent a neuron responds more strongly to its preferred input spike pattern than to random patterns. This discriminability turns out to be much higher for R-NoM than for the other codes, especially in the early phase of the responses. We also argue that R-NoM is much more hardware-friendly than the original ROC proposal, although NoM remains the easiest to implement in hardware because it only requires binary synapses.Spiking neural networks (SNNs) using time-to-first-spike (TTFS) codes, in which neurons fire at most once, are appealing for rapid and low power processing. In this theoretical paper, we focus on information coding and decoding in those networks, and introduce a new unifying mathematical framework that allows the comparison of various coding schemes. In an early proposal, called rank-order coding (ROC), neurons are maximally activated when inputs arrive in the order of their synaptic weights, thanks to a shunting inhibition mechanism that progressively desensitizes the neurons as spikes arrive. In another proposal, called NoM coding, only the first  Indeed, neural networks trained with Deep Learning are now state of the art in many domains. All such systems can be thought of as “neuromorphic” in that they involve large networks of neuron-like elements with connections that resemble the synapses of biological brains. However, there is currently an intense debate about whether future systems will need to include additional neuromorphic features. One key difference between these state-of-the-art AI systems and biology is how information is represented. Artificial systems typically perform calculations using floating-point variables to represent both the neuronal activation levels and the strength of synaptic connections. In contrast, real neurons send information as discrete all or none pulses—spikes. Is this difference important? Spiking Neural Networks (SNNs) are becoming increasingly popular, especially for low-power embedded systems. But many mainstream researchers consider that this difference is essentially irrelevant. Many assume that neurons send information using a firing rate code in which the neuron's activation level is represented by the number of spikes emitted in a given time window. If that was the case, replacing the firing rate with a floating-point number is a perfectly reasonable strategy. However, it has been argued that this sort of firing rate code would be intrinsically very inefficient because you would need a lot of spikes to encode information with any degree of accuracy —, there are a number of very interesting options that can be considered. In principle, you could use the latency at which a single neuron fires in response to an input to derive information about the intensity of the activation. For example, a neurophysiologist could use an oscilloscope to determine a neuron's latency. But this requires knowing precisely when the stimulus came on. Inside the brain, there is no way to know this. Hence, in this paper we consider an alternative strategy: looking across a population of neurons and determining the order in which they fire. Note that TTFS is not well-suited for dynamic inputs, since coding changes in the input requires additional spikes. We thus focus on static inputs, e.g., flashed images. For simplicity and hardware-friendliness, we also restrict ourselves to non-leaky neurons. A leak is useful to process dynamic inputs because the oldest inputs should be forgotten. Yet it is not required with the static inputs used in this paper.Historically, TTFS was first proposed to explain the phenomenal speed of processing in the brain for certain tasks, such as object recognition  and a combination of both (Ranked-NoM Coding).M afferents of a neuron fire a spike  was an early proposal  coding is another proposal, in which only the N first spikes among M afferents are propagated, but readout neurons can be selective to a particular order of the N spikes thanks to inhomogeneous weights, and a decreasing modulation function. Later on, we came across an article by Furber et al.  coding, and which incorporates features of both ROC and NoM coding : only thr et al.  where a r et al. .All these codes have been formalized in our unifying mathematical framework that involves:W non-zero weights.A set of weights, which can be homogeneous (as in NoM), or decreasing, either linearly , or geometrically. This set contains N spikes.A modulation function which can be constant (as in NoM), or decreasing, either linearly, or geometrically . This modulation can also have a cut-off, i.e., becomes zero after the first Our unifying framework allows comparing these codes in terms of discriminative power. We introduce a discriminability measure that quantifies how much more a neuron responds to its preferred pattern than to random inputs. The unifying mathematical framework also allows tuning the parameters of the codes in order to optimize their discriminative power.We conclude that Ranked-NoM Coding with linearly decreasing modulation and weights offer a particularly interesting compromise between discriminative power and hardware-friendliness.The paper is organized as follows: the Section 2 briefly introduces the unifying mathematical framework and the discriminability measure. Then, it gives the main analytical formulas for the discriminability of R-NoM, NoM, and ROC, but not their derivations, which can be found in the The goal is to measure the discriminability power of these codes. We define a measure of selectivity (Equation 2.7) which quantifies how much more the neuron responds to its preferred pattern than to random stimuli.M neurons  ≠ 0.By construction, each component X in a deterministic way by defining the affine transformation W = Φ(X):This input order is transformed into a vector of weights. For this, we transform the random variable Wi are determined from the probability distributions of Xi by the change of variables theorem in multivariate calculus. At this stage, the random experiment is fully defined by the random variable X, taking values in Λ, and the bijective function Φ.The marginal and joint probability distributions of the new random variable C, a vector-value function ΦC from Ω to ΩC and we use the term score vector to denote elements in ΩC.We denote Ω the set of the weights vectors. Ω is the base to establish the support of each coding scheme. For this, we define, for each scheme R), the function ΦR is the identity function and so its cardinality is M!.For ROC (denoted by H), we build the scores-support ΩH using a function which depends on the parameter For Ranked-NoM Coding , we define the scores-support ΩF from the scores-support ΩH by the compositions of the indicator function 1A with ΦH. Thus we haveFor NoM coding , and for the NoM scheme ΨF ≡Φ F (2.4).Next, we define, for each scheme, a Finally, we define an integration function — effectively equivalent to the membrane potential — which indicates how well the random scores vector matches the fixed modulations vector.I. Then, over the first I inputs, the integration function SC reads:To formally translate intermediate states , we first define the gate function I = M and we would have intermediate states for all values I < M.Given that Ranked-NoM and NoM are defined for values w is a random vector, then SC is a random function. Let SC,I denote the corresponding output random variable. Its distribution depends on the coding scheme. We compare the three coding schemes in terms of discriminative power, characterizing its distribution by the difference between its best possible value and its expected values, scaled by its variance.Since Definition 2.1. We define discriminability DC(I) as:I ∈ ℤ and takes values for ROC in the interval  and variance Var of integration at intermediate states of each scheme C depend on the mean μWC, variance VarWC and covariance CovC of the scores for the corresponding coding scheme C  affects discriminability  has a horizontal asymptote in Y≃5.2:For In light of these behaviors, we propose that Ranked-NoM and NoM are to be preferred over ROC.M = 31, how discriminability increases as more and more inputs become available .As shown above, discriminability saturates to the same value for Ranked-NoM and NoM  is based on the unconstrained membrane potential, i.e., ignoring the threshold. But of course, in a real scenario, a threshold is needed, especially for neurons in the hidden layers . When choosing a threshold, a high value:Ensures that the probability of reaching it with random input  is low.Causes a longer latency even when the preferred pattern is given as input.Conversely, a low threshold does the opposite (shorter latency but higher FA rate). This can be seen as a speed-accuracy trade-off.M = 20 and estimated the false alarm probabilities for ROC (m = 0.8), R-NoM  as well as new ones. The activation is maximal when the spikes arrive in the order of the weights: the first spike should arrive through the strongest weight, and so on. This defines the preferred input spike pattern of a neuron. Then, we defined discriminability, which measures how much more the neuron responds to its preferred pattern than to random inputs. Our framework allows us to compute this discriminability analytically. Thus various TTFS codes can easily be compared in terms of discriminability. The framework also allows the design of new codes that maximize this discriminability. In particular, we propose a new code that we dubbed “Ranked-NoM” (R-NoM), which makes use of integer modulation and weights that both decrease linearly. We demonstrated that R-NoM has much more discriminative power than ROC and NoM, especially in the early phase of the response, which is already very selective. Thus it allows detectors that are both accurate and reactive. In addition, the fact that R-NoM uses only integers makes it much more hardware-friendly than ROC, and the geometric modulation suggested in Furber et al. .There are however situations where NoM coding can be particularly interesting for hardware implementations. The advantages of R-NoM coding described here apply in situations where incoming spikes are processed one by one. However, in some designs, it is possible to process spikes as a packet. For example, you could define an input array with M bits that are initially all set to zero. As spikes come in, the corresponding input lines can be flipped on until a fixed number of bits (N) are set to one. At this point, it is easy to determine the level of activation of a target neuron by performing a logical AND operation between the array of input spikes and a second array of bits corresponding to the connected weights. Counting the number of “hits” and comparing the result to the neuron's threshold can be done in a single clock cycle with specialized FPGA or ASIC hardware. Similar results can be obtained using memristor-based cross-bar arrays.That said, the current analysis provides a strong argument for using implementations that process incoming spikes in order since it is the only way to take advantage of the remarkable early discriminative power of R-NoM coding. Such an approach goes a long way toward ensuring that computations can be done with the minimum number of spiking events.One important issue that we did not address in this paper is learning. We plan to address it in future work. Only then we will be able to confront the different coding schemes with real-world data  and compare their performance, possibly using the methodology of Guo et al. . For unsThe original contributions presented in the study are included in the article/ST and TM designed the project. LB and JG performed the mathematical derivations. JG and TM did the numerical simulations. All authors wrote the paper. All authors contributed to the article and approved the submitted version.The authors gratefully acknowledge financial support from the Colombian non-profit foundation COLFUTURO and the partial funding of Le Centre de Recherche Cerveau et Cognition (CerCo).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.


