Calcium signals have distinct organization and underlying mechanisms in different astrocytic compartments thus allowing complex spatiotemporal signaling. Signals mediated by fluctuations in cytosolic Na+ are instrumental for coordination of Na+ dependent astrocytic transporters with tissue state and homeostatic demands. Astroglial ionic excitability may also involve K+, H+, and Cl−. The cyclic AMP signalling system is, in comparison to ions, much slower in targeting astroglial effector mechanisms. This evidence review summarizes the concept of astroglial intracellular excitability.Classic physiology divides all neural cells into excitable neurons and nonexcitable neuroglia. Neuroglial cells, chiefly responsible for homeostasis and defense of the nervous tissue, coordinate their complex homeostatic responses with neuronal activity. This coordination reflects a specific form of glial excitability mediated by complex changes in intracellular concentration of ions and second messengers organized in both space and time. Astrocytes are equipped with multiple molecular cascades, which are central for regulating homeostasis of neurotransmitters, ionostasis, synaptic connectivity, and metabolic support of the central nervous system. Astrocytes are further provisioned with multiple receptors for neurotransmitters and neurohormones, which upon activation trigger intracellular signals mediated by Ca This was an inert tissue, forming a filling or basic substance that surrounds and covers all components of the organism being in a way a predecessor of the connective tissue of Rudolf Virchow.The concept of physiological excitability and the definition of excitable and nonexcitable tissues was formulated by Albrecht von Haller, who exposed different organs or their parts to injury, by squeezing and stinging, by sprinkling with cold, hot, or corrosive substances or by electrocuting. Analyzing responses to such interrogations, von Haller proposed to classify all organs into sensible (cerebellulas (minute brains) that are connected by nerve fibers, which receive sensations from or project motor impulses into the peripheral organs. Swedenborg described these structures as the substrates for brain function. He wrote “From each cortical gland proceeds a single nerve fiber; this is carried down into the body, in order that it may take hold of some part of a sensation or produce some action.”globules” or “kugeln” both denoting spheres) from the microscopic observations were made by Christian Gottfried EhrenbergAlthough the cellular theory had not yet been established by the time of von Haller’s work the notion of the cell as an elementary living entity has been considered; the term being invented by Robert Hook in the 1650s.Kugeln der Belegungsmassen) and nerve fibers (Primitivfasern), whereas the passive substance was defined as intermediate substance (Zellgewebescheide) made from fibers and threads. The ideas about specific brain connective tissue were further developed by Carl von Rokitansky,bona fide connective tissue, glial cells have been visualized and identified by many neuroanatomists These considerations changed the role of glia from being regarded as simply a connective tissue to an active counterpart of neurons in executing brain functions.A pupil and assistant of Purkinje, Gabriel Gustav Valentin was the first to contemplate two types of nervous elements, which he called nervous masses. One of these masses representing “the creative, active, higher principle” while the second “the receiving and guiding, passive, lower principle.”,,The advent of electrophysiology and intracellular recordings led to a detailed characterization of electrical excitability of nerves, muscle, and neurons. The very first observation of experimentally evoked muscle contractions were made in the 1660s by Jan Swammerdam, who designed the classic frog neuromuscular preparation.in vivo, in organotypic cultures, in isolated optic nerve preparations from amphibians or in the isolated ganglionic chain of the leech+ accumulation in the extracellular space.Some 150 years later, the seminal discoveries of Hodgkin and Huxley provided the first quantitative description of the ionic conductance changes underlying the action potential,,in vivo expression of these receptors was tightly regulated by the neurochemical environment: the neuroglial receptor pattern is tailored to neurotransmitters operating in a particular brain region.2+indicators, it turned out that chemical or mechanical stimulation of glia almost invariably triggered complex cytoplasmic Ca2+ signals, which, in a form of Ca2+ waves, could propagate over long distances through the gap-junction connected glial syncytium. Thus, the concept of calcium excitability of neuroglia was developed.