At the onset of plaque deposition, somatosensory cortical astrocytes of AD female mice exhibit a drastic reduction of Ca2+ signaling, closely associated with decreased endoplasmic reticulum Ca2+ concentration and reduced expression of the Ca2+ sensor STIM1. In parallel, astrocyte-dependent long-term synaptic plasticity declines in the somatosensory circuitry, anticipating specific tactile memory loss. Notably, we show that both astrocyte Ca2+ signaling and long-term synaptic plasticity are fully recovered by selective STIM1 overexpression in astrocytes. Our data unveil astrocyte Ca2+ hypoactivity in neocortical astrocytes as a functional hallmark of early AD stages and indicate astrocytic STIM1 as a target to rescue memory deficits.Calcium dynamics in astrocytes represent a fundamental signal that through gliotransmitter release regulates synaptic plasticity and behaviour. Here we present a longitudinal study in the PS2APP mouse model of Alzheimer’s disease (AD) linking astrocyte Ca 2+ signaling is involved in the pathogenesis of Alzheimer’s disease. Here, the authors show Ca2+ hypoactivity in astrocytes at plaque deposition onset related to reduced expression of the Ca2+ sensor STIM1 and impaired synaptic plasticity that was rescued by STIM1 overexpression in astrocytes.Altered Ca In this searching for a cure, a fundamental, albeit neglected, aspect is that brain function depends on dynamic interactions between neurons and astrocytes, a rather heterogeneous type of glial cells3. Indeed, besides representing fundamental neuron-supportive elements in the control of brain tissue homeostasis, astrocytes are specifically recruited to neuronal circuits by neurotransmitters and neuromodulators that evoke cytosolic Ca2+ rises, essentially mediated by G-protein coupled receptors (GPCRs)4. Activated astrocytes release gliotransmitters that contribute to regulate brain functions, including memory mechanisms based on short- and long-term synaptic plasticity, neurovascular coupling and behavior6. The importance of gliotransmission in brain physiology and the widely recognized alteration of Ca2+ homeostasis in neurons and astrocytes from AD mice8 demand targeted studies to unveil novel therapeutic targets and early AD hallmarks related to astrocyte Ca2+ signaling.Alzheimer’s disease (AD) is a progressive, incurable neurodegenerative disorder characterized by a long preclinical phase and a relentless worsening of cognitive functions, most often starting with mild memory impairments. The molecular and cellular mechanisms underlying AD pathogenesis and the early events that anticipate the cognitive decline remain poorly understood. According to the “neurocentric” view of AD9. According to studies in AD mouse models based on APP and PS1 mutations, both Ca2+ hyperactivity and hypoactivity have been described at the neuronal level, with hyperactivity dominating the early stage of the disease, being brought by soluble Aβ13. In AD mice, occurrence of amyloidosis has generally been associated with an increase of astrocyte Ca2+ activity14. In APPPS1 mice, this hyperactivity involves abnormal purinergic Ca2+ signaling16, although a recent work reported a diminished sensory-evoked astrocyte responsiveness in AD mice also based on APP and PS1 mutations17. In line with this, a reduced astrocyte Ca2+ response to locomotion has been found also in the neocortex of awake-behaving 15-month-old tg-ArcSwe mice18, while a causal relationship has been demonstrated in APPNL-F mice between network hyperactivity and defective Ca2+ signaling in the cingulate cortex astrocytes at early stages of the disease19. However, it has not been established yet how Ca2+ activity evolves in astrocytes as the disease progresses and how it relates to alterations in memory encoding and retrieval.In the attempt to recapitulate the sequence of altered events occurring in brain circuits along with AD progression, a common strategy is to employ transgenic (tg) AD mouse models expressing human proteins carrying mutations associated to familial AD (FAD), especially those involved in Aβ production: the amyloid precursor protein (APP), presenilin 1 (PS1) and presenilin 2 (PS2)21), which is characterized by neuronal hyperexcitability22. In these mice, changes in brain circuitry, relevant to the development of AD, are detectable at both 3 and 6 months of age25, earlier than the onset of spatial memory deficits revealed at 8 months of age with the Morris Water Maze (MWM) test26. Specifically, PS2APP mice at 3 and 6 months of age are marked by alterations in the spontaneous electrical brain activity linked to slow waves, i.e., those oscillations that are responsible for global connectivity28 and are closely linked to defective long range Ca2+ signaling in AD mice29. Given that FAD-linked PS2 mutations are coupled to alterations of Ca2+ homeostasis30 and considering that the mutated PS2 is also expressed in astrocytes, the PS2APP mouse model is expected to be highly informative on the role of astrocyte Ca2+ dynamics in AD. In the present study, we investigate the alterations of astrocyte Ca2+ signals along with AD progression as well as their impact on long-term potentiation (LTP) in the somatosensory cortex (SSCx), a brain region where astrocytes play a fundamental role in synaptic plasticity32. Furthermore, we shed light on the mechanisms underlying these changes and provide a proof-of-principle approach to rescue astrocyte-dependent synaptic plasticity, suggesting the astrocyte Ca2+ sensor stromal interaction molecule 1 (STIM1) as a therapeutic target in the context of AD.Here, we take advantage of the double tg PS2APP mouse model of AD  and PS2APP female mice, at 3 and 6 months, i.e., before and after the appearance of Aβ plaques and gliosis in PS2APP mice25 , adenosine triphosphate  evokes global Ca2+ elevations in WT astrocytes at 3 and 6 months of age , together with the astrocyte marker sulforhodamine 101 (SR101)   microscopy to study astrocyte Caage Fig. . The ATPage Fig. . In parties Fig. , indicat2+ events are also affected in PS2APP mice. Spontaneous Ca2+ events in astrocytes frequently occur at microdomains, whereas they are very rare at soma and proximal processes. In the presence of TTX (1 μM), the spontaneous Ca2+ microdomain activity of 3-month-old PS2APP mice shows a significant increase in mean amplitude and a trend towards a higher frequency, measured as number of events per minute per astrocyte, with respect to age-matched WT mice , while it is higher albeit not significantly at the level of proximal processes, in terms of both percentage of recruited processes and Ca2+ signal amplitude with respect to age-matched WT mice . In 6-month-old PS2APP mice, spontaneous signal amplitude is significantly reduced with respect to age-matched WT mice at proximal processes, both ex vivo and in vivo, while it shows a trend toward a reduction at the somatic level .We next evaluated whether astrocyte spontaneous Caice Fig. . In contude Fig. . In vivoude Fig.  confirm ice Fig. . We exte2+ alterations in PS2APP mice are influenced by the distance from Aβ plaques, 6-month-old PS2APP mice were i.p. injected with methoxy-X04 (M04), an Aβ-plaque vital marker, 12 h before Ca2+ imaging experiments14  or the APP Swedish (APPSwe line) mutation under the same promoters used in PS2APP mice26. In PS2.30H mice, Aβ plaques are not detectable in the SSCx at 6 months of age, consistently with the observation that Aβ42 does not accumulate in these mice up to 12 months22. Although APPSwe mice express the same APP level as PS2APP mice22, they accumulate Aβ42 at much lower rate24 because of the lack of a mutated presenilin. We carried out experiments in SSCx slices from 6-month-old PS2.30H and APPSwe mice and evaluated both evoked and spontaneous astrocyte Ca2+ activity. The ATP-evoked response in astrocytic territories is not significantly changed between any of the two single tg lines and WT mice  of excitatory synaptic transmission in SSCx layer II/III31. This form of LTP depends on the recruitment of astrocytes by neuronal noradrenaline (NA) and the subsequent astrocytic release of ATP which activates neuronal post-synaptic receptors37. We firstly assessed the Ca2+ response to NA (10 μM), in the presence of TTX (1 μM), of SSCx layer II/III astrocytes in brain slices from 6-month-old PS2APP mice and found that it is significantly impaired with respect to age-matched WT mice 39 would have been more effective than the standard one in detecting possible impairments related to SSCx in PS2APP mice.Driven by the results on the altered synaptic plasticity in SSCx circuits of PS2APP mice, we investigated the mnemonic capabilities related to this brain region. We tested the PS2APP mice with a modified version of the Object Recognition Test (ORT), originally designed to evaluate the ability of recognizing previously presented stimuli2+ defects observed in SSCx astrocytes from 6-month-old PS2APP mice could therefore be the harbinger of the upcoming loss of long-term memory retention in tORT. It is worth noticing that discrimination index analysis shows that both 4 and 6-month-old PS2APP mice display a worse performance than age-matched WT mice in remembering the explored objects at 24 h retention interval, suggesting an initial decline in tORT that becomes a full memory deficit in 8-month-old AD mice , has significant impairments in working memory at 6 and 8 months  signaling pathway. A reduction in free endoplasmic reticulum (ER) Ca2+ concentration ([Ca2+]ER) and the consequent reduction in IP3-mediated Ca2+ release, may account for this defective Ca2+ response. To address this hypothesis, we monitored [Ca2+]ER in situ and in vivo with the ER-targeted Ca2+ probe G-CEPIA1er40  in astrocytes from 6-month-old PS2APP mice with respect to age-matched WT mice, while no significant difference is observed between 3-month-old WT and PS2APP mice  containing TTX (1 μM) and cyclopiazonic acid , an inhibitor of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA). The reduction of the G-CEPIA1er signal (ΔF) observed upon CPA perfusion provides a rough estimate of the starting level of [Ca2+]ER -trisphosphate , which is mediated by the interaction of the ER transmembrane protein STIM1 with plasmalemmal Ca2+ permeable channels41. It is well known that SOCE activation strongly relays on the abundance of STIM142. To