In flow, MAPK signaling required intracellular Ca2+ release to activate integrin within 2 min. Integrin activation via non-MAPK signaling occurred first locally in the vicinity of ligated P-selectin glycoprotein ligand-1 (PSGL-1) within sub-seconds, and then over the entire cell surface within 1 min in an extracellular Ca2+ influx-dependent manner. The transition from a local (but rapid) to global (but slow) activation mode was triggered by ligating the freshly activated integrin. Lipid rafts, moesin, actin, and talin were involved in non-MAPK signaling. Fluid loads had a slight effect on local integrin activation with a second-level timescale, but served as enhancers of global integrin activation.Activation of integrins is crucial for recruitment of flowing leukocytes to inflammatory or injured vascular sites, but their spatiotemporal characteristics are incompletely understood. We discovered that β Lym(GP) Ibα , 11, 14.(GP) Ibα .2 integrins have “low”, “moderate”, and “high” affinity states corresponding to three distinct conformations: bent, extended, and head-opened  signaling pathway. The latter comprises an Src family tyrosine kinase , an immunoreceptor tyrosine-based activation motif (ITAM)-containing protein [Fc receptor γ-chain (FcRγ) or DNAX-activating protein of 12kDa (DAP12)] and tyrosine kinase (Syk) in the spleen  signaling of neutrophils on immobilized P-selectin requires a stimulus time of ~75 s under a wall shear stress of 0.2 Pa and may be via the MAPK pathway, but not in a lipid raft-dependent manner .In a hemodynamic microenvironment, the fluid load on adhered cells serves as a regulator for activation of βselectin , 32, whintegrins , 33. In affinity , 34. Howselectin  suggest 2+ bursts, and activation of β2-integrin signaling of human neutrophils on functionalized substrates through a parallel plate flow chamber (PPFC) experiment using immunofluorescence. We discovered two signaling pathways for β2-integrin activation of neutrophils on immobilized P-selectin in flow: a fast  pathway and a slow  pathway. Engagement with ICAM-1 or GPIbα mediated the transition from local integrin activation to global integrin activation. This action was followed by accelerated integrin activation over the entire cell surface. The flow load triggered Ca2+ bursts and activation of β2-integrin signaling of neutrophils on P-selectin in flow. Lipid rafts, moesin, actin, and talin-1 were involved in fast signaling, but Syk, lipid rafts, and moesin were involved in slow signaling. These results provide novel insights into β2-integrin activation, a key early event in the recruitment of flowing leukocytes to inflammatory sites of vascular surfaces.Here, we examined cell tethering, Ca2-integrin activation of firmly adhered human neutrophils on immobilized P-selectin (10 μg/mL) with or without co-immobilized ICAM-1 (5 μg/mL) or GPIbα (40 μg/mL). This P-selectin density of 10 μg/mL was selected to support specific firm adhesion of neutrophils on substrates  and KIM127 (Alexa Fluor 594-labeled β2-subunit extension reporter antibody) was used to identify the β2-integrin extension on cells. Alexa Fluor 488-labeled IBL-6/2 and M1/70 (CD11a (αL-subunit) and CD11b (αM-subunit) recognition antibodies) were used to recognize the β2-integrin family members LFA-1 and Mac-1, respectively.First, we examined βbstrates . The fir2 integrin phases (red) in the absence of ICAM-1 or GPIbα that formed over the cells shared topologic structures with anti-CD11a or CD11b antibody-ligated β2-integrin phases (green), respectively. These phases were weak initially and then became intensively dose-dense as the stimulus duration increased. Engagement with ICAM-1 or GPIbα enhanced formation of KIM127-ligated β2-integrin phases (red), especially under a fluid load for 0.5 min  conformation emerged in integrins over the entire cell surface that involved ICAM-1- or GPIbα-mediated enhancement which was weak initially but which strengthened as the loading time increased.The KIM127-ligated β2-integrin conformations. We used two parameters: (i) relative density of extended-β2 integrins (KIM127-related fluorescence intensity of the cell over that of the background minus one); (ii) cell activation ratio , which was normalized through dividing it by its value in the absence of a mechanical load. Plots of the normalized cell activation ratio and relative density of extended-β2 integrins against the stimulus time at a wall shear stress of 0.2 dyne/cm2  low levels of 1.0 and 16 initially, then climbed rapidly to plateaus of 2.1 and 24, and the phase switch occurred at a stimulus-time threshold of 1–2 min, whereas engagement with ICAM-1 or GPIbα halved these stimulus-time thresholds and heightened the plateaus considerably  reduced the cell activation ratio significantly in the absence of ICAM-1 or GPIbα, but decreased it slightly in the presence of ICAM-1 or GPIbα, whereas blockade of extracellular Ca2+ influx by LaCI3 had the opposite effect. Also, blockade of extracellular Ca2+ influx and intracellular Ca2+ release reduced the cell activation ratio more significantly in the presence of ICAM-1 and GPIbα, but did not do so in the absence of ICAM-1 and GPIbα  first and then triggers Ca2+ signaling through extracellular Ca2+ influx to quicken extension of integrins over the entire cell surface  and Mg2+-treated-neutrophils  under a wall shear stress of 0.2 dyne/cm2 for loading times of 0.0, 0.5, 1.0, 2.0, 4.0, and 6.0 min. The cellular Ca2+ burst ratio  remained at a low (but stable) level in a static state, increased steeply first and then moderately with wall shear stress, but showed no significant difference within various substrates at each given wall shear stress  under a wall shear stress of 0.2 dyne/cm2  (2), the delay times of Ca2+ bursts for Mg2+-treated neutrophils on ICAM-1 alone and GPIbα alone were almost identical to those for neutrophils on P-selectin with ICAM-1 or GPIbα, respectively, but significantly shorter than those on P-selectin only  and neutrophils (on P-selectin plus ICAM-1 or GPIbα)  initially and then remained at a constant (but lower) level when wall shear stress ≥0.2 dyne/cm2 for neutrophils on ICAM-1 alone or with P-selectin, whereas wall shear stress shortened the delay time of Ca2+ bursts slightly for neutrophils on GPIbα, P-selectin alone, or in combination with each other  before perfusing a cell suspension into the flow chamber. Under a wall shear stress of 0.2 dyne/cm2 and a loading time of 6 min, 2-APB pretreatment reduced the Ca2+ burst ratio and prolonged the delay time of Ca2+ bursts prominently for cells on P-selectin alone, but not upon LaCl3-pretreatment. In contrast, LaCl3 pretreatment reduced the Ca2+ burst ratio and led to a significant increase in the delay time for Ca2+ bursts on cells (on P-selectin plus ICAM-1 or GPIbα) and Mg2+-treated cells (on ICAM-1 or GPIbα), but not upon 2-APB pretreatment (2+ bursts were derived dominantly from P-selectin/PSGL-1 axis-mediated cytosolic Ca2+ release or integrin/ICAM-1 (or GPIbα) axis-mediated extracellular Ca2+ influx. Also, the Ca2+ burst ratios and delay times for Ca2+ burst cells on P-selectin plus ICAM-1 or GPIbα had a negligible difference from those of Mg2+-treated cells on ICAM-1 or GPIbα. These data suggested that integrins on the cell-contact area were activated rapidly by P-selectin initially and then contributed to meditating extracellular Ca2+ influx through binding with ICAM-1 and/or GPIbα under WSS of 0.2, 0.6, or 2.0 dyne/cm2.Whether Ca2 to 0.45 dyne/cm2  of neutrophils on substrates coated with P-selectin (30 ng/mL) alone or with either ICAM-1 (500 ng/mL) or GPIbα (20 μg/mL) under fluid wall shear stress from 0.1 dyne/cmdyne/cm2 . Hence, dyne/cm2 .versus wall shear stress  of tethering events at a given time t were introduced and evaluated using N/NT and (PE −P)/PE, where NT denotes the total number of events and N is the number of the events with a lifetime ≥t, and PE and P are the survival ratios of tethering events in the presence and absence of ICAM-1 or GPIbα, respectively. The IIF represents the possibility that a cell remained tethered by binding integrins to ICAM-1 or GPIbα instead of P-selectin to PSGL-1 at a given tether lifetime. Plots of the survival ratio versus tether lifetime , methyl-β-cyclodextrin , 3,4-methylenedioxy-beta-nitrostyrene , cytochalasin B (actin disruptor), staurosporine (moesin inhibitor), dimethyl sulfoxide (DMSO) or no agent (control), under a wall shear stress of 0.2 dyne/cm2.Cellular Ca in flow , but did in flow 5. These2-integrin signaling via signal transducers such as lipid rafts, moesin, actin and talin. None of these treatments could regulate the tether lifetime (~0.7s) for cells on P-selectin alone. Hence, binding of PSGL-1 to P-selection was responsible for most of the tether events (The tether lifetime (~1.25s) of cells on P-selectin combined with ICAM-1 or GPIbα was shortened (~0.7s) by depolymerizing actin with cytochalasin B, disrupting lipid rafts with MβCD, blocking moesin with staurosporine, and inhibiting talin with MNS Figure 6r events , moesin r events , 39, ther events , 41, andr events , 43.2+ bursts. Treatment with MβCD, staurosporine, or cytochalasin B significantly reduced the cellular Ca2+ burst ratio in the absence and presence of ICAM-1 or GPIbα, as did piceatannol treatment in the absence of ICAM-1 or GPIbα, and MNS treatment only in the presence of ICAM-1 or GPIbα  β2-integrin axis because their treatment had no effects on the Ca2+ burst ratio or delay time of Ca2+ bursts  to a global (but slow) mode of integrin activation ignaling Figure 7liocytes  or plateliocytes . Also, bntegrins , 48, 49.2+-dependent global integrin activation via MAPK or non-MAPK pathways, in which cytosolic Ca2+ release or extracellular Ca2+ influx is involved  axis-mediated extracellular Ca2+ influx  instead of rolling, and measured integrin activation over the entire cell surface in non-real-time, but observed cell tethering and Ca2+ bursts in real-time. This strategy led to underestimation of the loading-time requirement for integrin activation over the entire cell surface and lack of information on integrin activation for rolling cells. For example, in cell rolling, integrins close to ligated PSGL-1 would extend one-by-one. These freshly extended integrins would make cell roll slower by binding with their immobilized ligands  and in vivo in our future works.We herein just examined P-selectin-induced β ligands  and woulf ICAM-1 S4. Thus rolling . Besides2-integrin activation for circulating human neutrophils. In this model, MAPK and non-MAPK signaling pathways participated in β2-integrin activation of neutrophils via the PSGL-1 axis. This involved a transition of signal transduction from a local (fast) mode to a global (slow) mode in the non-MAPK signaling cascade, and which might be a requirement for recruiting circulating leukocytes to inflammatory vascular sites. Our findings provide novel insights into β2-integrin activation but also cues in the development of molecular-targeted agents.We demonstrated a model of P-selectin-induced β2-subunit was purified from a hybridoma supernatant . Bovine serum albumin (BSA), Histopaque 1077, Histopaque 1119, Ficoll PM 400, DMSO, LaCl3, 2-APB, MβCD, piceatannol, staurosporine, and MNS were from MilliporeSigma . An actin inhibitor, cytochalasin B, was sourced from Aladdin . ACK lysing buffer and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were obtained from Gibco . Recombinant human P-selectin/Fc chimera, recombinant human ICAM-1/Fc chimera, and recombinant human CD42b (GPIbα) were from R&D Systems . Hank’s balanced salt solution (HBSS) and phosphate-buffered saline (PBS) were obtained from Gibco. NaCl, KCl, CaCl2, and MgCl2 were from purchased DM Reagents . Glucose was sourced from GBCBIO Technologies . All reagents were of the highest purity available.Two CD11a monoclonal antibodies (TS1/22 and IBL-6/2), goat anti-rat IgG (H+L) cross-adsorbed secondary antibody (Alexa Fluor 488), Hoechst 33342, and Fluo-4 acetoxymethyl (AM) ester were purchased from Invitrogen . M1/70 and 2LPM19c  were obtained from eBioscience  and Santa Cruz Biotechnology , respectively. Purified mouse anti-human CD162 clone KPL-1 was sourced from BD Biosciences . Anti-CD11+CD18 antibody (mAb24) was from Abcam . An Alexa Fluor 594-labeled IgG kit was purchased from Invitrogen. The KIM127 monoclonal antibody to the human βThe study protocol was approved by the Research Ethics Committee of Guangzhou First People’s Hospital within South China University of Technology  and aligned with the Declaration of Helsinki 1964 and its later amendments. Written informed consent was provided from all healthy volunteers.g for 30 min at room temperature. Neutrophils were collected from the appropriate density layer (granulocyte layer) and washed with HBSS (10 mL) without Ca2+ or Mg2+. After washing, the tube was centrifuged at 400 × g for 10 min at room temperature. The supernatant was removed, and erythrocyte-lysing buffer  was used for resuspension. Subsequently, the tube was shaken gently for 10 min at 37°C. Then, neutrophils were washed with HBSS (10 mL). After centrifugation at 400 × g for 10 min at room temperature, the supernatant was discarded, and the neutrophil pellet was resuspended in HBSS at room temperature to test cell viability based on the exclusion of trypan-blue dye. The obtained preparations were >95% pure and viable.Sodium-citrated whole human blood was obtained from healthy volunteers. Human neutrophils were isolated by a density-gradient separation method, as described previously . Briefly3, see w/v) to remove excess unabsorbed proteins and then incubated within the same solution for 1 h at room temperature to block nonspecific adhesion of cells. Selected densities for P-selectin, ICAM-1, and GPIbα of 10, 5, and 40 μg/mL supported firm adhesion instead of a transient tether stop. Of all five functionalized substrates of PPFC, each was used to test the firm adhesion and Ca2+ response of cells under fluid shear stress . Three substrates coated with P-selectin alone or with either ICAM-1 or GPIbα were used to test integrin activation over the entire surface of firmly adhered cells in flow.Five protein solutions were created by dissolving their respective dry powders into PBS (40 μL): P-selectin/Fc (10 μg/mL); P-selectin/Fc (10 μg/mL) plus ICAM-1/Fc (5 μg/mL); P-selectin/Fc (10 μg/mL) plus GPIbα (40 μg/mL); ICAM-1 (5 μg/mL); GPIbα (40 μg/mL). Another five protein solutions were mixed by dissolving P-selectin (30 ng/mL) alone or combining it with ICAM-1 (500 ng), GPIbα (20 μg/mL), KIM127 (1 μg/mL), or mAb24 (1 μg/mL) into PBS (40 μL), respectively. Each of these solutions was used to functionalize the parallel plate flow chamber (PPFC) (length × width × height = 20 × 5 × 0.254 mmw/v) and Ca2+ (1.5 mmol/L) at a final cell concentration of 1.0×106 cells/mL. To test whether cell adhesion was specific for P-selectin, ICAM-1, and GPIbα, each of the five substrates mentioned above was also used in the PPFC experiment. Neutrophils were pretreated with MgCl2 (10 mmol/L), mouse anti-human CD162 clone KPL-1 (20 μg/mL), monoclonal anti-LFA-1 monoclonal antibody , and anti-Mac-1 monoclonal antibodies . In each specific experiment, a neutrophil suspension (5×105 cells/mL) was perfused over each substrate for 1 min at 0.2 dyne/cm2. Images were recorded at 100 fps by a digital complementary metal oxide semiconductor (CMOS) camera  coupled to an inverted microscope. A “firm cell-adhering event” was defined as a cell movement of distance <10 μm in 1 min. All adhesion events in the view window over a 7-min observation were counted. The number of firmly adhered cells and the total number of all cells observed near the flow-chamber floor were counted by Image-Pro Plus 6.0  and Excel™ 2010 . The ratio of firmly adherent cells was calculated by dividing the number of firmly adherent cells by the total number of all cells on the flow-chamber floor.Isolated neutrophils were resuspended in HBSS containing 2% BSA  was dropped gently onto the functionalized PPFC bottom for 0.5 or 3.0 min for the static condition. In an inhibition experiment, neutrophils were preincubated with a chemical inhibitor of inositol 1,4,5-trisphosphate (IP3) receptors, 2-APB (100 μmol/L), for 8 min or an inhibitor of Ca2+ channels on cell membranes, LaCl3 (10 μmol/L), for 30 min. This was done to detect the effect of Ca2+ signals on integrin activation. The firmly adherent cells on the PPFC bottom were washed with HBSS containing 2 % BSA (w/v) to remove free cells. Next, they were fixed in 4 % paraformaldehyde for 30 min at room temperature, followed by blockade with HBSS containing 2 % BSA overnight at 4°C. To identify extended integrins, fixed cells on substrates coated with P-selectin alone or with ICAM-1 were incubated in turn with the monoclonal antibody CD11a (IBL-6/2) (5 µg/mL), goat anti-rat secondary antibody Alexa Fluor 488 (10 µg/mL), and Alexa Fluor 594–labeled KIM127 monoclonal antibody (20 µg/mL) at room temperature for 1 h. Meanwhile, fixed cells on substrates coated with P-selectin alone or with GPIbα were incubated in turn with Alexa Fluor 488-labeled monoclonal antibody CD11b (M1/70) (10 µg/mL) and Alexa Fluor 594-labeled KIM127 monoclonal antibody (20 µg/mL) for 1 h at room temperature. Nuclei were stained with Hoechst 33342 (12.5 µmol/L) for 30 min at room temperature. Images were acquired under the objective lens (60 × 0.70 NA) on an inverted microscope  using the digital CMOS camera. To quantify assays, ten random fields of each group were analyzed by ImageJ, and the relative KIM127-relative cellular fluorescence intensity was evaluated by (FIC-FIB)/FIB = FIC/FIB -1, where FIC and FIB were the KIM127-related cellular and background fluorescence density per unit surface, respectively  under a wall shear stress 0.2 dyne/cm2-integrin extension if its KIM127-related fluorescence intensity was high in comparison with its background. Two parameters were used to quantify the temporal characteristics of β2-integrin extension via different durations of mechanical stimuli: (i) the relative density of extended-β2 integrins (KIM127-related fluorescence intensity of a cell over that of the background minus one); (ii) cell activation ratio , which was normalized by dividing it by its value in the absence of a mechanical load.A cell had β2+ response was done as described previously (w/v), HEPES (20 mmol/L), and glucose (20 mmol/L) in PBS (pH 7.4) at 1.0×106 cells/mL. The monochrome Ca2+-sensitive dye Fluo-4 AM was applied to detect intracellular Ca2+ at longer times. For real-time monitoring of changes in intracellular Ca2+ levels, Fluo-4 AM (1 μmol/L) was added to a neutrophil suspension for 30 min at 37°C. After centrifugation at 400 × g for 10 min at 37°C, the neutrophils were resuspended in loading buffer without Fluo-4 AM for an additional 30 min at 37°C until use. To block IP3 receptors and Ca2+ channels in cell membranes, neutrophils were incubated with 2-APB  or LaCl3 , respectively. Also, neutrophils were incubated with