+ fluxes that generate cytoplasmic Na+ signals as well as with highly organized changes in cytosolic second messengers such as InsP3 and cAMP; the former being linked to Ca2+ signaling whereas the latter being connected with numerous intracellular enzymatic cascades and influenced by Ca2+. Consequently, a coherent concept of intracellular astroglial excitability is in need of definition.When the technique for making purified glial cell cultures was developedAppearance of the central nervous system (CNS), which emerged early in evolution, was accompanied by division of neural cells into neuron, which represent executive arm responsible for sensory input, information processing, and initiation of peripheral responses and homeostatic and defensive neuroglia. Neuroglial cells of the brain and the spinal cord are classified into macroglia  and microglia, which are scions of fetal macrophages invading the CNS early in the development. Neurons are universally considered as the only excitable cells of the nervous system; they generate fast action potentials, which are conducted over large distances and initiate neurotransmitter release responsible for synaptic connectivity. Nonetheless, fast signaling (in addition to relatively slow ones) does also occur in glial cells, which respond to physiological stimulation with transient fluctuations in their ionic content; these ionic signals are the substrate for rapid stimulus-induced glial excitability.l-serine.,,Astrocytes are the principal homeostatic cells of the CNS, which constantly adapt operation of elaborated homeostatic molecular cascades to neuronal activity and brain state. Astrocytes control CNS homeostasis at many levels. First and foremost astrocytes are responsible for CNS ionostasis—the ionic composition of the interstitial fluid, which are tightly associated with changes in brain state, such as sleep and arousal.i), which regulate astroglial physiological activity. Similarly to neurons, astrocytes, are activated in response to sensory stimulation; numerous experiments in vivo in anesthetized and awake animals have demonstrated synchronous cytosolic [Ca2+]i transients engulfing groups of astrocytes in the sensory cortex.,2+ signals originating from endoplasmic reticulum (ER) Ca2+ release and/or from Ca2+ entry across the plasmalemma,+-dependent transporters, generating a massive Na+ influx, which triggers cytosolic Na+ signals.,,Sensing the neural tissue environment involves the stimulation of astroglial membrane receptors. Activation of these receptors does not trigger regenerative transmembrane depolarization, instead it produces changes in intracellular ion activity reflecting changes in free ion concentration , which represents the prime neuronal plexus localized in the brain stem; projections of the LC neurons synchronously release NA in various brain and spinal cord regions. In the adult human brain, the LC consists of only around 50 000 neuronsAstrocytes are major targets of NA in the CNS; mature astrocytes express adrenoceptors of both α and β varieties while the density of adrenoceptors in astrocytic processes seems to be significantly higher than in neurons.Maintenance of cellular ionic homeostasis is one of the most fundamental conditions for life; all living organisms on planet Earth are keeping the ionic composition of cytosol and organelles under tight control at the expense of considerable energy. Ionic gradients between the extracellular space and the cytosol are driving ion fluxes. These ionic fluxes originate from opening of ion channels following for example an environmental stress in unicellular organisms or due to release of chemical messengers in multicellular ones. Thus, from the very beginning of life, ions evolved as dynamic intracellular signalers coupling extrinsic challenges to intracellular processes. Conceptually, living cells are constantly balancing the preservation of their ionic composition with generation of ionic fluctuations organized in space and time. To achieve this steady-state, evolution selected transport systems moving ions along and against concentration gradients. In essence, all changes in the cytosolic concentration of any ion, di- or monovalent, can regulate/modulate various cellular events, and hence may act as second messengers in biological systems. Ionic signalingis shaped by dynamic interactions of diffusion  and primary or secondary ion transport , which requires energy. All these molecular cascades are in operation in astrocytes.2+ concentration acts as a ubiquitous physiological signal, operating in most  cells and tissues. Changes in the Ca2+ concentration in various cellular compartments trigger or regulate a wide variety of cellular processes. The Ca2+ homoeostatic and signaling system involves relatively few molecular elements  which, by operating in concert, shape Ca2+ signals in the cytosol and in the organelles while