investigate whether the reduction in [Ca2+]ER is linked to STIM1 availability, we evaluated STIM1 expression in SSCx astrocytes of PS2APP mice through immunohistochemical staining. We observed a punctate staining of STIM1 in S100β-positive astrocytes widely distributed in all astrocytic territories  expressing both mouse STIM1 (mSTIM1) and the cytosolic reporter tdTomato under the control of the GFAP short promoter (mSTIM1 LV), in the SSCx of 6-month-old PS2APP mice. As a control, a different group of 6-month-old PS2APP mice was injected with a LV encoding only for tdTomato under the same promoter (CTRL LV). We observed that the LV expression is restricted to a small SSCx area, achieving a penetrance of ~50% to S100β-positive astrocytes in the infected area  fibers projecting to different brain areas51. For instance, the release of NA during locomotion is known to enhance the sensitivity of visual cortex astrocytes to local circuit activity during visual stimulation52. Astrocytes are also recruited by NA in the synchronous Ca2+ response induced by transcranial direct current stimulation53, a protocol that has been shown to have positive effects on memory in humans and animal models54. In 6-month-old PS2APP mice, we show that SSCx astrocytes exhibit a reduced response to NA and an impairment of a peculiar form of SSCx LTP that is strictly dependent on the recruitment of astrocytes by NA31.Astrocyte Ca55. Furthermore, noradrenergic enhancement of memory is also described in different behavioral tests56. Here, we provide evidence that the loss of astrocyte-dependent LTP in the SSCx of 8-month-old PS2APP mice temporally correlates with the full impairment in the consolidation of tactile recognition memory, which relies on SSCx functionality and plasticity. Although in humans the somatosensory function was believed to be spared from the disease, it has recently been shown in AD patients that a reduced functionality of primary sensory cortices can be unmasked when considering the variability of cognitive decline among individuals57. Noteworthy, the classic version of ORT could not detect a memory deficit even at this age. On the one hand, such observation could spring from different pattern separation abilities required by the different pairs of stimuli utilized in the ORT with respect to tORT, taking into account that in FAD pattern separation is impaired in both mice58 and humans59. On the other hand, the stimuli utilized in the classic ORT activate different sensory areas, i.e., they are objects with different shapes, colors, and textures, with respect to tactile-only stimuli. Therefore, multisensory stimulation could help PS2APP mice in the choice during classic ORT, as it does in humans with mild cognitive impairment60.Growing evidence supports the role of NA in shaping neuronal response to sensory stimuli, facilitating information processing and transferring in sensory circuits2+ signaling specifically at the level of astrocyte Ca2+ microdomains15. We here provide information on this localized Ca2+ activity by analyzing whether and how different parameters, such as the frequency and amplitude of the events and the number of active microdomains, change along with AD progression, showing that both evoked and spontaneous Ca2+ activity of microdomains are drastically reduced in SSCx astrocytes from 6-month-old PS2APP mice. Calcium microdomains are emerging as fundamental elements in the contribution of astrocytes to different pathophysiological processes36. Astrocytic thin processes (30–50 nm) tightly enwrap the synapse and have been estimated to account for about 75% of astrocytic volume, with their Ca2+ events accounting for a similar fraction of total astrocyte activity61. The Ca2+ source for these signals relies on both release from ER and influx from the extracellular milieu through plasma membrane (PM) channels and transporters63. All these players can co-localize at the level of microdomains and contribute to both neurotransmitter-evoked Ca2+ signals and spontaneous Ca2+ activity, as modeled in different studies65. Although a functional role for astrocyte spontaneous activity has not been elucidated yet, we can speculate that these recurrent Ca2+ signals in astrocytes are actively involved in the modulation of synaptic transmission, through Ca2+-dependent gliotransmitter release. Accordingly, alterations of spontaneous Ca2+ activity in astrocytes during AD progression may affect brain circuits.Previous works on AD mouse models did not investigate astrocyte Ca2+ hypoactivity depends on the presence of both PS2 and APP mutations since present results show no significant difference between astrocyte Ca2+ activity in WT and PS2.30H or APPSwe mice at 6 months of age. Of note, 6-month-old PS2.30H mice showed no impairments in both LTP and astrocyte [Ca2+]ER in SSCx. Data obtained in cultured fibroblasts, derived from FAD patients, and mouse neurons66 show that the PS2 mutation is sufficient to cause a reduction in [Ca2+]ER. A reduced response to metabotropic glutamate receptors is already present also in neurons and astrocytes from hippocampal slices of two-week-old mice bearing the PS2 mutation21. Our observations that the reduction in [Ca2+]ER is not observed in SSCx astrocytes from 3-month-old PS2APP mice, while being evident in 6-month-old mice, and is absent also in SSCx astrocytes of PS2.30H mice at 6 months of age suggest that this phenomenon develops with variable speed and intensity in different brain regions, possibly because of the high functional and structural heterogeneity of astrocytes67. Noteworthy, both PS2.30H and APPSwe mice are characterized by a milder phenotype than PS2APP mice, due to either lack of Aβ accumulation (PS2.30H) or delayed plaque load (APPSwe)25. Previous studies reported a strong relationship between Ca2+ dysfunctions and a prolonged incubation period with Aβ oligomers (Aβo)68. In particular, the incubation of mouse cortical neurons with soluble Aβo is reported to disrupt Ca2+ dynamics at the store level, decreasing the response to different IP3-generating agonists69. In line with this, Ca2+ hypoactivity has been observed in Aβ-preconditioned astrocytes70, and a decreased response to metabotropic agonists was also found in cultured microglia obtained from samples of sporadic AD patients71, hinting at GPCR-mediated hypo-responsiveness as a possible common feature of glial cells in AD.In the SSCx of PS2APP mice, astrocyte Ca2+]ER level underlies Ca2+ dysfunctions in SSCx astrocytes from 6-month-old PS2APP mice. Standing the problem in [Ca2+]ER, the use of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to recover astrocytic Ca2+ signaling19 as a rescue strategy would be ineffective. An alternative strategy is to act on SOCE, a mechanism of Ca2+ influx across the PM to the ER operating in different cell types, including astrocytes72. It is well known that SOCE is necessary for ER-mediated Ca2+ signaling in astrocytes75 and it is also implicated in gliotransmitter release in the hippocampus63. SOCE machinery is localized at ER-PM junctions and involves the concerted action of STIM1, the Ca2+ sensor monitoring intraluminal [Ca2+]ER, and Orai and/or TRPC channels on the PM. STIM1 oligomerizes upon Ca2+ decrease within ER lumen and interacts in ER-PM junctional puncta with PM channels that mediate Ca2+ influx and ultimately lead to ER replenishment77. STIM1 availability is crucial for SOCE activation, with the ratio between STIM1 and Orai1 being largely in the favor of the former for maximal Ca2+ entry78. We here provide evidence that STIM1 expression is reduced in SSCx astrocytes from 6-month-old PS2APP mice, in parallel with the observed drastic impairment in astrocyte Ca2+ signaling. Importantly, STIM1 levels are reduced also in the brain of sporadic AD patients79. Considering that STIM1 is a substrate of the γ-secretase activity of PS180, a plausible explanation for the reduction of STIM1 reported in AD is that the mutated PS2 can induce excessive cleavage of STIM1 in astrocytes. An alternative explanation could reside in a specific effect of Aβ species and/or inflammatory signals that are present in PS2APP mice. Accordingly, a direct interaction between internalized Aβ species and STIM1 in neurons has recently been suggested81.We here show that a reduction in ER as well as astrocyte-dependent long-term synaptic plasticity. Further studies are needed to verify whether the rescue of astrocyte Ca2+ signaling positively impacts also on tactile memory retention.The present study supports the significance of STIM1 reduction in astrocytic Ca2+ hypoactivity as an early marker of defective somatosensory function in AD and point to STIM1 as a potential primary target for the development of novel AD therapies based on the modulation of astrocyte Ca2+ signaling.Altogether, our findings identify astrocyte CaβAPP751 carrying the Swedish double mutation  and the human PS2-N141I (under the prp promoter); the same transgenes and promoters were used to create the single tg lines APPSwe and PS2.30H20. Only female mice were used for all the experiments because in the human pathology women are most affected  and PS2APP mice follow this trend20. Mice were used from 3 to 8 months of age and C57BL/6J (WT) mice were used as control.All experimental procedures were performed according to the European Committee guidelines (decree 2010/63/CEE) and the Animal Welfare Act (7 USC 2131), in compliance with the ARRIVE guidelines, and were approved by the Animal Care Committee of the University of Padua and the Italian Ministry of Health . Mice were housed under a 12 h light–dark cycle (7 a.m. to 7 p.m. light), with a room temperature (RT) of 22 °C and humidity of 60%. The homozygous tg mouse lines PS2APP (B6.152H), PS2.30H, and APPSwe (BD.AD147.72H) were kindly donated by L. Ozmen . The B6.152H line expresses the human A LV vector for rescue experiments (mSTIM1 LV) was designed by inserting mouse Stim1 and tdTomato sequences separated by a self-cleaving T2A peptide, under the control of a GFAP short promoter in a pLV vector (pLV[Exp]-Puro-GFAP(short)>mStim1[NM_001374058.1]ns)*:T2A:{tdTomato} (Vector ID:VB200716-1209uve). A control LV vector (CTRL LV) was also generated containing only tdTomato under the control of the same promoter (pLV[Exp]-Puro-GFAP(short)>{tdTomato} (Vector ID:VB200719-1276txs). Vector