a disruptor of lipid rafts , actin disruptor , Syk inhibitor , moesin inhibitor , talin inhibitor , KCl (10 mmol/L), glucose (10 mmol/L), HEPES (30 mmol/L), CaCl2 (1.5 mmol/L), 1 % BSA (w/v), 12 % Ficoll (w/v), pH 7.4). A neutrophil suspension was perfused over no agent, 1 % BSA, or each of the above-mentioned PPFC substrates under different wall shear stress  for 7 min. Fluorescence images of firmly adherent cells were acquired at 20 fps by the CMOS camera coupled with the inverted microscope running NIS-Elements AR 5.01.00 for 64-bit software (Nikon). The fluorescence intensity of a cell was normalized using the equation FIN = (FIC – FIB)/FIB, where FIB is the mean background fluorescence intensity , and FIN and FIC are the normalized and mean cell fluorescence intensities, respectively. The Ca2+ burst ratio and delay time of Ca2+ bursts were used to characterize Ca2+ signaling in neutrophils. The Ca2+ burst ratio was the percentage of Ca2+ burst events in all firmly adherent cells in the viewing field over an observation period of 7 min. The delay time of Ca2+ bursts was the duration from emerging to Ca2+ bursts of firmly adherent cells on the substrate.Each of the five substrates mentioned above was used in the PPFC experiment for examining the Caw/v) and Ca2+ (1.5 mmol/L) at a final cell concentration of 1.0×106 cells/mL. The cell suspension was perfused over each of the five newly functionalized substrates under various values of wall shear stress (0.1–0.45 dyne/cm2) for 5 min. A tether event (stop between two consecutive movements of flowing cells) and the stop time of cells on substrates were recorded at 100 fps by the digital CMOS camera coupled to an inverted microscope  and analyzed using Image-Pro Plus 6.0 and Excel™ 2010. The lifetime of ≥100 tethering events was analyzed and averaged for each experiment. Meanwhile, as described above, neutrophils, which had been pretreated with MβCD, cytochalasin B, piceatannol, staurosporine, and MNS as well as DMSO (vehicle control), respectively, were also used to detect the actor in tether events. To examine if a tether event was specific for P-selectin, LFA-1, and Mac-1, neutrophils were pretreated by KPL-1 (20 μg/mL), TS1/22 (10 μg/mL), or 2LPM19c (10 μg/mL). The tethering adhesion ratio was calculated by dividing the number of tethered cells by the total number of all cells observed near the flow-chamber floor for 1 min, and the tether force was estimated using 125 pN/(dyne/cm2), a conversion from wall shear stress to tether force . All error bars represent the mean plus or minus standard error of the mean based on several independent experiments indicated in the figure legends.Statistical significance was analyzed by two-way (or one-way) analysis of variance (ANOVA) for multiple comparisons with the The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Research Ethics Committee, Guangdong General Hospital, Guangzhou, China. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.JW, YF, XS, BH conceived the experiments, which were performed by XS, BH, YP, HW and YJ. XS performed flow chamber assay. XS and BH performed calcium response measurement, with the helping of HW and YJ. XS and YP performed tether lifetime measurement and analysis. YF, XS, BH, YP, HW and YJ analyzed the data. JW, XS, BH, YP, JF, YJ, PG, JL, HW, YL, and QL wrote the manuscript. JW, YF and BH secured the funding. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China .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.


Long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic transmission in the hippocampus are also induced by mAChR. An AMPA receptor (AMPAR) trafficking model for hippocampal neurons has been proposed to simulate N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity in the early phase. In this study, we demonstrated the validity of the hypothesis that the mAChR-dependent LTP/LTD shares a common AMPAR trafficking pathway associated with NMDAR-dependent LTP/LTD. However, unlike NMDAR, Ca2+ influx into the spine cytosol occurs owing to the Ca2+ stored inside the ER and is induced via the activation of inositol 1,4,5-trisphosphate (IP3) receptors during M1 mAChR activation. Moreover, the AMPAR trafficking model implies that alterations in LTP and LTD observed in Alzheimer’s disease could be attributed to age-dependent reductions in AMPAR expression levels.The forebrain cholinergic system promotes higher brain function in part by signaling through the M  •1 mAChR- and NMDAR-dependent LTP and LTD share the common AMPAR trafficking pathwayM•2+-dependent exocytosis and endocytosis regulates LTP and LTDCompetition between Ca•A simple reduction in the number of AMPARs weakens LTP and strengthens LTD•The synaptic dysfunction is correlated with cognitive decline in Alzheimer’s disease Cellular neuroscience; Cellular physiology; Molecular neuroscience This finding provides further evidence supporting the validity of a unified molecular mechanism of Ca2+-dependent AMPAR trafficking in hippocampal bidirectional synaptic plasticity.Extracellular acetylcholine concentrations increase in the hippocampus by as much as 4-fold during a variety of hippocampal-dependent learning tasks,2+ into the spine cytosol via the NMDAR ion channel  and LTD (mLTD), in addition to the same Ca2+ leak and Ca2+-pump fluxes, Ca2+-pump flux from the cytosol to the ER, which is caused by sarcoendoplasmic reticulum calcium ATPase (SERCA),2+-leak flux from the ER to the cytosol, were taken into consideration  the phosphorylation/dephosphorylation dynamics of the tetrameric AMPAR ion channel subtype GluA1/A2, (3) the endocytosis/exocytosis dynamics of AMPARs, mediated by the Ca2+-sensors protein interacting with C-kinase 1 (PICK1) and synaptotagmin 1 (Syt1), and (4) the active transport of recycling endosomes containing AMPARs by molecular motor myosin Vb toward the perisynaptic/synaptic membrane  Camembrane . In hipp,33, and SAP97 has PDZ domains that interact with the phosphorylated S845 site of GluA1.2+-dependent PP2B,2+-dependent PKC,,,2+-sensor protein PICK1,,Phosphorylation and dephosphorylation of GluA1/A2 heterotetramers in postsynaptic membranes are regulated by the A-kinase anchoring protein 150 (AKAP150) signaling complex2+ concentration and mediates Ca2+-dependent endocytosis, together with clathrin and dynamin , synaptobrevin-2/VAMP2, and complexin.,,,,,Recycling endosome exocytosis is mediated by CaA network model consisting of ordinary differential equations (ODEs) was applied to mLTP and mLTD induction in adult hippocampal neurons. The flux of IP3 molecules generated by PLC activated by M1 mAChR-dependent signals was employed as the input for the mLTP and mLTD simulations. Details of the input flux of IP3 are provided in ,,,,,,,2+] is much higher than that for mLTD ASyt1], [(Ca2)ASyt1], and [(Ca2)A(Ca)BSyt1], gradually increased; however, [(Ca2)A(Ca2)BSyt1] and [(Ca3)A(Ca2)BSyt1] did not increase significantly, as shown in 2PICK1] increased with a slight delay following a rapid increase in [CaPICK1]  are recycling endosomes bound to the perisynaptic/synaptic membrane surface, namely A1A2psd(GRIP2)MyoVSyt1 MyoVSyt1] occurred after mLTP stimulation, until it was depleted by Syt1-mediated exocytosis (2)MyoVSyt1]. The decrease in the membrane AMPAR population following the peak (2)MyoVSyt1] following the depletion  and pep2-SVKI (YNVYGIESVKI), on the induction of hippocampal mLTD have been examined experimentally; pep2-EVKI had no significant effect, while the pep2-SVKI impaired mLTD.d to 80% A. Conseqd to 80% D. The red to 80% C. Such aThe inhibitory effects of PICK1 by pep2-SVKI were also examined for the NMDAR-dependent induction of LTD and LTP. The induction of LTD was impaired in a similar manner to that of mLTD, and the induction of LTP was not affected at all see , which wAs another demonstration to confirm the validity of the network model, the so-called occlusion experiment of mLTD induction was examined by utilizing the saturated inductions of NMDAR-dependent LTD . First, ,,2+ influx into the spine cytosol through NMDAR ion channels was introduced as the input for LTP and LTD simulations. In the present study, to verify the hypothesis that M1 mAChR-dependent induction of LTP and LTD shares the common AMPAR trafficking pathway with NMDAR-dependent synaptic plasticity, an M1 mAChR-dependent ER model for Ca2+ dynamics was developed and incorporated into the AMPAR trafficking network model.,,C-FOS and brain-derived neurotrophic factor (BDNF), which are regulated by CREB. However, protein synthesis due to gene expression was not considered in the present model. Nevertheless, the incorporated AMPAR trafficking pathway successfully reproduced the characteristic induction forms of LTP and LTD mediated by the M1 mAChR and NMDAR. Therefore, it can be concluded that the M1 mAChR-dependent induction of LTP and LTD shares the common AMPAR trafficking pathway with NMDAR-dependent synaptic plasticity, and new gene expression is not necessary, at least in the early stages of LTP and LTD.In our previous study,CREB, have been found in the brains from patients with AD ,,,Accumulating evidence has demonstrated that cognitive decline in AD is caused by amyloid β (Aβ), which induces synaptic dysfunction and loss via tau protein.,2+ influx into the spine cytosol that were used in our previous studyIn most AD animal models, LTP deficits are observed that vary among different AD models, and although the reports on LTD are limited, they indicate enhanced induction of LTD.b-mediated transport