at the same time preventing life-endangering Ca2+ overloads. Changes in [Ca2+]i are sensed by numerous Ca2+-binding proteins, which translate Ca2+ signals into cellular activity.It is universally acknowledged that an increase in the Ca2+ signaling is characterized by a complex spatiotemporal organization, which reflects the elaborate astrocyte architecture. Furthermore, different types of astrocytes seemingly have distinct [Ca2+]i dynamics with idiosyncratic underlying mechanisms. The morphological compartments of protoplasmic astrocytes (which are probably the most studied class of astroglia) are represented by (i) soma; (ii) main processes also known as branches; (iii) secondary to tertiary processes designated as branchlets; (iv) peripheral parenchymal and perisynaptic processes known as leaflets; and (v) perivascular processes, which terminate with end feet plastering blood vessels  domains of elevated [Ca2+]i. Focal [Ca2+]i transients can either be spontaneous, with no association with neuronal activity,2+ signals can result from neuronal activity and stimulation of astroglial receptors.2+ signals in the peripheral processes of protoplasmic astrocytes are shorter in duration than in the soma,2+ influx through Ca2+ permeable ionotropic receptors,,+/Ca2+ exchanger (NCX).,2+ events in branchlets and branches can be amplified by Ca2+ released from the ER and mitochondria. The higher surface-to-volume ratio of branchlets allows larger plasma membrane Ca2+ influx and hence larger [Ca2+]i fluctuations.2+]i fluctuations more frequently reach the threshold for Ca2+-induced Ca2+ release through InsP3 receptors. Hence, loss of fine astrocytic branchlets in pathological conditions such as epilepsy can be linked to reduced astrocytic Ca2+ activity.Numerous lines of evidence have demonstrated that Ca2+ signals in protoplasmic astrocytes, as well as [Ca2+]i transients in the primary processes are larger in amplitude and slower, are often synchronized with neighboring astrocytes .,3 receptor type II  eliminates Ca2+ signals in the soma and in the primary processes, leaving [Ca2+]i dynamics in branchlets and leaflets very much undisturbed or only partially suppressed.Somatic Cam the ER 53,85,86 2+ signalling  does not operate in all types of astrocytes. For example, in Bergmann glial cells  Ca2+ signalling microdomains are associated with specific morphological structures—the appendages. These appendages emanate from the primary radially oriented processes of the Bergmann glial cells; each appendage contains mitochondria and projects leaflets that contact 50–70 synapses formed by axons of granular neurons  and InsP3 receptor-mediated Ca2+ release, with subsequent activation of SOCE.,2+]i-induced Ca2+ release was shown to substantially contribute to α1-adrenoceptor-mediated Ca2+ signals2+]i dynamics in the peripheral fine branchlets of cortical mouse astrocytes  were reported to originate from mitochondrial Ca2+ release through the flickering mitochondrial permeability transition pore.2+ signals in branchlets (which possess ER) may involve combination of InsP3-induced Ca2+ release and plasmalemmal Ca2+ entry. Spatial restriction of [Ca2+]i increases could result from local mitochondria, which act as powerful Ca2+ buffers2+]i increases in astroglial processes.2+ signals can be associated with plasmalemma-ER junctions that have been described in cultured primary astrocytes; these junctions are rich in InsP3 receptors, SERCA pumps, and NCX being thus a substrate for focal Ca2+ signaling.2+ signaling, which most likely changes depending on the physiological context, astrocyte morphology, age, and environmental settings.This type of segregated Ca neurons . Activat2+]i dynamics in vivo, in awake animals, remain rather limited. It seems that sensory stimulation triggers large and global  [Ca2+]i elevations controlled mainly by noradrenergic stimulation of α1 adrenoceptors.,,2+]i increase in response to transcranial direct current stimulation.2+ signals evoked by acetylcholine release from projections of the nucleus basalis of Meynert; these [Ca2+]i responses are mediated through muscarinic ACh receptors and involve InsP3-induced Ca2+ release.,in vivo setting communicate through propagating Ca2+ waves, which were characterized in detail in vitro and in brain slices,,Data on astroglial i fluctuations remain largely unanswered questions. Similarly to other cells, astroglial Ca2+ signals regulate gene expression and provide for excitation-metabolic coupling; formation of [Ca2+]i microdomains in astroglial branchlets immobilize mitochondria thus securing local metabolic support2+ signals may trigger astrocyte morphological plasticity.,2+ signalling is implicated in secretion, both exocytotic and nonvesicular.,,2+ signals.,3-mediated Ca2+ signalling does not affect increases in local blood