cloning and LV packaging were performed by Vector Builder. Sequences are available upon request.er40 into the right SSCx to obtain an astrocyte selective sparse expression of the cytosolic marker tdTomato and the cytosolic Ca2+ indicator GCaMP6f or the ER-directed G-CEPIA1er. Injections were performed at postnatal days (P) 75 or P165 WT, PS2APP, PS2.30H and APPSwe mice under general anesthesia using continuous isoflurane . For rescue experiments, we injected mSTIM1 LV or CTRL LV together with the AAV encoding for GCaMP6f or GCEPIA1er into the right SSCx 1 month before imaging. After inducing anesthesia, mice were injected subcutaneously with carprofen (5 mg Kg−1) to reduce pain and inflammation following surgery. Depth of anesthesia was assured by monitoring respiration rate, eyelid reflex, vibrissae movements, and reactions to pinching the tail and toe. Briefly, the skin over the skull was disinfected with iodopovidone and a cut was performed along the sagittal line to expose the bone. Injections of the two viral vectors  were performed after drilling one or two holes  into the skull over the SSCx  using a pulled glass pipette in conjunction with a custom-made pressure injection system. Virus injections were performed over a period of 2–5 min. At the end, the pipette was left in place for 5 more min and then gently withdrawn. After injections, the skin was sutured, and mice were revitalized under a heat lamp and returned to their home cage. Animals were carefully monitored in the following days for recovery and used for experiments from 2 to 4 weeks after injection.We performed injections of AAV5.GfaABC1D.cyto-tdTomato.SV40  and AAV5.GfaABC1DcytoGCaMP6f.SV40  or AAV5-GfaABC1D-G-CEPIA13, 2.5 KCl, 2 CaCl2, 1 MgCl2, 25 glucose, pH 7.4 with 95% O2 and 5% CO2). Coronal slices were cut with a vibratome  in the following ice-cold solution . Then, slices were transferred for 1 min in a solution at RT . Finally, slices were transferred in ACSF at 32 °C for 20 min and then maintained at RT for the entire experiment. During the experiment, individual slices were perfused with the recording solution .Coronal SSCx slices of 350 μm were obtained from mice at P90–P97 or P180–P187. Animals were anesthetized with isoflurane and the brain was removed and transferred into an ice-cold ACSF . Animal pinch withdrawal and eyelid reflex were tested to assay the depth of anesthesia. After inducing anesthesia, mice were injected subcutaneously with Carprofen (5 mg Kg−1) to reduce pain and inflammation following surgery. Both eyes were covered with an eye ointment to prevent corneal desiccation during the experiment. We monitored the respiration rate, heart rate, and core body temperature (maintained at 37 °C through a heating pad) throughout the experiment. The mouse was head-fixed and a craniotomy of 3–4 mm in diameter was drilled over the SSCx and a glass cover slip  was gently placed over the craniotomy and fixed with cyanoacrylate. Mice were mounted under the microscope with a metal head-post glued to the skull and dental cement was used to stabilize all the implant. Imaging session lasted up to 3 h.For in vivo imaging experiments, P180 tdTomato/GCaMP6f or tdTomato/G-CEPIA12+ dynamics in astrocytes in both in vivo and brain slice preparations, we used a 2P laser scanning microscope  equipped with a pulsed IR laser  tuned at 920 nm. Power at the sample was kept in the range 10–15 mW in brain slices and 15–25 mW in vivo to avoid photostimulation and photobleaching. Images were acquired at 1.53 Hz, for 2 min in basal conditions or 3 min during drug perfusion in brain slices, through a water-immersion objective . The field of view ranged between 700 × 700 μm and 120 × 120 μm depending on the zoom factor. Ca2+ signal recordings were performed in cortical layers II/III in SSCx brain slices and in in vivo preparations . In a subset of experiments performed in SSCx brain slices to investigate astrocyte Ca2+ activity with respect to Aβ-plaque proximity, mice received an intraperitoneal (i.p.) injection of M04 5 mg kg−1  12 h before experiment. M04 was also visualized at 920 nm under the 2 P microscope. We used SciScan 1.2 as acquisition software.To image CaDrugs applied to the slice perfusion solution were ATP 100 µM , TTX 1 µM , NA 10 µM  and CPA 50 µM .2+ elevations was performed with ImageJ 1.51n in a semi-automated manner using the GECIquant plugin83. The software was used to identify ROIs corresponding first to the soma , then proximal processes  and finally microdomains . All pixels within each ROI were averaged to give a single time course of fluorescence values, F(t). Analysis of Ca2+ signals was performed with ImageJ 1.51n (NIH) and AstroResp, a custom code developed in MATLAB 7.6.0 R2008 A  and originally described in Mariotti et al.84. To compare relative changes in fluorescence between different cells, we expressed the Ca2+ signal for each ROI as ∆F/F0 = (Ft − F0)/(F0). F0 was defined as the 15th percentile of the whole fluorescent trace for each ROI and considered as a global baseline. For each ROI we then defined as baseline trace the points of the ∆F/F0 trace with absolute values smaller than twice the standard deviation of the overall signal. Significant Ca2+ events were then selected with a supervised algorithm as follows. Firstly, a new standard deviation was calculated on the baseline trace, and all local maxima with absolute values exceeding twice this new standard deviation were identified. Secondly, among these events, we considered significant only those associated with local Ca2+ dynamics with amplitude larger than fourfold the new standard deviation, whereas in the original paper the threshold used was threefold the new standard deviation. The amplitude of each Ca2+ event was measured from the 20th percentile of the fluorescent trace interposed between its maximum and the previous significant one. Essentially, this procedure combines a threshold measured from the global baseline with a stricter threshold computed from a local baseline. We adopted this method to reduce artefacts from the recording noise superimposed on the slow astrocytic Ca2+ dynamics and from slow changes in baseline due to physiological or imaging drifts. All Ca2+ traces were visually inspected to exclude the ROIs dominated by noise. In the analysis of spontaneous microdomain activity, for each astrocyte we calculated the number of active ROIs, defined as the ROIs displaying at least one significant Ca2+ event, the frequency, i.e., the total number of Ca2+ events per minute and the mean amplitude of the Ca2+ events. For each parameter, we then calculated the mean value among all analyzed astrocytes. In the analysis of evoked responses, for each astrocyte we calculated the amplitude of the Ca2+ response for soma and the mean amplitude for its proximal processes and microdomains, the mean percentage of responsive proximal processes and the number of active microdomains. We then calculated the percentage of responsive soma and for the other parameters, i.e., amplitude, percentage of proximal processes and number of active microdomains, the mean value among all analyzed astrocytes.For experiments in GCaMP6f-expressing astrocytes the detection of Regions Of Interest (ROIs) displaying Caer-expressing astrocytes, the detection of ROIs was manually performed with ImageJ 1.51n. Somatic regions were free-hand drawn from individual astrocytes in time-lapse series for experiments in slices, in which a single focal plane was selected according to the focus of tdTomato signal and cells were followed also during CPA application. Mean ROI gray value was measured for each astrocyte throughout 1 min of basal conditions and during 10 min of CPA application. Basal and post-CPA fluorescence values were calculated and separately averaged among all analyzed astrocytes. The same procedure was applied for the analysis of G-CEPIA1er during NA perfusion. In in vivo experiments, in which CPA application was not performed, 40–50 μm deep Z-series were acquired along layer II/III of SSCx to maximize the number of sampled astrocytes. In these experiments, ROIs were free-hand drawn from individual astrocytes in focal planes selected according to tdTomato signal, and their fluorescence signal was obtained by averaging the signal from three successive focal planes centered at the best tdTomato focus. Basal fluorescence values were then averaged among all analyzed astrocytes.For experiments in G-CEPIA12+-sensitive indicator fura-2  and Pluronic F-127  in a 30 mm Petri dish containing 2 ml of ACSF. After loading, slices were recovered in ACSF at RT for at least 40 min.Coronal SSCx slices of 350 μm were obtained as described above for GCaMP6f imaging experiments, but from non-injected WT and PS2APP mice at P180-P187. To selectively stain astrocytes, slices were transferred in ACSF containing the astrocyte-specific fluorescence marker SR-101  at 32 °C for 15 min and then returned to ACSF. Slices were then loaded for 40 min at RT with the fluorescent Ca2+ imaging experiments, and time series were acquired for 60 s at 1 Hz.Imaging experiments with fura-2 were performed in ACSF at RT on a Leica THUNDER Imager - Live cell and 3D assay inverted microscope, equipped with a HC PL FLUOTAR (340) ×40/1.30 oil objective  and a HC PL FLUOTAR ×4/0.13 dry objective . Excitation light at 340 and 380 nm was obtained through a CoolLED pE-340fura Illumination System . The filter cube consisted of a dichroic at 400 nm and an emission filter BP510/80 . Images were acquired upon alternative illumination for 100 ms at 340 nm (100% LED power) and 40 ms at 380 nm (8% LED power) through an ORCA-Flash4.0 V3 Digital CMOS camera . After the acquisition of the fura-2 signal, SR101 images of the same field of view were obtained upon illumination at 555 nm obtained through a LED8 light source . Images were acquired with Leica Application Suite (LAS) software and were analyzed offline using ImageJ 1.51n software; somatic ROIs were manually drawn on cells positive for both SR101 and fura-2 and the ratio of the fluorescence emitted upon excitation at 340 and 380 nm (F340/F380) under resting conditions was calculated. During the experiment, individual slices were perfused with the recording solution already described for Ca2, 2.5 MgCl2, 1 NaH2PO4, 25 NaHCO3, 15 glucose, pH 7.4 with 95% O2 and 5% CO2), slices were