between the WT and the AD model during the inductions of mLTP and mLTD , show significantly reduced expression after the age of 40 years.GRIA1, suggesting its effectiveness in preventing AD.Aging of the human brain causes cognitive decline in the elderly and is a major risk factor for AD. In fact, several neurotransmitter receptors that are centrally involved in synaptic plasticity, including GluA1 , namely, mAChRs, mGluRs, aARs, cannot be distinguished. The activation signals of mAChRs,In the presented models, parameter changes other than the number of AMPARs caused by gene expression were not taken into consideration. Thus, the alterations in synaptic plasticity observed in AD, especially for NMDAR-dependent LTP, could be attributed to the influence of the expression of other genes, and further experimental and theoretical studies to identify them are necessary.sumi@okayama-u.ac.jp) upon reasonable request.The COPASI input data used to generate the data for the current study are available from the lead contact Tomonari Sumi  upon activation of M1 mAChR is used as the input of simulations for M1 mAChR-dependent LTP (mLTP) and LTD (mLTD). The concentration [IP3] is given by the following ordinary differential equation:2+ concentration in spine cytosol, 2+ sensitivity of PLC,2+ influx from ER into the spine cytosol is given as the sum of Ca2+ release flux through IP3 receptor from ER toward the spine cytosol and Ca2+ leak flux from ER into the spine cytosol,2+ concentration inside ER, 2+ release through IP3 receptor, and 2+ leak. Sarcoendoplasmic reticulum Ca2+–ATPases (SERCA) works in ER as an ATP-dependent calcium pump, and causes Ca2+ outflux from the spine cytosol into ER, which is given as2+ sensitivity of SERCA.2+ influx from the ER to the spine cytosol is given byThe concentration 2+ pulses are used to induce NMDAR-dependent LTP and LTD as the input of simulations. A model of Ca2+ influx is responsible for regulation of [Ca2+], thus plays an important role in the network model of NMDAR-dependent LTP and LTD. Ca2+ influx into spine cytosol is used to elevate [Ca2+] and is modeled with two sigmoid functions:CaWe used the parameters shown in the table below for LTP and LTD stimulation.The following explanations on the AMPAR trafficking model are same as that provided in our previous work on NMDAR-dependent LTP and LTD.2+ extrusion used in this study (see below) is based on previous works.,2+ into the cytosol as a zero order reaction, which counteracts the extrusion of Ca2+ from the cytosol, so that the system sustain a basal Ca2+ concentration of ∼50 nmol/L.2+ dynamics model together with either M1 mAChR-dependent Ca2+ dynamics model or NMDAR-dependent Ca2+ dynamics model.The model of Ca,cAMP is synthesized by calcium-calmodulin bound adenylyl cyclase 1 (AC1). The related model used in this study (see below) is based on previous works.,,cAMP is degraded through multiple pathways by phosphodiesterase type 1 (PDE1),,,,,PKA binds to A-kinase anchoring protein 150 (AKAP150),,2+-binding affinity and PP2B is activated by calcium-bound CaM.2+-dependent activation of PP2B used in this study (shown below) is provided by previous works.,,,Protein phosphatase 2B  binds to AKAP15029 at the postsynaptic plasma membrane. PKC has three C2 domains that bind Ca2+ ions,2+-bound PKC phosphorylates the S880 site of GluA2 at the postsynaptic membrane.2+-binding to PKC used in this study (shown below) is provided by previous works.2+-bound PKC is according to a previous workProtein kinase C (PKC) also forms a signaling complex with the AKAP150 2+-sensor protein that has two Ca2+-binding sites and Ca2+-bound PICK1 triggers endocytosis of AMPARs by binding to phosphorylated S880 of GluA2.,,2+-binding constants, K1b and K2b, have been experimentally determined to be 1.80 and 0.27 L/μmol, respectively.K1b and K2b.k1f and k2f, to be 21.6 and 10.8 L/(μmol s),k1b and k2b, were determined to be 12 and 40 s−1, respectively, by using k1b = k1f/K1b and k2b = k2f/K2b. These forward reaction constants were adjusted so that NMDAR-dependent inductions of LTP and LTD were reproduced by the AMPAR trafficking model with these parameters.Protein interacting with C-kinase 1 (PICK1) is a Ca2+-sensor protein that has two Ca2+-binding domains, C2A and C2B.2+-bound Syt1 triggers exocytosis of AMPARs by cooperating with synaptic vesicle protein synaptobrevin-2/VAMP2, synaptic membrane protein synaptotagmin 7 (Syt7), and amongst others.,,,,2+-binding site mutations of Syt1 in both the C2A and C2B domains block hippocampal LTP.2+-binding constants of Syt1 to model a Ca2+-dependent regulating factor on the exocytosis mediated by Syt1 together with Syt7, synaptobrevin-2/VAMP2, and complexin, amongst others.,,,,,2+ ions, respectively. Ca2+-binding constants for the C2A domain, KA1, KA2, and KA3, have been experimentally determined to be 2.04 x 10−2, 2.04 x 10−3, and 3.21 x 10−4 L/μmol, respectively, and that for the C2B domain, KB1, has been determined to be 7.04 x 10−3 L/μmol.Synaptic vesicle protein, synaptotagmin 1 (Syt1) is a CakA1f, kA2f, kB3f, kB4f, and kA5f, to be 2.0, 1.0, 1.0, 1.0, and 0.5 L/(μmol s),kA1b, kA2b, kB3b, kB4b, and kA5b, were determined to be 97, 488, 142, 142, and 1560 s−1, respectively, by using the experimentally determined binding constants, KA1, KA2, KA3, and KB1. These forward reaction constants were adjusted so that NMDAR-dependent inductions of LTP and LTD were reproduced by the AMPAR trafficking model with these parameters.If we assumed these forward rate constants, ,We incorporated GluA1/A2 heterotetramer, which is the most dominant subtype at hippocampal neurons,2PICK1-binding to the AMPARs with two dephosphorylated GluA1s and two phosphorylated GluA2s  mediates exocytosis of AMPARs, and the AMPARs are internalized into the cytosol as recycling endosomes. The recycling endosomes containing AMPARs undergo diffusion in the cytosol and those bound to molecular motor myosin Vb are transported to the peri-synaptic and synaptic membrane surface. On the basis of an analogy with Syt1-mediated synaptic vesicle exocytosis at peri-synaptic and synaptic membrane that results in neurotransmitter release,,,,,2+-binding Syt1 species are included in the recycling endosomes together with AMPARs with various states. In the present work, for simplification, we introduce the following approximation: the Syt1 proteins are artificially remained at the postsynaptic membrane when AMPARs are internalized as the recycling endosomes; the Syt1 proteins left at the postsynaptic membrane bind to the recycling endosomes as immediately as myosin-Vb molecules transport the recycling endosomes to peri-synaptic and synaptic membrane surface.Ca


NPM1, also known as nucleophosmin-1, B23, NO38, or numatrin, are seen in approximately one-third of patients with acute myeloid leukaemia (AML). A plethora of treatment strategies have been studied to determine the best possible approach to curing NPM1-mutated AML. Here, we introduce the structure and function of NPM1 and describe the application of minimal residual disease (MRD) monitoring using molecular methods by means of quantitative polymerase chain reaction (qPCR), droplet digital PCR (ddPCR), next-generation sequencing (NGS), and cytometry by time of flight (CyTOF) to target NPM1-mutated AML. Current drugs, now regarded as the standard of care for AML, as well as potential drugs still under development, will also be explored. This review will focus on the role of targeting aberrant NPM1 pathways such as BCL-2 and SYK; as well as epigenetic regulators (RNA polymerase), DNA intercalators (topoisomerase II), menin inhibitors, and hypomethylating agents. Aside from medication, the effects of stress on AML presentation have been reported, and some possible mechanisms outlined. Moreover, targeted strategies will be briefly discussed, not only for the prevention of abnormal trafficking and localisation of cytoplasmic NPM1 but also for the elimination of mutant NPM1 proteins. Lastly, the advancement of immunotherapy such as targeting CD33, CD123, and PD-1 will be mentioned.Mutations in  NPM1-mutations, targeting NPM1 is a constant and ongoing area of research, and the wide variety of drugs available are dependent on the types of co-mutations present in NPM1-mutants. Despite various clinical trials and research of new agents targeting NPM1-mutants, about 50% of patients either relapse or unfortunately die due to the progression of the disease, and this is often the case in patients who are unable to withstand the clinically aggressive nature of AML, nor the harshness of intensive chemotherapy required for disease remission. Seeing as various cellular pathways are affected by NPM1 mutations, various targeted approaches are being investigated to tackle the challenges in treating relapsed and refractory AML patients. Some examples of NPM1-targeting approaches include DNA modulation, ribosome biogenesis, protein deregulation, mitochondrial-mediated cell death, and antibody-mediated treatments.Due to the large proportion of AML patients exhibiting NPM1-mutant detection, haematologists and medical governing bodies are implementing the use of MRD, also known as a measurable residual disease, for AML patients. The use of MRD can act as a prognostic tool for patients’ treatment, especially during the post-transplantation stage, allowing for a better understanding of the disease progression and prediction of survival outcomes.With the current understanding of NPM1-mutated AML risk stratification, monitoring of NPM1 mutants by MRD, and finally targeting NPM1-mutations for treatment of AML patients.Within this review, an overview of the structural changes influencing the functionality of cells is given, followed by NPM1 is localised to chromosome 5q35 and consists of 12 exons. Specifically, NPM1 contains the N-terminal core domain, acidic domain, and nuclear localisation signal domain are found to be highly conserved among each protein of the NPM family, indicating that all three proteins have a similar functional role. However, the less conserved C-terminus, on the other hand, suggests an alternative role for NPM1. It is highly conserved between metazoans and when mutated can result in leukaemic-initiating activity .  