flow in response to sensory stimulation.2+]i transients occurring in the end feet shortly before vasodilatation.,2+-dependent slow tonic regulation of the vascular tone through continuous release of prostaglandins.2+ signals were also reported to undergo changes in sleep, while suppression of astroglial [Ca2+]i dynamics by knocking out the InsP3 receptor type II affects slow-wave sleep with an increase in the number of micro-arousals, abnormal brain rhythms, and an increased frequency of slow-wave sleep state transitions and sleep spindles.How Ca2+ signaling controls reactive astrogliosis, the archetypal astrocytic defensive program. Challenge of astrocytes with damage-associated molecular patterns, such as ATP or β-amyloid triggers [Ca2+]i rises,2+ signaling by genetic ablation of InsP3 receptorsIn a pathological context, Ca+]i are of particular significance for astrocytic homeostatic function, because the absolute majority of plasmalemmal transporters engaged in maintaining various aspects of molecular homeostasis are driven by transmembrane Na+ gradient. These transporters not only utilize Na+ gradients; their operation produces Na+ fluxes thus transporters being simultaneously the sensors and modifiers of [Na+]i. The resting [Na+]i in astrocytes lies in the range of 15–20 mM,,,+]i, which may last for tens of seconds.,,,,+]i transients were shown to spread in the form of propagating [Na+]i waves through individual cells  led to the concept of astroglial Na+ signaling.,Changes in [Na2+]i and [Na+]i dynamics in astrocytes, the underlying mechanisms are quite distinct. Astrocytic Na+ signals rely on plasmalemmal Na+ movements only, as no Na+ storing structures exist in the cells  or Na+-dependent bicarbonate symporter (NBCe1). The NKA in astrocytes incorporates the α2 subunit; which is not expressed in neurons (which possess α 1 and α 3 subunits). As a result, the affinity of astroglial NKA to K+ is substantially lower than in neurons. The EC50 for K+ for astroglial NKA composed from α2/β1 subunits is ∼3.6 mM, while EC50 for K+ in neuronal NKA  varies between 0.25 and 0.65 mM.+] (∼3–3.5 mM) the neuronal NKA K+ binding sites are fully saturated; whereas for astrocytic NKA half of the K+ binding sites remain unoccupied. Consequently, an increase in interstitial [K+] activates astroglial NKA, which is the main mechanism for extracellular K+ sensing and buffering. Neuronal NKA is activated solely by an increase in [Na+]i. The NKA-dependent transport of K+ and Na+ in astrocytes maintains ion gradients critical for operation of homoeostatic transporters; in essence, the NKA acts as the master regulator of astroglial homoeostatic physiology. Increases in NKA transport, which accompany neuronal activity (to buffer K+ or to expel excessive Na+ entering the cell in the course of glutamate uptake) are also linked to astroglial l-lactate production and hence are central for the operation of astrocyte-to-neurone-lactate shuttle (ANLS).,Extrusion of Na+ from astrocytes is mediated solely by Na+/K+ ATPase (NKA). Inhibition of NKA in cultured astroglia by ouabain or by removal of extracellular K+ results in an increase in [Na+]+ signaling is represented by the NCX; astrocytes express all three subtypes of this exchanger . These subtypes are quite similar from the functional point of view, exchanging Na+ and Ca2+ with a 3:1 stoichiometry.,+]i or small depolarizations turn NCX into the reverse mode when it brings Ca2+ into the cell in exchange for Na+; in this mode, the NCX generates a [Ca2+]i rise while accelerating recovery to resting [Na+]i.,,2+]i rises the NCX is forced into the forward mode in which it assists the recovery of [Ca2+]i transients while producing Na+ influx.2+ and Na+ signaling.The second key player of Na2+]i dynamics, astrocytic Na+ signals may be confined to microdomains. Such local subcellular [Na+]i transients have been characterized in Bergmann glia and in hippocampal protoplasmic astrocytes.,+ buffers/binding sites, which may account for the localization of [Na+]i rises. The Na+ transporters in the plasmalemma endowed with Na+ binding sites may act as some sort of highly localized and relatively immobile Na+ buffers. Alternatively, Na+ (and other cations) may be trapped in tiny leaflets by binding sites associated with the inner side of the plasma membrane.+ signals can propagate from cell to cell by diffusion through gap junctions; the speed of these waves in [Na+]i may reach 100–150 mm/s.,Similar to i is represented by the SLC membrane transporters, which fulfill an astroglial homoeostatic function. Changes in [Na+]i may affect not only the efficacy of transports but also change their operational direction. The reversal is well documented for NCX (see above) and can also occur to some other transporters, such as, for example, GABA or glycine transporters, which have been shown to reverse in physiological settings following an increase in [Na+]i.