cut with a vibratome  using the same ice-cold solution. Then, slices were transferred in the following recording solution  and maintained at RT.Coronal SSCx slices of 280 μm were obtained from mice at P90-P97 or P180-P187. Animals were anesthetized with isoflurane and the brain was removed and transferred into an ice-cold ACSF  with the recording solution maintained at a temperature between 30 and 32 °C. Recordings of field excitatory postsynaptic potentials (fEPSPs) were obtained using a multiclamp-700B amplifier . Signals were filtered at 1 kHz and acquired at 10 kHz sampling rate with a Digidata 1440 A interface board and Clampex 10.5 software. fEPSPs were measured trough a recording-filled glass micropipette pulled on a vertical puller  placed in neocortical layer II/III. Square current pulses (duration 0.2 ms) were applied every 30 s (0.033 Hz) using a S-900 stimulus generator connected through a stimulus isolation unit to a bipolar coaxial electrode  placed in layer V close to layer IV border. The stimulus magnitude was set 2-3 times higher than the minimal stimulus necessary to elicit a response in layer II pyramidal neurons. Twelve min baseline responses at 0.03 Hz were recorded before LTP-induction protocol. In order to induce LTP of EPSPs, five episodes of theta-burst stimulation (TBS) were delivered with 10 s start-to-start interval; each TBS episode consisted of five pulses of 5 ms duration each given at 100 Hz, repeated 10 times with 200 ms start-to-start interval . Analysis was performed using Clampfit 11.1 software.A single slice was transferred in a recording chamber and continuously perfused ).Two-to-4 weeks after AAVs injection, mice were deeply anesthetized with isoflurane and transcardially perfused with phosphate saline buffer (PBS), followed by 4% paraformaldehyde (PFA) in 0.1 M PBS, pH 7.4. Brains were fixed in PFA overnight, washed and cut into 70 μm coronal sections in PBS with a vibratome (Leica Vibratome VT1000S). Floating coronal sections were incubated for 1 h in the Blocking Serum  and 0.3% TritonX-100. Then, primary antibodies were diluted in PBS and 0.03% TritonX-100 (16 h at 4 °C). Primary antibodies used were: anti-S100β ; anti-GFAP ; anti β-Amyloid 17-24 ; anti-STIM1 ; anti-NeuN ; anti-RFP . After washing with PBS, slices were incubated at RT for 2 h with specific secondary antibodies conjugated with Alexa Fluor-488 , Alexa Fluor-556 , Alexa Fluor-555 , Alexa Fluor-633 (F (ab’)2-Goat anti-Mouse, RRID: AB_2535720, 1:500, A21053, Invitrogen Thermo Fisher Scientific, Lot n. 431664). Nuclei were stained with To-Pro-3 . Floating sections were then washed and mounted on glass slides with an Elvanol mounting medium. Negative controls were performed in the absence of the primary antibodies. Immunofluorescence images were obtained with a Leica SP5 confocal microscope equipped with a ×20 objective. Images were acquired with LAS software. Single images were taken with an optical thickness of 1 µm and a resolution of 1024 × 1024 pixels. Analysis was performed with ImageJ 1.51n software. STIM1 and G-CEPIA1Specificity of LV infection was assessed by αRFP immunolabeling to reveal tdTomato protein expression, and by calculating the percentage of tdTomato+ astrocytes displaying S100β or NeuN immunoreactivity. Penetrance was quantified as the percentage of S100β+ astrocytes displaying tdTomato immunoreactivity in the infected area (FOV 456 µm × 456 µm × 8 µm depth).Three different batches of animals, not involved in other experiments, were utilized for behavioral tests: one for ORT and Y-Maze, a second for tORT and a third one for MWM.ORT was performed in a Y-apparatus with high, homogenous white walls, 30-cm high: one arm was used as start arm and had a sliding door to control access to the arena; the other two arms were used to display the objects. The start arm is 31 cm in length with the sliding door placed at 13 cm, and the lateral arms are 18-cm long. All arms are 10-cm wide. On the first day (habituation day) mice explored the empty arena for 10 min. The following day, the learning session (sample phase) was performed: for 5 min the animals were free to explore the arena, which contained two identical objects, one for each arm, placed at the end of the arm. At 1 or 24 h after the sample phase, the test phase was run; the animals were free to explore the arena containing two objects, the familiar object and a novel one. Arena and objects were cleaned up between trials to stop the build-up of olfactory cues. A video camera was mounted above the apparatus to record trials with the EthoVision XT14 software (Noldus). The exploration time was taken as the time during which mice approached the objects with muzzle and paws.85. We used a Y-shaped maze which was constructed with three symmetrical gray solid plastic arms at a 120-degree angle . Mice were individually placed in the center of the maze. The mouse was allowed to freely explore the three arms for 5 min. Arm entry was defined as all four limbs within the arm. A triad was defined as a set of three arm entries, when each entry was to a different arm of the maze. The number of arm entries and the number of triads were recorded in order to calculate the alternation percentage . The maze was cleaned with 10% ethanol between sessions to eliminate odor traces.The first 5 min of the ORT habituation phase were analyzed for spontaneous alternation, as described in ref. TNew − TOld)/(TNew + TOld), with TNew and TOld being the time spent exploring the novel and the familiar objects, respectively.tORT was performed in a square chamber , made by gray acrylic Plexiglas. Three types of cubes covered by sandpapers with various grits were used for the two-step tORT . The objects were located at the positions with equal distance from the center of the chamber and from the walls of the chamber. The first day animals were allowed to explore the empty arena for 10 min (habituation phase). The day after, during the learning phase, mice were exposed and allowed to freely explore two identical 400-grit cubes for 5 min. After 1 h of retention period in a separate cage, each mouse was re-exposed to the same chamber with a new cube of 400 grit  and a 240-grit cube and allowed to explore the cubes for 5 min. Then, after 24 h from the learning phase, the subjects were reintroduced in the arena, for 5 min, with one cube of 400 grit and a second one of 80 grit. The discrimination index was calculated as follows: , filled to a depth of 25 cm with water (23 °C) rendered opaque by the addition of a small amount of nontoxic white paint. Four positions around the edge of the tank were arbitrarily designated North (N), South (S), East (E), and West (W), which provided four alternative start positions and also defined the division of the tank into four quadrants: NE, SE, SW, and NW. A circular clear Perspex escape platform  was submerged 0.5 cm below the water surface and placed at the midpoint of one of the four quadrants. The hidden platform remained in the same quadrant during training, while the start positions  were randomized across trials. Mice were allowed up to 60 s to locate the escape platform, and their escape latency was automatically recorded by the Ethovision system. On the last trial of the last training day, mice received a single probe trial, during which the escape platform was removed from the tank and the swimming paths were recorded over 60 s while mice searched for the missing platform; the swimming paths were recorded and analyzed with the Ethovision XT14 software (Noldus).As described in ref. t test was applied on normally distributed data sets and Mann-Whitney test on data sets that were not normally distributed. Two-way RM ANOVA, followed by Holm–Sidak multiple comparison procedure, was used to compare normally distributed data differing for two independent factors. For comparison between percentages we used Fisher’s exact test. Correlation index was computed by using Pearson’s  and Spearman’s  correlation coefficient. Results were considered statistically significant at p ≤ 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. In the figures, means are shown and error bars represent standard error of the mean (SEM). Statistical analysis was performed with Origin 2018 and Microsoft Excel 2016 software.Before statistical analysis, data were tested for normality by using Shapiro–Wilk test and for equal variance. Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Our experiments have demonstrated that this preparation has the normal active tension and elementary steps of the CB cycle. Furthermore, we investigated the effect of Ca2+ on the rate constants and found that the rate constant 2+] when it is <5 μM. This observation suggests that the activation mechanism can be described by a simple second order reaction. As expected, we found that magnitude parameters including tension and stiffness are related to [Ca2+] by the Hill equation with cooperativity of 4–5, consistent to the fact that Ca2+ activation mechanisms involve cooperative multimolecular interactions. Our results are consistent with a long-held hypothesis that process C  represents the CB detachment step, and process B (phase 3) represents the force generation step. In this report, we further found that constant The use of frozen and cryo-sectioned cardiac muscle preparations, introduced recently by , offers  The advantage of these preparations is that contractile proteins are packed in the high  concentrations, hence allosteric cooperativity among participating macromolecules is present and intact. Another advantage is that experiments can be performed in the physiological ionic strength solutions.The exploration of active muscle mechanics and kinetics has undergone a progressive evolution, with a trend towards preparations of increasing simplicity to unravel the intricate workings of muscle function. Early investigations employed intact muscles  and intact single fibers  study, the Ca2+ study, and the rigor study. When tension reached a steady value, sinusoidal analyses (range: 1–100Hz with 13 frequencies at 0.2% amplitude) were carried out. To test the reproducibility, the standard activation was repeated, and if the tension reproducibility was <80%, the data were not used.On the day of experiments, a small bundle was removed and mounted in the experimental apparatus. One end was connected