Seen in  . TModulation of the NPM1 structure is governed mainly by post-translational modifications including phosphorylation, SUMOylation, and ubiquitination. NPM1 proteins are comprised of three different regions: the N-terminal domain (NTD), the central region, and the CTD, each with a distinct functional role  with their negative charge properties allowing mimicry of nucleic acids and thereby influencing the nuclear localisation signal (NLS). NLS regions interact with importin alpha and beta causing nuclear translocation of the NPM1 protein. The acidic central region of NPM1 facilitates histone binding to both core and linker histones for the formation of perinuclear heterochromatin . AdditioThe CTD contains predominantly basic and positively charged amino acids influencing its role in binding to nucleic acids. At the CTD, the nucleolar localisation signal (NoLS) domain allows the localisation of NPM1 to the nucleolus with aromatic residues (tryptophan 288 and 290) forming a three-helix structure and stabilises NoLS due to its hydrophobic core. The lack of aromatic residues is common in NPM1 mutant proteins and results in the unfolding of its helical structure which inhibits nuclear import and nucleolus import of NPM1. Aberrant localisation of NPM1 is typical amongst AML patients.NPM1 is a multifunctional protein influencing processes involved in ribosome biogenesis, mRNA transcription, embryonic development, DNA repair, chromatin remodelling, and apoptosis. The most well-studied function of NPM1 is its involvement in directing ribosome biogenesis .A mechanism known as liquid-liquid phase separation (LPS), has been proposed to explain the effects of NPM1 localisation in the nucleolus on ribosome biogenesis. Modulation of LPS occurs via homotypic and heterotypic interactions of the three key regions  of the NPM1 protein . Three tThe nucleolus subcomponent—the fibrillar centre (FC), is responsible for rRNA transcription. In the initiation of rRNA gene transcription, two components of the FC are formed: the dense fibrillar component (DFC) and the granular component (GC) . The GC NPM1-mutated AML.DNA damage causes defects in mRNA processing, which leads to potential translation errors downstream; however, this is often remedied by translesion synthesis which is employed as a DNA repair mechanism by Polymerase Eta (Poly eta), a low-fidelity polymerase ,24. NPM1A recent study also noted the impact of long noncoding RNA (lncRNA) increasing NPM1 mutant samples. Analysis using genome-wide RNA sequencing concluded that the introduction of lncRNA led to an increase in cell differentiation proteins; THSB1, MAFB, and ASB2. In addition, in vivo experimentation on mice confirmed decreased survival and accelerated AML pathogenesis in the presence of lncRNA. Decreased levels of lncRNA increases sensitivity to the chemotherapy drug, cytarabine, thus affecting treatment outcome . OverallNPM1 mutations. Assembly of the histone octamers, removal of DNA from histones, and transfer of histones from other chaperone proteins or enzymes are all roles of histone chaperones that are associated with NPM1 [Centrosome duplication influences the activation of chromatin and thus microtubule distribution. Correct chromatin remodelling allows a smooth transition of the cell cycle from interphase to metaphase . The forith NPM1 . Collectith NPM1 ,33.ARF by NPM1 allows binding to MDM2 via rpL5. ARF-binding partners, such as MDM2 and NPM1, associate with ribosomal proteins, which implicates its role in p53 homeostasis [NPM1 mutation-positive AML patients [Within healthy cells, NPM1 has the capacity to trigger either cell proliferation or apoptosis. However, a shift in homeostasis can affect NPM1 translocation to the cytoplasm, which in turn affects protein interactions in the cytoplasm profoundly. NPM1-induced activation of p53 stress-response mechanisms leads to a mitochondrial-associated release of extrinsic factors such as caspase and cytochrome c . Mouse deostasis . Studieseostasis . Therefoeostasis . Recent patients . TherefoNPM1-mutated AML, and this increases its activity as a chromatin-binding factor by upregulating proliferating cell nuclear antigen (PCNA), a NER protein. Subsequently, PCNA forms a homo trimer, interacts with cell cycle regulator p21 in the nucleus, and promotes DNA repair [DNA repair response mechanisms are key to maintaining proper cell differentiation and genome stability. Post-translational modification of NPM1 regulates cell cycle proteins and promotes stability. DNA double-stranded lesions caused by UV results in the initiation of the nucleotide excision repair pathway (NER) which is overexpressed in A repair ,41. ReleA repair . NPM1-muA repair . As mentA repair ,45.NPM1-mutated AML has been acknowledged as a distinct entity [FLT3-ITD , DNMT3A , IDH1/2 (isocitrate dehydrogenase ½), and TET2 (Tet Methylcytosine Dioxygenase), raising speculation of synergistic interactions that promote AML transformation and progression [NPM1 is a driver mutation and often a secondary event to pre-existing mutations generated by clonal hematopoiesis of indeterminate potential (CHIP) [NPM1-mutated AML, without FLT3-ITD, belongs to a favorable risk group. Cytogenetically normal NPM1-mutated AML with FLT3-ITD belongs to an intermediate risk group exhibiting adverse outcomes, especially in the presence of the aforementioned co-mutations [NPM1-mutated AML cases exhibit normal karyotypes, with the remaining cases showing various cytogenetic abnormalities e.g., del (9q), +21, +8, and +4 in AML [According to the current WHO classification of hematopoietic malignancies, t entity . Nonethegression ,48,49. Il (CHIP) ,47,50. Tutations . It must4 in AML .NPM1 insertional mutations have also been found in myeloid neoplasms including myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), and chronic myelomonocytic leukaemia (CMML). In lymphoid neoplasms, translocation events are reported. Accounting for about 2% of NPM1-mutant-related diseases, NPM1-mutation in MDS has been associated with a higher risk of secondary AML and is most commonly found in MDS with an excess blast (MDS-EB) [JAK2-V617F and associated diseases include essential thrombocythemia (ET) and myelofibrosis (MF) of which NPM1-mutations have been found on a rare occasion [NPM1-mutant cells seen in MPN and possible regimens without the need for stem cell transplantation. Contributing to 3% of NPM1 mutant cases, CMML with NPM1 mutation shows chemosensitivity to anthracyclines and cytarabine (7 + 3 induction chemotherapy), and hypomethylating drugs have promising effects in maintaining remission [NPM1 mutations outside of AML can be found.(MDS-EB) ,54. Moleoccasion ,56. Thisemission ,57. On temission ,59. It mMRD− to CRMRD+, or the increase of PB/BM-MRD by ≥1log10 between two positive samples [NPM1-mutated AML data, log reduction of MRD should be the preferred choice for reporting, as multiple studies have shown that MRD kinetics are reliable prognostic indicators of the outcome, but not absolute MRD levels [With the advent of modern technology and improved molecular techniques, MRD has become indispensable for AML patient management and monitoring of mutant NPM1 before, after, and during the course of treatment ,61,62,63 samples . For betD levels . TechnicD levels .Conventional technologies for MRD detection include flow cytometry and molecular MRD assessments by qPCR and ddPCR. In AML patients receiving hematopoietic stem cell transplantation (HSCT), MRD positivity at pre-HSCT by flow cytometry is strongly correlated with worsened overall survival and leukemia-free survival (LFS) than MRD-negative patients. Pre-HSCT MRD negativity is also associated with a reduced incidence of relapse supporting the predictive significance of MRD monitoring on survival and post-HSCT relapse . SimilarNPM1-mutated patients, a unique immunophenotype of AML patients was identified [NPM1 mutation strongly expressed myeloperoxidase (MPO) and CD33 with dim expression of other myelomonocytic antigens commonly expressed in AML including CD13, CD65, CD15, and CD14. Its utility in the MRD monitoring of NPM1-muated AML in general is yet to be confirmed. Therefore, molecular MRD strategies targeting these specific mutations are crucial for more specific analyses of MRD kinetics and clonality.When evaluating MRD using multiparametric flow cytometry specifically in entified . LeukaemNPM1-mutated AML should be monitored by qPCR and ddPCR; the implication of both approaches have been extensively published and reviewed [NPM1 mutations are commonly monitored using RNA normalized to ABL transcripts, a technique that is used to monitor other fusion genes in a similar fashion. Meanwhile, recent studies and in-house data (data not published) have shown that quantification of NPM1 mutation allele frequency at the