+]i may translate to various biochemical and cellular responses though action on enzymes; in an astroglial context Na+ regulates glutamine synthetase thus affecting availability of glutamine for the glutamate (GABA)-glutamine shuttle.+ ions are also known to modulate or open various types of ion channels, such as, for example, Na+-dependent K+ channels or Kir4.1 inward rectifying K+ channels.,+ signaling therefore was proposed as a mechanism for rapid tuning of astroglial homeostatic cascades to neuronal activity.There are surprisingly large varieties of Na−. Changes in [Cl−]i regulate plasmalemmal channels  serine/threonine protein kinases.,−]i contribute to the regulation of several fundamental cellular processes such as cell differentiation and death.,Chloride, the major inorganic anion in the living tissues, is a likely contributor to astroglial ionic excitability. There are multiple indications for signaling role of intracellular Cl− is the central ion for mediating inhibitory currents in neural cells, and hence fluctuations in [Cl−] in the interstitial fluid are of paramount importance for balancing neurotransmission. Experiments on cultured astrocytes demonstrated that astrocytes maintain high [Cl−]i ranging between 20 and 50 mM, which corresponds to ECl = −35 mV.in situ in acute brain slices. In Bergmann glial cells of the cerebellum Cl− imaging indeed revealed high [Cl−]i of around 50 mM in newborn and 35 mM in mature mice.−]i at 3–4 mM.−]iin vivo is of pressing importance; as it may reveal either regional or state-dependent differences.At the same time, Cl− homeostasis depends on Cl− diffusion through several sets of anion channels that include (i) GABAA and glycine receptors; (ii) inwardly rectifying chloride channels ClC-1, -2, and -3; (iii) Ca2+-dependent Cl− channels; (iv) anion channels of the Bestrophin (Best) family and by (v) volume-regulated anion channels VRAC or SWELL1.− efflux or influx depending on the [Cl−]i; at the same time molecular mechanism(s) for Cl− accumulation into astrocytes remains to be identified. The only known Cl− accumulating transporter, Na+/K+/Cl− co-transporter NKCC1/SLC12A1, has been frequently identified in astrocytes in culture, however, whether NKCC1 operates in situ or in vivo remains controversial.,− fluxes.,− signaling in astrocytes are yet to be fully characterized; the role of astrocytes as a source for Cl− to maintain inhibitory transmission has been proposedAstrocytic Cl+-rich Primordial Ocean. Surprisingly, the cytoplasm of most cells has high K+ and low Na+ concentrations. Several hypotheses explaining this phenomenon have been developed. For example, protocells could have emerged in the K+ enriched vents at the bottom of the ocean; or they may have appeared in the inland basins molded from K+ rich clay and filled with rainwater.+ plays a vital role in cellular life. High [K+]i is required for protein synthesis and sets the cell membrane potential, while K+ efflux repolarizes the cell membrane following action potentials, excitatory postsynaptic potentials, and dendritic spikes in neurons.Life on Earth is believed to have emerged around four billion years ago in a Na+ fluxes across astroglial membranes. Astrocytes remove excess K+ at the peak of neuronal activity and then return K+ back to restore neuronal ionic gradients; in pathology, astrocytes are capable of redistributing K+ through the syncytial networks. Notably, most of the K+ removed by astrocytes from the synaptic cleft during neuronal activity comes from K+ efflux through ionotropic glutamate receptors, predominantly of NMDA type.+ into astrocytes is mainly mediated by NKA (discussed in the previous section), while K+ efflux is mediated by inwardly rectifying K+ channels.+ microdomains in perisynaptic astroglial processes, but whether these domains exist remains to be experimentally seen. The mechanisms of formation of K+ microdomains are similarly unknown. Recently, the role of intramembrane negative charges preventing K+ diffusion+]i have a signaling role and directly modulate cellular functions similarly needs to be tested. One testable possibility is that K+-mediated depolarization can affect voltage-dependent steps of glutamate transporter cycle, hence, affecting glutamate uptake.Hence, neuronal activity is associated with substantial K+]i. This phenomenon is known as “depolarization-induced alkalinization” and results in accumulation of H+ in the extracellular space.