to a linear motor (length driver) and the other end to a tension transducer via stainless-steel wires with a minute amount of nail polish. They were then stretched to just before resting tension was visible, which generally yielded a sarcomere length of 2.1–2.2 μm in myocardium as determined by optical diffraction at longer sarcomere length . The thiThis was performed as described , and theProcess B Process Cescribed . In briekinetics . Processkinetics , but it kinetics . When ch2+ activated state(s), hence not much is lost by prolonging the data collection period or increasing the oscillation amplitude. In contrast, fiber run down is much more severe when the fiber is activated with Ca2+ in the presence of ATP. This part of experiments was usually carried out after the ATP and the Pi studies.This was performed as shown in  earlier . In addi2+ . Because the relaxing solution contained 2 mM MgATP, the first solution change was used to wash out excess MgATP. Tension quickly developed to reach a plateau. The fiber length was then oscillated in a series of sinewaves ranging 1 Hz and 100 Hz. Time course data from both the length 2+]=28 μM). The experiments were also carried out in the decreasing order of [ATP] in the next preparation, and the results were averaged to minimize the effect of fiber run down.The ATP study was carried out as in escribed . Y(f) waxed in R . These exed in R  and negl2PO4 and K2HPO4  and 80 unit/ml CK (creatine kinase). For the pCa study, the ratio [Na2CaEGTA]:[Na2K2EGTA] was adjusted to achieve the desired pCa value by keeping the total [EGTA] constant (6 mM) and by using our homemade computer program, ME.exe (multiple equilibria). The rigor solution contained (mM:) 6 mM CaEGTA, 1.55 mM MgAc2, and 8 mM Pi. In all solutions, [Mg2+]=1 mM, the ionic strength was 200 mM, and pH was adjusted to 7.00 by KOH. All experiments were carried out at 25°C.This is listed in All data are expressed as the mean ± standard error (SEM).This is depicted in nterface  and analnterface . The comat 17 Hz . This frs |Y(f)| . The EM requency .vs. VM in the ordinate 2πrsal see . The datperiment , and we periment  as the  is increased (pCa<6.5), and it reaches a saturation by pCa 5.3–4.55. The pCa-tension curve was fitted to Hill equation  . Consequ2+] activations, and the kinetic parameters are summarized in 2+] scale in μM. The straight lines are entered by eye to show the trend of the data. The data in 2+]. This inflexion divides the data in two sections: 5 points for [Ca2+] < 5 μM, the rising phase, and 3 points for [Ca2+] > 5 μM, the saturation phase. The rising phase of 2+]→0 (see below). All the magnitude parameters We have carried out sinusoidal analysis during partial  < 5 μM and approached saturation at [Ca2+] > 5 μM; this latter effect was similar to magnitude 2+] point at 1.58 μM. 2+]. In principle, these magnitude parameters 2+]→0 (no activation), similar to pCa-tension plot . Our results demonstrate excellent cross-bridge kinetics with reduced noise and well-defined exponentials, which are better than skinned fibers, and easier to handle than single myofibrils. An advantage of the cryosection method is that the strips are uniform in cross sectional area and uniform perfusion effect for accurate and reproducible mechanical measurements in contrast to the traditional trabeculae preparations. This feature is especially valuable for kinetic studies. This preparation was introduced recently by  compared to fiber studies (~200 mM) which may also make a difference.One positive aspect is that we were able to deduce exactly the same CB model  whether 6 54±6%,  that inc6 54±6%,  measuredis 11±1% , and do eriments . It is p2+ sensitivity and cooperativity results , a plot of 2+] is linear and it appears to go through the origin: 2+] . These in turn result in a steep pCa-tension (or stiffness) relationship (2+] and the rate constant of force generation 2+ activation mechanisms.What is interesting is that, at low Cao [Ca2+] . As discn step 4 , Fig. 4.t < 5 μM  indicateposed by  as an “aturation . Actual tionship  with theg to 4–5 . Compare2+] on , we conclude that the major effect of Ca2+ is on process B, and its effect on process C may be secondary and can be caused by the interaction with its effect on process B. Thus, we should not state here that [Ca2+] affects CB detachment step 2, and that the detachment is lessened as [Ca2+] is increased up to 5 μM. The fact, that their sum 2+] studied (2+ on process C may be indeed secondary. The fact, that 2+] is at variance with the model proposed by (The apparent rate constant t step 2  & 4 (Kawt step 2 . Becausely small  comparedall (2πb , we conc studied , may sugposed by  that pha2+] sensitive (The magnitude parameter al value  amountinue (Yact . Furtherensitive  and beco(B and C  and 9B. Magnitude l fibers . While pl fibers . It is lCB cycle . BecauseCB cycle .2+ in a cooperative manner to result in the large Hill factor of 4–5. The effect of Ca2+ on the rate constant of the force generation step can be approximated by a second order reaction (We conclude that magnitude d states . These mreaction . We foun


Ion channels and transporters are ubiquitously expressed on cell membrane, which involve in a plethora of physiological process such as contraction, neurotransmission, secretion and so on. Ion channels and transporters is of great importance to maintaining membrane potential homeostasis, which is essential to absorption of nutrients in gastrointestinal tract. Most of nutrients are electrogenic and require ion channels and transporters to absorb. This review summarizes the latest research on the role of ion channels and transporters in regulating nutrient uptake such as K+ channels, Ca2+ channels and ion exchangers. Revealing the mechanism of ion channels and transporters associated with nutrient uptake will be helpful to provide new methods to diagnosis and find potential targets for diseases like diabetes, inflammatory bowel diseases, etc. Even though some of study still remain ambiguous and in early stage, we believe that ion channels and transporters will be novel therapeutic targets in the future. Establishing and maintaining the driving forces  is essential for maximum uptake. Firstly, it was found that changes in membrane potential (Em) affecting nutrient absorption. Membrane potential is primarily controlled by voltage‐gated K+ channel (Kv) activity through electrophysiological experiments, fluorescence detection, RT‐PCR, QPCR and immunohistochemistry. Reducing Kv channel activity by applying Kv channel inhibitors leads to membrane depolarization. Membrane depolarization or hyperpolarization can modulate the activity of various nutrient transporters on intestinal epithelial cell (IEC).+/H+ exchangers or an anion exchangers to reduce epithelial acidification.2+ regulates many intracellular physiological activities and plays an important role in regulating intestinal nutrient absorption through perfusion techniques in the small intestine of rabbits and mice.2+ ([Ca2+]i) homeostasis is finely controlled by Ca2+ channels and transporters which may ultimately affect nutrient absorption  tract is to digest and absorb nutrients from the diet and to excrete unabsorbed or harmful substances. Meals are digested enzymatically into absorbable nutrients such as monosaccharides, dipeptides, free amino acids and fatty acids. Absorption of the above nutrients requires specific transporters accompanied by cotransport or reverse transport of ions  remains the preferred option, findings from epidemiology study, animal study and clinical study have proved that the treatments targeted at IEC malabsorption may be more effective and safer.22.1+ to epithelial cells through SGLT1 of brush border membrane (BBM). GLUT2 of the basolateral membrane (BLM) transports glucose from IEC to mesenteric veins. In addition to the classical pathways described above, active glucose transport saturates in the presence of high concentrations of glucose (> 30 mM) in the intestinal lumen, possibly involving intercellular glucose transport and GLUT2 can rapidly bind to the brush border membrane of enterocytes and participate in promoting glucose diffusion across the membrane.Dietary carbohydrates must be broken down into monosaccharides  before they can be transported and absorbed. Intestinal monosaccharide uptake mainly depends on sodium‐glucose cotransporter 1 (SGLT1) and glucose transporter 2 (GLUT2). SGLT1 and GLUT2 were detected primarily in the small intestine  but not in the oesophagus, stomach, colon or rectum.2.2+‐dependent peptide transport system on the basement membrane. At the same time, FAA is mediated by several specific amino acid transporters. Most of the FAA in the lumen are absorbed by Na+‐dependent cotransporters , while a small portion of the FAA enters intestinal cells with other ions such as H+, protein associated with topoisomerase (PAT), Cl− or other amino acids.Dietary proteins are cleaved into oligopeptides in the lumen and further processed into small peptides (dipeptides and tripeptides) and free amino acids (FAA). Ma GG et al. constructed a mouse model, and conducted haematoxylin and eosin (HE) staining, enzyme‐linked immunosorbent assay (ELISA) and other experimental studies to show that PEPT1 (SLC15A1), a high‐capacity/low‐affinity peptide transporter, can achieve proton coupling absorption of more than 8000 different dipeptides and tripeptides, a variety of peptide‐like compounds are also PEPT1 transport substrates, including specific immune stimulants, angiotensin‐converting enzyme inhibitors and β‐lactam antibiotics.2.33−). The colon takes a fraction of undissociated SCFAs through passive diffusion into the mesenteric venous blood and then the portal vein.Hydrolysis products of dietary lipids, such as fatty acids, glycerol esters and cholesterol, do not dissolve in water. They form water‐soluble micelles with bile salts before reaching the microvilli through the hydrostatic layer on the mucosal surface of the small intestine. Then fatty acids, glycerol esters, etc., are released from micelles and enter the intestinal epithelium through the membrane, while bile salts return to the lumen. In vivo and in vitro studies on weaned piglets and the IPEC‐J2 cell line indicate that fatty acid translocase (CD36) and fatty acid transporter 4 (FATP4) actively transport medium‐chain and long‐chain fatty acids (MCFA and LCFA) across the apical membrane of intestinal cells.33H] or [14C] isotopes.133I] ‐triolein and [75Se] ‐glyceryl triether, and cholesterol absorption was measured with radiolabeled dietary plant sterols (e.g. sitosterol or sitosterol) as a nonabsorbable reference marker.Isc) or mucosal‐to‐serosal fluxes (Jms) induced by the addition of nutrients to the apical chamber of tissues, or epithelial cells would represent the process of nutrient absorption.