DNA level is a good alternative to RNA-based assay. In spite of lower sensitivity than RNA-based assays, clinical correlation with DNA mutation allele frequencies are also observed. This permits flexibility of the MRD protocol and alleviates the need to normalize to ABL1 which has a significantly slower expression rate than NPM1 [As per the latest MRD guidelines from the ELN, molecularly defined groups such as reviewed ,60,61,68reviewed . NPM1 muhan NPM1 .NPM1 mutations months prior to the onset of de novo AML even though coverage is generally lower than gene panel sequencing [NPM1 mutations as non-type-A mutations that are reported to confer shorter survivals and inferior clinical outcomes than wild-type or type A mutations [NPM1 type A mutants but not other NPM1 mutation subtypes.Application of NGS for MRD monitoring can be conducted at bulk tumour level and single-cell level, although none of them are routinely applied. Bulk tumour sequencing is most commonly adopted at the early stages of AML as screening tools that allow simultaneous screening of a vast number of mutations. Whole exome sequencing (WES) can detect quencing . NGS alsutations ,72,73. DNPM1 mutated AML patients [NPM1 mutant clone has been revealed, and serial monitoring has shown that the population changes dynamically with the acquisition of NPM1 and FLT3 mutations during the intermediate stages of the disease. Clonal switch of NPM1 co-mutation with PTPN11 to WT1 is also resolvable at relapse, demonstrating the benefits of high resolution NGS. The ultra-sensitivity of single-cell technology is demonstrated with <1% of mutant cells carrying NPM1 or other SNPs out of as few as 7000 cells being sequenced per time point [low patient achieved CR, the MRD positivity observed in a few p-ERK1/2low patients is due to the presence of NPM1 mutations. This provides a novel method that deciphers the effect of intracellular signalling on MRD dynamics [Similarly, the application of single-cell technology is continuously under development for MRD monitoring for improved resolution. Both DNA and conventional RNA single-cell sequencing have been employed in MRD monitoring in AML but are not routinely used due to cost and the high requirement for sample preparation ,75. Withpatients . In thesme point . When sime point . In a codynamics . This alNPM1-mutated, so developing treatment approaches for this population is becoming crucial. Upon initiation of treatment, a risk assessment is recommended by the ELN for diagnosis and management [NPM1-mutated AML patients involves induction with intensive chemotherapy followed by consolidation chemotherapy. Co-occurrence of FLT3-ITD and/or DNMT3A mutation is found in approximately 66% of NPM1-mutated AML, which portends an inferior prognosis [NPM1 mutant transcripts following induction or consolidation chemotherapy is associated with high relapse rates [NPM1-mutant transcripts in the peripheral blood after the second cycle of chemotherapy were associated with a higher risk of relapse compared to those with absent NPM1 mutant transcripts (82% versus 30%) after three years of follow-up [NPM1-mutated AML receiving allogeneic HSCT, the estimated two year overall survival was 83%, 63%, and 13% in patients with negative, low , and high levels of MRD respectively after a median follow-up of 4.9 years [NPM1-mutated AML patients undergoing allogeneic HSCT, positive measurable residual disease (MRD) prior to transplantation was associated with high risk of post-HSCT relapse [NPM1-mutated AML  [0 years) ,89. Anth0 years) ,91. The 0 years) . The mos0 years) . A stabl0 years) . Next, d0 years) ,95. Duri0 years) . HoweverAnother mechanism of anthracyclines is via the immunostimulatory pathway STING, which is a common immune response pathway upon pathogen entry . STING mNPM1-mutated AML individuals. Notably, older adults showed longer remission rates in the CPX-351 arm and had a similar safety profile as the intensive standard chemotherapy arm [NPM1-mutated AML patients, cytarabine efficacy is comparable to anthracycline efficacy due to cytarabine’s role in inhibiting DNA replication and the capacity of NPM1 to regulate transcriptional processes. As a result, it is highly effective against rapidly diving cells regardless of the subtype of mutated NPM1 [Cytarabine is used with anthracycline (7 + 3 regimen) during the remission induction. High-dose cytarabine (HDAC) is used in the consolidation of AML. LDAC is prescribed for patients unfit for intensive induction chemotherapy. Cytarabine  is a pyrimidine analogue that competes with the base pair cytidine in both DNA and RNA. Therefore, by limiting DNA polymerase beta and base excision repair, inhibition of DNA replication occurs, particularly at the S phase ,104. In rapy arm . This suted NPM1 ,107.NPM1-mutated patients carry FLT3 mutations, initial screening of patients with FLT3-ITD or FLT3-TKD (tyrosine kinase domain with missense point mutation) benefit greatly from FLT3 inhibitors in combination with various chemotherapy [As a large proportion (about 25%) of otherapy . Under notherapy . Subsequotherapy ,110. Theotherapy ,112.FLT3-ITD mutants exhibit persistent activation in the absence of ligand binding, resulting in the formation of its dimeric form and downstream cell proliferation [FLT3-ITD positive AML cases [FLT3-ITD-positive MRD pre-transplant has been associated with higher rates of relapse. A recent study has shown better overall survival within double positive FLT3-ITD and NPM1 mutations in both younger and older patients, signifying NPM1’s influence on using FLT3-ITD inhibitors [FLT3-ITD mutants play in NPM1-mutated AML patients. Some studies also show combination with venetoclax show molecular clearance of FLT3 mutations [feration . Midostaferation . Second-ML cases . CollectML cases . Second-hibitors . Therefoutations .®, Philadelphia, PA, USA) is the next FDA approved drug for AML. According to the national comprehensive cancer network (NCCN), GO administration should be given to either CD33-positive patients as post-remission therapy or patients placed in the favourable and intermediate cytogenetic risk groups [NPM1-mutated AML. In terms of NPM1 influence on GO treatment effect, CD33 cells are abundant among NPM1-mutated AML patients [NPM1-mutated/FLT3-WT genotype [The CD33 antibody-drug conjugate (ADC)—gemtuzumab ozogamicin (GO (CMA-676); Wyeth Laboratories, Mylotargk groups . In combpatients . Severalpatients ,121,122.genotype .NPM1-mutated AML [GO is useful due to its conjugated cytotoxic antibiotics, calicheamicin. Calicheamicin  inflicts double-stranded cleavages on DNA which result in apoptosis . The CD3ated AML . Calicheated AML . Next, gated AML .NPM1-mutated AML patients who are unfit and/or elderly (>60 years), is a combination of venetoclax and HMA or LDAC. The small FDA approved molecule, venetoclax, targets the NPM1 apoptotic pathway by inhibiting B-cell leukaemia/lymphoma-2 (BCL-2). BCL-2 is a key anti-apoptotic protein that sequesters BAX/BAK, thereby regulating mitochondrial outer membrane permeabilization (MOMP) [NPM1 mutation reveal an increase in sensitivity to chemotherapy drugs through suppressing NF-κB activity and BCL-2/BAX expression, which is pertinent to the efficacy of venetoclax seen in NPM1-mutated AML [FLT3-ITD or loss of TP53 causing a mitochondrial defect in NPM1-mutated AML patients. Additionally, IDH1/2 mutations occur in NPM1-mutated AML with increasing age influencing the efficacy of venetoclax-based therapy [The standard treatment protocol for newly diagnosed n (MOMP) . BAX/BAKn (MOMP) ,129. Venn (MOMP) . Studiesated AML . Predictated AML . However therapy .DNMT3A) and NPM1. DNMT3A mutants in hematopoietic stem cells (HSC) and commonly associated as a concomitant gene for NPM1-mutated AML, and precursors for de novo adult AML cases. Cooperation between the two genes shows the activation of promoters influencing preleukaemic mechanisms such as PI3K/Akt/mTOR signalling, stem cell pathways, and HSC cycling. Thus, showing the significance of using demethylating drugs such as azacytidine and decitabine in treating NPM1-mutated AML [The use of HMA drugs in standard protocols is due to their proven efficacy and effect in MDS patients, but it must be noted that AML contains specific clinical characteristics, making it distinct from MDS. Mechanistically, the influence of NPM1 on DNA methylation was studied in mouse models with mutant DNA methyltransferase (ated AML . Azacitiated AML . HMA’s hNPM1-mutated cells. Actinomycin D (or dactinomycin) is a low-cost chemotherapy drug that induces nucleolar stress by targeting RNA polymerase I to inhibit transcription elongation. The structure of Actinomycin D resembles the ring structure of nitrogenous bases in DNA, therefore enabling its intercalation into DNA. At low doses, actinomycin D induces stress responses in the cell by increasing ROS through the activation of cyclic GMP-AMP synthase signalling and the release of mitochondrial DNA [Another potential target is inducing nucleolar stress on rial DNA . Mechanirial DNA . Therebyrial DNA .ATO and ATRA are popular molecular targeted treatment strategies that inhibit downstream phosphorylation pathways and have been implicated in acute promyelocytic leukaemia (APL) treatment. The myeloid leukaemia cell differentiation protein (Mcl-1) inhibits apoptosis by promoting the growth of AML cells. Studies have suggested ATO inhibits AKT and the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade thereby upregulating Mcl-1. Usually, Mcl-1 is downregulated