+/+ is represented by phosphofructokinase; activation of the latter is perceived as a key step in stimulation of astrocyte-neuronal lactate shuttle.Neuronal activity is accompanied by a transient decrease in astroglial i.Given the relatively complex design of cAMP nanosensor, it is not surprising that the first single-cell measurements of [cAMP]i, consistent with the action of β-adrenergic receptors.i was characterized by a monoexponential rise to a plateau with a time-constant of ∼15 s, much slower than the agonist-induced increases in [Ca2+]i in astrocytes.i represents the balance between the production of cAMP by adenylyl cyclases (AC) and its enzymatic degradation by phosphodiesterases.i were recorded and were considered to be due to an interaction with Ca2+ signaling,Stimulation of astrocytes with adrenaline at 29 nM induced a half-maximal increase in [cAMP]2+ signaling operate in different time domains in astrocytes, there is an interaction between these pathways.i is tonic, lasting several minutes, the swift changes in [Ca2+]i are phasic, often exhibiting oscillations . Glycogen-derived l-lactate exits astrocytes through monocarboxylate transporters (MCTs) 1 and 4 and/or yet unidentified ion channelsl-lactate can also act as an extracellular signal where it binds to l-lactate metabotropic receptors or to yet unknown receptors.Astroglial glycogen represents an energy reserve, which is used during increased activity to support many CNS functions, including memory formation and consolidation.2+- and/or cAMP-pathways, such as ARs and purinoreceptors. Upon stimulation of LC neurons, NA is released, with subsequent activation of metabotropic adrenoceptors and increases in astrocytic [Ca2+]i and [cAMP]i.,2+ and cAMP as second messengers to the regulation of aerobic glycolysis and glycogenolysis in astrocytes remains unclear and even controversial. It is thought that aerobic glycolysis and glycogenolysis are primarily elevated through the cAMP-dependent pathway in astrocytes,,2+ signals might also be involved.Aerobic glycolysis together with glycogenolysis is regulated in astrocytes by a variety of receptors on the surface of astrocytes that are linked to intracellular Ca2+ and cAMP regulates many cellular processes affecting the function of astrocytes and neighboring neurons in health and disease. This intracellular excitability provides regulatory clues in distinct space and time domains, which underlies the capacity of adapting to dynamic and life-long changes that occur during the function of the CNS in health and disease.In conclusion, astroglial noradrenergic signaling, involving CaAstrocytes are an indispensable part of the nervous tissue, which together with neurons and other neural cells produce a cellular fabric responsible for brain function. Homoeostatic cascades in the astrocytes, which support the most fundamental functional properties of the CNS, are tightly correlated with neuronal activity and tissue demands. This coordination is a function of astroglial excitability mediated through spatiotemporal fluctuations of intracellular ions and second messengers.A.S. work was supported by Russian Science Foundation (grant number 20-14-00241); R.Z. is supported by grants from the Slovenian Research Agency , CIPKEBIP, COST Nanonet, COST Mouse Ageing, and COST CM1207 – GLISTEN.None declared.


SITS). The rate of SHTSITS was higher in the higher GA group than in the lower GA group . A higher GA level (GA ≥ 16.0%) significantly increased the risk of SHTSITS  in the logistic regression analysis. The predictive ability of the GA level for SHTSITS was good , and the cutoff value of GA in SHT was 16.3%. GA was a reliable predictor of SHT after IVT in acute ischemic stroke in this study.We evaluated the impact of prestroke glycemic variability estimated by glycated albumin (GA) on symptomatic hemorrhagic transformation (SHT) in patients with intravenous thrombolysis (IVT). Using a multicenter database, we consecutively enrolled acute ischemic stroke patients receiving IVT. A total of 378 patients were included in this study. Higher GA was defined as GA ≥ 16.0%. The primary outcome measure was SHT. Multivariate regression analysis and a receiver operating characteristic curve were used to assess risks and predictive ability for SHT. Among the 378 patients who were enrolled in this study, 27 patients (7.1%) had SHT as defined by the Safe Implementation of Thrombolysis in Stroke-Monitoring Study (SHT The occurrence rates of symptomatic HT (SHT) after IVT ranged from 2.4 to 8.8%, and the rates of asymptomatic HT after IVT ranged from 4.5 to 39.6% in several major clinical trials4. Because the use of IVT has increased, the rate of HT after IVT may also be increased5.Hemorrhagic transformation (HT) is a devastating complication that leads to severe disability and high mortality after intravenous thrombolysis (IVT) in acute ischemic stroke patients4. Several previous studies showed that chronic hyperglycemia estimated by glycated hemoglobin (HbA1c) increased the risk of HT and poor functional outcomes after IVT by inducing microvascular injury9. A recent experimental