+ and dipeptide transport accompanied by H+.Various animals and their isolated intestinal segments, brush border membrane vesicles, cultured organoids or cell lines are used in vivo and in vitro to study nutrient absorption. The material balance test is an intuitive method to measure the absorption of nutrients in the intestine. The concept of the ‘ingestion minus output’ technique measures the amount remaining at a point in the digestive tract, for example, in ileostomy output or stool. Although the concept is simple, the practice in vivo is difficult because of the need to measure dietary composition and consumption accurately. It also requires consideration that bacterial consumption is also involved in the loss of nutrients after ingestion. Radioisotope labelling is used to study the absorption of various nutrient‐like substances in vivo and in vitro experiments better than mass balance measurement, usually with i) transport is mainly mediated by calcium‐binding proteins, including calbindin‐d9k and calmodulin (CaM), which may play a role in the buffering system to prevent [Ca2+]i overload and transfer Ca2+ to the serosal side. The [Ca2+]i are actively excreted by plasma membrane Ca2+‐ATPase (PMCA) and other transporters such as Na+/Ca2+ exchanger (NCX).2+ influx, such as TRPV1/4 and store‐operated Ca2+ channels (SOC).2+]i homeostasis through the GPCR‐PLC‐IP3R and GPCR‐PLC‐TRPV6 pathway.2+]i signalling through the above pathways regulates intestinal physiological functions, including barrier protection, enteral hormone secretion and motility. And nutrient transport is usually accompanied by [Ca2+]i movement, [Ca2+]i signalling more or less mediated the nutrient's absorption.Ca2+‐PKCβII dependent mechanism. The Ca2+ influx in this process mainly originates from the Cav1.3 activated by depolarization.2+ influx through Cav1.3 activates the ryanodine receptor (RyR1) to release the endoplasmic reticulum (ER) Ca2+ storage, thereby inducing store‐operated Ca2+ entry (SOCE) mechanism. The increased [Ca2+]i caused by the above transports further activates SGLT1 to mediate intestinal glucose uptake.2+‐dependent manner.+‐glucose absorption by inhibiting TRPV4 channel.Hyson, D H et al. applied calcium channel blockers (nisoldipine) in rabbit jejunum. They found that D‐glucose uptake was decreased, suggesting that calcium channel blockers could affect the active transport of glucose through SGLT1 to different degrees.2+ levels stimulated Na(+)/H(+) exchanger 3 (NHE3) activity, thereby maintaining the high electrochemical driving force of PEPT1 to drive peptide transport.2+]i activated Na+‐glutamine absorption in ileal epithelial cells via several Ca2+ permeable channels and transporters, including mucosal Cav1.3, serosal TRPV1/2, SOC channels and NCX.2+ ‐sensing receptors (CaSR) and serve as allosteric modifiers to nutrient signal supply to intestinal epithelial cells (IEC).2+]i stimulates the positive feedback pathway of peptide absorption. The absorbed Gly‐Sar stimulates basolateral CaSR, leading to the activation of phospholipase C (PLC) and the increase of [Ca2+]i, which then activates IKCa to provide the driving force for PEPT1 transepithelial uptake.Studies in Caco‐2 cells showed that reducing Ca2+ increases the absorption of LCFA in rat jejunum; there is a potential for that Ca2+ transport and LCFA transport to be correlated in the brush border and the basolateral membrane of IEC.2+ reduces plasma cholesterol by downregulating intestinal (NPC1L1) and MTTP, upregulating hepatic cholesterol‐7α‐hydroxylase (CYP7A1) and intestinal ABCG 5/8.2+‐dependent manner in Caco‐2 cells, which is associated with AHR ligand‐mediated upregulation of membrane Ca2+ channels encoded by transient receptor potential cationic channel (TRPV6) gene.In the study of lipids absorption, early research studied that luminal Ca2+‐PKCβII mechanism mainly coordinated glucose absorption and PEPT1‐mediated peptide absorption. Glucose decreased the expression of PEPT1 in the jejunal membrane of rats, and nifedipine as an L‐type voltage‐gated calcium channels (VGCC) blocker could attenuate the effect of glucose using perfused rat jejunum in vivo.2+ signalling pathway linking nutrients uptake. [Ca2+]i binds CaM as a compound that activates calmodulin‐dependent protein kinase β (CaMKK β). Previous literature has shown that AMP‐activated protein kinase (AMPK) is a substrate for CaMKK2 in mammalian cells from rat brain or expressed in E. coli phosphorylates,2+ signalling could regulate the activities of NHE, K+ channels and targeted transporters, which are involved in nutrient transport. It can be seen that the mechanism is complex and still needs to be further studied.Hiroshi Arakawa et al. found that the nutrient absorption and energy supply network composed of the Cav1.3‐Ca52+]i and [Na+]i in nutrient absorption is recognized; in addition to the above channels, there are also transporters to maintain ion homeostasis. There are currently three subtypes of Na+‐Ca2+ exchangers (NCX) expressed in various cells, including IEC. The transport ratio of NCX is 3:1; in forward transport mode: 3 Na+ are transferred in and 1 Ca2+ is moved out of the cell, while in reverse transfer mode is the opposite. The direction depends on the gradient of Na+ and Ca2+ concentrations and membrane potential (Em).  secretion.+ and Ca2+ influx can affect the direction of NCX transport through blot analysis, immunofluorescence analysis, expression profile and qPCR were conducted in cell experiments.2+]i and promote glucose and glutamine absorption.The importance of i, promote phosphorylation of ASCT1 through the PKC‐α pathway, and reduce affinity to inhibit Na+‐alanine cotransport in IEC during chronic intestinal inflammation.+‐glutamine cotransporter B0AT1 in villus cells was inhibited secondary to reduced cotransporter numbers and reduced NKA activity. SNAT5/SN2 in crypt cells was stimulated secondary to increased transporter affinity and NKA activity.2+]i through leukotriene receptors, stimulating NKA in intestinal crypt cells via the PKC pathway.− secretion and promotes Na+ ‐glucose absorption by blocking TRPV4 channels,Inflammatory bowel diseases (IBD) include ulcerative colitis (UC) and Crohn's disease (CD), which can cause damage to the whole digestive tract. Its pathogenesis is thought to be a complex combination of the genetic, gut microbiome, immune response and environmental factors, leading to excessive and abnormal immune responses to the symbiotic microbiota. Diarrhoea is a very common debilitating symptom in patients with IBD, and several causes, including malabsorption, can cause it. One reason for disrupting nutrient absorption is that the activity of Na7− secretion, motility, hormone secretion, etc. They are ideal drug targets for treating several GI diseases, including IBD, colorectal cancer and metabolic syndrome. Ion channels and transporters in IEC have been shown to regulate luminal nutrient uptake in physiological or pathophysiological states, excluding neurological and endocrine roles.Ion channels and transporters play key roles in various physiological processes in the gastrointestinal tract, including Cl+ channels are involved in nutrient absorption, especially in the positive regulation of carbohydrate and protein hydrolysate absorption, by affecting Em after activation. There is still a lack of studies on the involvement of intestinal K+ channels or transporters in lipid absorption. Nutrient absorption is often accompanied by Ca2+ movement. In turn, increased [Ca2+]i caused by activation of Ca2+ transporters regulate nutrient absorption from multiple directions, and the following two are considered: firstly, the activity of K+ channels or NHE can be controlled to affect the transmembrane potential, and secondly, the movement of nutrient transporters can be directly affected. Therefore, the therapeutic strategy of selecting Ca2+ channels or transporters' drugs to adjust nutrient absorption must be reconsidered. A series of ion exchangers  regulate nutrient uptake, especially proteins and lipids hydrolysates, mainly by influencing pHi. Taken together, although, in recent years, research on the intestinal ion channels and transporters regulating nutrient absorption to achieve considerable progress, targeting ion channels in disease treatment has promising prospects. In the development of ion channel targeting allows it to be more effective personalized therapy before, we still need to dig deeper into the mechanics.It is proved that KXianmin Lu: Writing – original draft (lead). Chen luo: Investigation . Jiangbo Wu: Resources (supporting). Ya Deng: Resources (supporting). Xingyi Mu: Resources (supporting). Ting Zhang: Resources (supporting). XiaoXu Yang: Formal analysis . Qi Liu: Resources (supporting). Zhuo Li: Resources (supporting). Siqi Tang: Resources (supporting). YanXia Hu: Resources (supporting). Qian Du: Methodology . JingYu Xu: Funding acquisition (supporting); project administration (supporting); resources (supporting); supervision (supporting); writing – review and editing (supporting). Rui Xie: Funding acquisition (supporting); writing – review and editing (supporting).This study was supported by research grants the National Natural Science Foundation of China  and Collaborative Innovation Center of Chinese Ministry of Education (2020–39). This study was supported by Guizhou Science and Technology Department ); This study was supported by Zunyi Science and Technology Bureau ); This study was supported by Guizhou Science and Technology Department (Qiankehe foundation‐ZK (2021–major project 004)); This study was supported by Graduate Education and Teaching Innovation Program of Zunyi Medical University (ZYK163).The authors declare that they have no competing interests.We have obtained consents to publish this paper from all the participants of this study.