via the AKT (protein kinase B) pathway by GSK-3β inhibiting Mcl-1 phosphorylated at Ser159 . In addiNPM1-mutated AML cells [NPM1-mutated AML cells with cysteine 288 are highly sensitive [NPM1-mutated AML, a possible explanation for its efficacy is its synergy with ATO as seen in both APL and AML cases. Notably, daunorubicin sensitivity increases NPM1-mutated AML cells’ post-treatment sensitivity to ATO-ATRA, showing promise as possible sensitisers for leukemic cells before chemotherapy [As a curative frontline treatment for APL, ATO-ATRA molecularly targets the oncogenic fusion protein; promyelocytic leukaemia–retinoic acid receptor α (PML::RARA) . By applML cells . ATO posensitive . Arsenicotherapy .XPO1 is a key protein that interacts with the NES regions of NPM1, resulting in an increase of cytoplasmic NPM1 proteins due to increased NES signalling. Therefore, targeting chaperone proteins of NPM1 will limit its activity in the cytoplasm, thereby sequestering it to the nucleolus/nucleus. Examples of XPO1 inhibitors include leptomycin, selinexor, and eltanexor. Leptomycin B has shown severe toxicity in vitro as it binds irreversibly to XPO1, therefore selinexor monotherapy was implemented in NPM1-mutated AML patients; however, selinexor has shown only temporary and insignificant clinical benefits due to its high toxicity . The knoNPM1-mutant AML cases, overexpression of HOXA and HOXB genes and cofactor MEIS1 are often observed. Inhibition of MLL-Menin complex binding results in the downregulation of HOX cluster genes, specifically HOXA9 (leukemogenic homeobox A9) and MEIS1 (myeloid stem progenitor homeobox transcription factor) [Menin is a scaffold protein involved in many protein-protein interactions and most notably, it can act as an oncogenic cofactor when bound to KMT2A (histone-lysine-N-methlytransferase 2A (MLL1)) complexes, resulting in gene transactivation . KMT2A i factor) . This HO factor) . As provCurrently, a few drugs are undergoing early-phase clinical trials on relapsed/refractory AML patients with KMT2Ar/NPM1 to evaluate efficacy as either an MLL-Menin complex inhibitor (KO-539 (NCT04067336), SNDX-5613 (NCT04065399), and JNJ-75276617 (NCT04811560)) or menin inhibitor (DS-1594b (NCT04752163) and BMF-219 (NCT05153330)). However, due to the targeted nature of therapy, the careful identification of biomarkers indicating patient suitability for the use of menin inhibitors would be beneficial and a key strategy in overcoming unwanted leukemic activity. The clinical trial, SNDX-5613 (NCT05326516), is currently underway to evaluate the potential of using menin inhibitors with chemotherapy drug cocktails .NPM1-mutated AML, with the driving factor being an oncoprotein, immunotherapy is an ideal approach that focuses on either targeting mutant NPM1-derived neoantigens or genetically engineering T cells specific for NPM1-mutated peptides. Various studies have attempted to determine ideal NPM1-derived immunogenic peptides suitable for HLA- (Human leukocyte antigen) induced T cell response. Efficient HLA presentation has been seen targeting 11 residues of the NPM1-mutated C-terminus due to the distinct amino acid sequence not normally found in human tissues. Specifically, HLA class I ligandomes: AIQDLCLAV, AIQDLCVAV, AVEEVSLRK 9-mer, CLAVEEVSL, LAVEEVSLR, and CLAVEEVSLRK have been studied [Another targeted-therapy approach on the rise is cancer immunotherapy treatments. As mentioned previously, NPM1 is a highly specific and stable mutation with relapses usually detected by increasing MRD . Due to  studied ,151,152.Another immunotherapy approach targeting NPM1 is the use of chimeric-antigen receptor T cells (CAR-T) and TCR-engineered T cell therapies. The most successful antibody therapy is the monoclonal antibody, GO, which shows promise in targeting the myeloid-specific transmembrane CD33. From this knowledge, bispecific CAR-T cells were designed that targeted CD33 as well as CD123, an IL3 receptor found overexpressed in leukemic neoplasms for example AML. Dual CAR targeting the CD33 and CD123 allows specificity to AML leukemic cells; this strategy is effective as a single expression of CD33 or CD123 by healthy cells will result in no or attenuated CAR-T cell response. The anti-CD123 first-generation CAR is attached to a CD3ζ domain, whilst anti-CD33 contains only CD28 and 41BB costimulatory domains .NPM1-mutated patients and absence in NPM1-WT. The ligand PD-L1, which inhibits the activation of T cells via PD-1 receptors downstream, is commonly overexpressed in tumour cells and is frequently associated with poor clinical outcomes [+/NPM1mut patient subgroups.Another T-cell immune response strategy involves targeting the programmed death 1 antibody (PD-1). PD-1/PD-L1-directed immunotherapy shows potential due to its expression in outcomes . Nivolumoutcomes . Nivolumoutcomes . Studiesoutcomes . AccordiNPM1-mutated AML are currently undergoing clinical trials, and these include entospletinib (GS-9973) and mivavotinib (TAK-659). The second-generation SYK inhibitor, entospletinib, is a reversible competitive small molecule with higher sensitivity to JAK-2, c-KIT, FLT 3, and RET [NPM1-mutated AML patients are still ongoing. Mivavotinib, a heteroaromatic SYK inhibitor, inhibits both SYK and FLT3 by competitively binding at the adenosine triphosphate-binding site. To note, AML patients with HOXA9/MEIS1 overexpression show improved survival with entospletinib, suggesting its involvement with menin-MLL complex inhibition [FLT3-ITD more sensitive to SYK inhibition, further supporting the screening of FLT3-ITD as an important determinant of prognosis in AML patients [NPM1-mutant AML with a better-predicted response rate for FLT3-ITD positive patients [Another target of interest is non-receptor spleen tyrosine kinase (SYK). Under normal circumstances, Lyn (tyrosine kinase protein) auto-phosphorylates Syk at Tyr518 and generates binding sites for the BCR signalling pathway regulators CBL, VAV1, and phospholipase C-gamma . SYK inh and RET . Clinicahibition . In vitrpatients . Thus, cpatients .NLRP3 mRNA and further downstream analysis on AML cell lines saw the presence of high mobility group box 1 (HMGB1) protein. This suggests the influence of stress on the HMGB1-NLRP3 axis and is a potential target for preventing AML progression [NPM1 and DNMT3A shows an increased expression of HMGB1, similar to what is hypothesised on mice exhibiting chronic stress. Thus, showing an overlap between the various suggested concepts. Another suggested mechanism for targeted therapy involves diverting immune escape by preventing the release of the immune suppressor protein galectin-9, which limits the release of cytokines. The stress-related hormone cortisol triggers the upregulation of neuronal receptor latrophilin 1 (LPHN1), and the upregulation of fibronectin leucine-rich transmembrane protein 3 (FLRT3). Subsequently, the release of galectin-9 causes immune escape [Recent studies show the significance of stress in the promotion of AML due to the accumulation of IL-1β. One possible mechanism is via the activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasomes leading to the progression of AML cells. Studies conducted on chronically stressed mice showed upregulation of gression . In addie escape . With ree escape ,163. ThoNPM1-mutated AML should be examined in terms of its mutational landscape and clonal evolution for better patient-specific treatment needs. Likewise understanding the immunophenotypic markers  of patients can better understand immune resistance and escape mechanisms in AML [Altogether, it is evident that the genetic diversity of s in AML ,165. Ones in AML . With coNPM1-mutated AML. Research focusing on understanding the functional biology of mutated NPM1 showed numerous mechanisms contributing to disease progression. However, the underlying molecular pathways driving NPM1-mutated AML pathogenesis have yet to be elucidated. Therefore, conducting further research into the molecular drivers of NPM1-mutated AML pathogenesis can lead to new targeted strategies. As recommended, mutational screening of FLT3-ITD, DNMT3A, IDH1/2, and TET2 could help guide clinicians in selecting the appropriate targeted treatment strategies for patients.Targeted therapy has become an important treatment strategy in targeting NPM1 mutants, treatment options will differ. Although the standard of care currently for AML is the “7 + 3” regimen, consideration is needed for NPM1-mutated AML co-mutated with; FLT3-ITD, for which FLT3 inhibitors are used or SYK inhibitors may also be used as in vivo studies have inferred SYK involvement in FLT3 activation. Other current treatment strategies include venetoclax and hypomethylating agents, Actinomycin D, and GO. Novel treatment strategies worth investigating is the use of daunorubicin, a popular anthracycline, in tandem with ATO and ATRA, menin inhibitors (due to the commonly seen overexpression of HOX cluster gene and XPO1 inhibitors linked to prolonged survival in mice studies. Lastly, immunotherapeutic approaches such as HLA-dependent T cell, CAR-T, and anti-PD-1 immunotherapy, should also be explored.Currently, there are many targeted therapies, but due to the heterogeneity of In addition, due to the wide array of suggested treatment strategies potential unwarranted side effects can occur. Therefore, further research on MRD monitoring strategies such as single-cell NGS and CyTOF on top of the typical qPCR and ddPCR may benefit in overall survival of patients and treatment management.