The global production of macadamia nuts has witnessed a significant increase, resulting in the accumulation of large quantities of discarded nutshells. These nutshells possess the properties of remarkable hardness and toughness, which are comparable to those of aluminum. Incorporating natural fillers to enhance the properties of composite materials for various applications, including light duty, structural, and semi-structural purposes, is a common practice. Given their inherent hardness and toughness, macadamia nutshells present an intriguing choice as fillers, provided that the manufacturing conditions are economically viable. With the urgent need to shift toward natural fillers and reduce reliance on synthetics, exploring macadamia nutshells as components of natural fiber composites becomes imperative. This review aims to comprehensively examine the existing body of knowledge on macadamia nutshells and their bio-synthetic polymer composites, highlighting key research findings, achievements, and identifying knowledge gaps. Furthermore, the article will outline prospective areas of focus for future research endeavors in this domain, aligning with the universal goal of minimizing synthetic materials. The former is preferred due to its higher oil content and improved nut edibility after roasting, making it the dominant species for commercial plantations [In recent times, growing concerns regarding environmental issues, health considerations, and the urgent need for sustainable manufacturing methods have amplified the focus on the research on and development of green materials for engineering applications. Natural fibers derived from sources such as bamboo, flax, coir, hemp, etc., have been employed as reinforcements in the production of bio-composites . Effectintations . In Austntations . The glontations . Unfortuntations . As a bontations . MacadamAlthough research on macadamia nutshell-reinforced composites is limited, no comprehensive review has been published thus far. Therefore, this article aims to provide an overview of bio-synthetic polymer composites utilizing macadamia nutshells, encompassing their chemical properties, mechanical characteristics, manufacturing methods, fire retardancy, water absorption behavior, and thermal properties.Researchers have long been exploring opportunities to incorporate natural fibers into various industries, a trend that gained momentum with the emergence of polymers in the early 19th century. Synthetic polymers and composites have become ubiquitous worldwide; however, their production and recycling processes contribute to environmental pollution, prompting the need for alternative solutions utilizing natural fibers. Bio-synthetic composites, composed of natural materials and artificially prepared or synthesized polymers, offer strength and structural integrity to the final product. Cellulosic fibers derived from sources such as flax, alpaca, hemp, jute, and wood are biodegradable and commonly used as reinforcement in composites with different thermoplastic matrices. Bio-composite materials offer numerous advantages over conventional materials, including higher specific strength, stiffness, and fatigue resistance, thereby enabling more adaptable structural design. Additionally, bio-composites are biodegradable, possess superior tensile strength, exhibit low specific gravity, and are recyclable. Consequently, these materials find application in diverse product manufacturing and innovative fields. Green composites, compared to their synthetic counterparts, offer various advantages, including reduced tool wear  and biodNutshells, which are agricultural waste, consist of lignocellulosic materials. The strength and stiffness of agro-fibers are primarily attributed to cellulose content. Traditionally, nutshells have been limited to low-end applications. However, recent research has focused on exploring enhanced and value-added uses for these nutshells. Some potential applications include utilizing them as carriers for insecticides and pesticides, employing them as abrasives for cleaning and polishing purposes, utilizing them as thickeners in paint formulations, and utilizing them as raw materials for activated carbon production. Moreover, in recent years, researchers have extensively investigated the potential of incorporating nutshells as fillers in polymer composites .Macadamia nutshells offer a promising opportunity to develop innovative green composites via the incorporation of them as fillers with compatible polymeric resins. Nutshells fall into the category of three-dimensional cells, characterized by cell walls with a random orientation. From the early stages after flowering, the nuts develop and remain within the shells until they reach their final lifecycle stage . MacadamMacadamia nutshells possess various anatomical features, including the hilum, micropyle, outer suture, inner suture, vascular bundles, sclerenchyma fiber layer, sclereid, and testa layers . These tIn a study investigating the production of composite panels using macadamia nutshells and waste plastic from the automotive industry, the researchers conducted an analysis to assess the elemental content, moisture content, volatile carbon, and fixed carbon of the nutshells. This analysis involved the use of X-ray fluorescence (XRF) spectroscopy, along with proximate analysis  analysis, which revealed the presence of numerous functional groups in the FTIR spectrum . The she−1 indicate C-H stretching, while the peak in the region of 1720 cm−1 may be attributed to the presence of acetyl groups in hemicellulose. Peaks around 1600 and 1510 cm−1 suggest the presence of aromatic structures, and the strong peak at 1000 cm−1 corresponds to stretching vibrations from the functional groups present in hemicellulose and cellulose structures.Notably, the peaks observed at 2910 and 2930 cmTo investigate the thermal behavior of macadamia nutshells, Sagar et al.  performeThe thermal analysis results confirm that macadamia nutshells exhibit thermal stability up to 250 °C. Furthermore, the TGA results provide confirmation of the presence of cellulose (~35%), hemicellulose (~30%), and lignin (~35%) in macadamia nutshells.The usability of any material in terms of engineering applications highly depends upon its mechanical attributes, such as flexural properties, tensile properties, fracture toughness, stress and strain curves. These characteristics mostly determine the probable behavior of a material in a specified application. Unlike man-made composites, biological materials do not rely on simply one fiber–matrix interface, but rather on interactions of the multiplicity of interfaces between the different structural levels of molecules, cells, and fibers. Studies have been conducted to determine the mechanical characteristics of macadamia nutshells, such as flexural properties, tensile properties, hardness, and compressive strength. They are known to have approximately the same fracture toughness as that of common ceramics and glass; when compared based on specific strength or modulus, they exceed those of these materials due to their low density.Wang, Zhang and Mai  reported3 and 1.5 × 103 kg/m3, and macadamia nutshells are heavier than most hard and soft woods.It is shown from The stress–strain curve and elastic stress distribution curve were evaluated . StresseThe unique characteristics of macadamia nutshells, such as their low density, high mechanical strength ,18, biodChensong et al.  conducteIn a study by Xiaohui et al. , the addIn another report, the authors studied such composites made of four kind of nutshells such as walnut, almond, macadamia (MSP) and wild almond . The PLASevda et al.  conducteExposure to weathering conditions led to a decrease in flexural strength and an increase in the modulus of elasticity of 62%. Color changes and a loss of gloss were predominantly observed in the MCC/nutshell reinforced composites, along with an increase in surface roughness. Laert et al.  conducteLucas et al.  studied Nycolle et al.  investigWechsler et al.  conducteEucalyptus saligna and macadamia nutshells [Derrick et al.  investigutshells . Urea foOmid et al.  also invv/v) of macadamia biomass in its natural form had minimal impact on the glass transition temperature, modulus of elasticity, and linear thermal expansion coefficient of the benzoxazine matrix.A study focused on the thermal behavior of benzoxazine composites reinforced with macadamia biomass was conducted . DiffereA study focused on the production of wood plastic composite (WPC) panels via a combination of waste automotive plastics with macadamia nutshells as a matrix material was conducted . The stuThe comprehensive modulus of the panel board increased as the proportion of macadamia shells increased, reaching a value of 548 MPa for a board containing a 75% macadamia shell mixture. However, in the flexural test, the incorporation of macadamia shell did not exhibit a reinforcing effect and led to a slight decrease in flexural strength . The repMicrostructure analysis of the fractured surfaces under compression revealed that the addition of macadamia shells partially transformed the brittle failure of the waste automotive plastic into a ductile failure. In terms of thermal properties, the WPC panel exhibited favorable flame-retardant properties compared to panels made solely from 100% plastic.2. The newly developed composites exhibited a performance improvement of over 30% compared to that of their previously developed adsorbents. The introduction of phenolic resin resulted in an enhanced efficiency of CO2 adsorption. Yingge et al. [2/g), larger pore volume (2.2 cm3/g), and higher mesoporous content (23.85%), providing significant technical advantages. Macadamia biomass was effectively used as carbon resource in cleaner production of iron [Jun et al.  developee et al.  utilized of iron  and mate of iron .The physical characteristics of polymers and cellulosic fibers are influenced by various internal factors, including molecular weight, crystallinity, crystal morphology, crosslinking, branching, copolymerization, plasticization, molecular orientation, and residual stress. Additionally, external variables such as ambient atmosphere, the nature of deformation, thermal history, weathering, time (aging), and the frequency of stressing also play a role. The viscoelastic nature of polymers and their composites further demonstrates a strong dependence of mechanical properties on time and temperature. As a result, understanding the intricate relationships between polymer structure and properties is a complex and ongoing area of research, offering intriguing avenues for exploration.By controlling the nucleation and growth of mineral phases, as well as manipulating the microstructure in materials such as synthetic polymers and biomaterials, it becomes possible to significantly alter their morphology and unlock advanced properties. This knowledge opens up new possibilities for developing novel processing methods and creating useful objects with enhanced functionalities. The mechanical properties such as strength, etc., of macadamia nutshell fibers are naturally high, which is a prime vantage. To be able to utilize such a naturally grown high strength fiber is a great scientific achievement. Furthermore, the fiber and its composites do not inherit the characteristics, e.g., high moisture absorption, that discourage its uses. In this context, exploring innovative approaches becomes crucial in order to fully harness the potential of natural fibers and fillers, such as macadamia nutshells, in the development of functional and structural composite materials.


Recently, the 5th edition of ‘The World Health Organization (WHO) Classification of Tumors of Haematopoietic and Lymphoid Tissues’ WHO2022) was released in beta version . In WHO2022 was rA total of 861 newly-diagnosed AML patients aged ≥18 years, according to the revised 4th WHO classification (WHO2016) , were inChi-square, Fisher’s exact, Mann–Whitney U, and Kruskal–Wallis H tests were used for comparison. Kaplan–Meier analysis with log-rank test was applied to plot overall survival (OS) curves. Prism version 9.5.1 for Windows  and MedCalc 20.121  were used.RUNX1::RUNX1T1, PML::RARA, CBFB::MYH11, DEK::NUP214 and BCR::ABL1 fusions) between WHO2016 and WHO2022  and AML not otherwise specified (AML-NOS) (n = 15). A total of 14 fusion partners were detected, with AFDN , SEPT9 , and ELL  being the most frequently observed. Furthermore, updated classification introduced two new categories: NUP98 rearrangement (6 patients) and other genetic alterations (2 patients). The number of patients diagnosed as ‘AML with CEBPA mutation’, including both biallelic mutations and single mutations located in the basic leucine zipper (bZIP) region, was increased to 65 (7.5%) according to WHO2022, representing an additional 8 patients compared to 57 (6.6%) who were diagnosed as ‘AML with biallelic mutations of CEBPA’ according to WHO2016. AML-MR employed significantly new essential diagnostic criteria in WHO2022. A total of 243 patients were diagnosed as AML-MR. Majority  of them were included in AML-MRC by WHO2016 while a considerable proportion were previously classified as ‘AML with RUNX1 mutation’  or AML-NOS  by the same system. The DDX41 germline mutation, the most common genetic predisposition to MDS and AML, was identified in 31 patients, including 27 with two mutations (germline and somatic), and 4 with a single mutation. We found an additional 4 patients with a single somatic DDX41 mutation.There was no change in the classification for 205 patients with defining genetic abnormalities  were classified as ‘other rare recurring translocations’, whereas the WHO2022 classified them as AML-Diff (Supplementary Table TP53 (AML-TP53), AML with myelodysplasia-related gene mutations (AML-MR-M), and cytogenetic abnormalities (AML-MR-C) , followed by RUNX1 (19.3%), BCOR (15.2%), TP53 (14.4%), TET2(14.0%), DMNT3A (12.8%), SRSF2 (12.8%), IDH2 (11.9%), and U2AF1 (11.1%).Of 243 AML-MR patients classified by WHO2022, 75 had only cytogenetic abnormalities, 79 had only mutations, and 12 had only a history of MDS or MDS/MPN, while 77 patients fulfilled at least two of the essential diagnostic criteria and seven patients fulfilled all three criteria Fig. . RegardiDMNT3A (23.2%) was the most frequently mutated gene, followed by DDX41 (15.5%), RUNX1 (15.5%), IDH2 (14.1%), and NRAS (10.6%). The ICC includes RUNX1 mutation as an additional molecular abnormality in the classification of AML-MR-M, resulting in the classification of 22 patients under this category. Only eight patients of AML-Diff did not possess any genetic abnormalities.Among the 142 AML-Diff patients diagnosed by WHO2022, 124 had molecular mutations, 53 showed cytogenetic abnormalities Fig. , and 43 KMT2A::MLLT3’ and ‘AML with KMT2A rearrangement other than KMT2A::MLLT3’     . Three-year OS was 42.5% (95% CI: 39.0–45.9%). The prognostic accuracy of the 2022 ELN criteria was demonstrated in our dataset Fig. . No sign01) Fig. . AML-Dif61) Fig. . Accordi01) Fig. . In addi64) Fig. .KMT2A rearrangement and an additional 23 with other genetic abnormalities [RUNX1 mutation’ and 15.2% from AML-NOS. The redefined AML-MR appears to provide a clear and simplified diagnostic approach as it removes morphology alone as a diagnostic criterion [Using WHO2022, 154 patients were reclassified from WHO2016, including 23 with riterion , 8. Furtriterion . Among AThis study, along with previous studies, has demonstrated that patients with history of MDS or MDS/MPN had a poor prognosis, likely due to the failure of hypomethylating agent treatment –12. SimiIn conclusion, our evaluation supports the refinements made in the WHO2022 classification for AML, and additionally incorporates the recommendations from ICC. Clinical, hematopathological, and genetic characteristics accumulated over the past two decades have contributed to the refinement of these classifications and the identification of new entities.Supplemental infromation


Neither clotrimazole nor senicapoc altered the intracellular Ca2+ concentration. These results suggest that the effects of IK blockers on cancer cells are not strictly dependent on a robust presence of the channel in the plasma membrane, but they might be due to off-target effects on other cellular targets or to the blockade of IK channels localized in intracellular organelles.Many studies highlighted the importance of the IK channel for the proliferation and the migration of different types of cancer cells, showing how IK blockers could slow down cancer growth. Based on these data, we wanted to characterize the effects of IK blockers on melanoma metastatic cells and to understand if such effects were exclusively IK-dependent. For this purpose, we employed two different blockers, namely clotrimazole and senicapoc, and two cell lines: metastatic melanoma WM266-4 and pancreatic cancer Panc-1, which is reported to have little or no IK expression. Clotrimazole and senicapoc induced a decrease in viability and the migration of both WM266-4 and Panc-1 cells irrespective of IK expression levels. Patch-clamp experiments on WM266-4 cells revealed Ca The gating mechanism became clear with the cryo-EM structure solved by Lee and MacKinnon i alterations, we performed Ca2+-imaging experiments on Panc-1 and WM266-4 cells testing whether clotrimazole or senicapoc can have some effects on [Ca2+]i. [Ca2+]i was monitored over time with the fura-2 fluorescent probe before and after the application of 30 µM clotrimazole or 30 µM senicapoc. In 2+]i values from these experiments are reported. Neither WM266-4 cells nor Panc-1 showed a significant variation of [Ca2+]i after the application of the two molecules; rather, there were only small fluctuations that were never larger than 15 nM. The average Δ[Ca2+]i values (with respect to the standard bath solution) after the perfusion of clotrimazole and senicapoc on WM266-4 cells were only 4 ± 1.3 nM and 5 ± 2.5 nM, respectively . Phalloidin staining did not appear to modify both WM266-4 and Panc-1 cells after 24 h of treatment . However2+, while 60 µM BA6b9 inhibited around 80%, with the residual currents being at least partially unspecific leak. The degree of inhibition is line what has been reported by Burg et al. i due to an increase in the electrochemical driving force. Conversely, the inhibition of IK might lead to a decrease in [Ca2+]i. However, in addition to these driving-force mediated effects, we know from our previous experience that K+ channel modulators can alter the intracellular calcium concentration of cancer cells in more unspecific ways i after acute exposure to clotrimazole and senicapoc. Calcium imaging experiments on WM266-4 and Panc-1 cells did not show any significant increase in [Ca2+]I after the perfusion of the two compounds, excluding that they can have secondary effects similar to those observed, for example, for BK modulators i) were performed using the fluorescent indicator fura-2 AM. Before the experiments, cells were incubated for 45 min at 37 °C with 5 μM fura-2 AM (Sigma-Aldrich) dissolved in the same extracellular solutions of patch-clamp experiments, adding 0.1% pluronic acid in order to improve dye uptake. Coverslips were then transferred on the stage of an inverted Nikon TE200 fluorescence microscope. Cells were excited at 340 and 380 nm at 0.5 Hz with a dual excitation fluorometric Ca2+ imaging system . Fluorescence emission was measured at 510 nm and was acquired with a digital CCD camera (Hamamatsu C4742-95-12ER). To monitor [Ca2+]i, the fluorescence ratio F340/F380 was used. Monochromator settings, chopper frequency, and data acquisition were controlled by a dedicated software . [Ca2+]i was calculated according to Grynkiewicz et al. [2+/fura-2 complex of 140 nM.Measurements of cytosolic calcium  and DAPI for 1 h and washed with PBS three times. Then, each sample was examined by confocal microscopy using a Leica STELLARIS 8 Falcon τSTED  inverted confocal/STED microscope. The fluorescence image (1024 × 1024 × 16 bit) acquisition was performed using an HC PL APO CS oil immersion objective 100× (1.40 NA).t-test or ANOVA followed by Tukey tests for mean comparison . For statistical analysis, we used OriginLab . Figures were prepared using Sigmaplot . The chosen significance thresholds of 0.05 and 0.01 are indicated by an asterisk (*) and double asterisk (**), respectively. Cells to be patched, imaged or used for any purpose were chosen randomly.Data are reported as mean ± SE. When bar charts are depicted, also individual data points are superimposed. When data are normalized, it is stated in the text or figure legends. Differences between data groups reported in the same graph were checked for statistical significance by means of a paired-sample KCNN4 gene has been performed, which would provide more direct evidence of KCNN4 independent effects of KCa3.1 inhibitors on cancer cell viability and migration.In the present study, no genetic knock-out or knock-down of the