study and clinical studies showed that glycemic variability led to more oxidative stress and microvascular injury than chronic hyperglycemia13. If glycemic variability prior to stroke worsens microvascular injury, then exploring the role of glycemic variability in acute ischemic stroke treated with IVT in clinical practice is important. However, evidence demonstrating whether glycemic variability increases HT and functional outcomes after IVT is sparse.The pathophysiology of HT is disruption of the cerebral microvasculature by acute ischemic events15. Using a multicenter registry database, we investigated the effects of glycemic variability estimated by GA on SHT and functional outcomes in patients treated with IVT.Glycated albumin (GA) reflects glycemic variability within 4 weeks prior to stroke and may be a useful marker for predicting recent prestroke glycemic variabilitySITS, and 48 patients (12.7%) had any HT. The baseline characteristics of patients receiving IVT only and combined IVT and EVT are described in Supplementary Tables Among the 2894 consecutive patients with acute ischemic stroke, 401 patients (13.9%) received recombinant tissue plasminogen activator (tPA) during the study period. Of the 401 patients, 378 patients who met the inclusion criteria were enrolled. Of those 378 patients, 128 patients (33.9%) were in the higher GA group (GA ≥ 16.0%). To perform subgroup analysis, we stratified the 239 of the 378 patients (63.2%) who received only IVT. The higher GA group was more likely to be older, female, have hypertension, diabetes mellitus (DM) and higher initial random glucose, fasting blood glucose and HbA1c levels than the lower GA group. The stroke mechanism (TOAST), acute stroke management and tPA dose were not different between the higher GA and lower GA groups  significantly increased the risk of SHTSITS was close to good . However, there were no significant differences in the prediction of SHTSITS between GA and HbA1c levels . The cutoff values of GA and HbA1c were 16.0% and 6.3%, respectively, for SHTSITS  curve showed that the predictive ability of GA and HbA1c levels for SHTels Fig. . The cutSITS and any HT. GA/HbA1c was significantly associated with poor functional outcomes at 3 months  The higher GA group had more occurrences of SHT and poor functional outcomes in acute ischemic stroke patients receiving IVT. (2) GA was a strong independent marker for predicting SHT and poor functional outcomes after receiving IVT. (3) The predictive ability of GA for SHT was reliable and the cutoff value of GA was 16.3%.17. Therefore, we hypothesized that GA, which measures recent prestroke glycemic variability quickly and easily in an acute stroke setting, would be a reliable parameter for predicting postthrombolytic bleeding. This multicenter cohort study is the first to evaluate the association between GA and SHT in patients receiving IVT, and the results support this hypothesis. The rates of SHTSITS in our study were comparable to those in previous clinical studies18, and a high GA level increased the risk of SHTSITS and any HT. The sensitivity analysis revealed that the impact of the GA/HbA1c ratio and GA in patients treated with IVT and DM on SHT improved the robustness of our results. We also showed that GA and HbA1c increased the risk of SHT  in our study was comparable to that in a previous study 6. We identified that GA and HbA1c levels may be useful markers for predicting SHT in patients receiving IV tPA using multivariate analysis and ROC curves.Our study revealed the predictive ability and cutoff point of GA for SHT after IVT. The ROC curve analysis showed that GA and HbA1c were reliable for predicting SHT20. However, the association between HbA1c and functional outcomes remains controversial22. In contrast, previous clinical studies on prestroke glycemic variability showed consistent results, and glycemic variability was associated with poor outcomes in stroke patients23. Similar to previous studies, we showed that a high GA level increased the risk of poor functional outcomes in patients treated with IVT. This result may be explained by the high incidence of SHT in the high GA group, despite the similarity in infarct volume between the two groups3. The SHT group in this study had greater stroke severity and eventually had a high proportion of poor functional outcomes  and coefficient of variation of the mean poststroke blood glucose level. If the poststroke glucose level is not reliable for predicting SHT after IVT, then the clinical use of the GA test, which estimates prestroke glycemic variability easily with one blood sample, may be used in an acute stroke setting.Notably, blood glucose levels on admission  were not associated with SHT in our multivariate analysis, which is different from several studiesOur study has several limitations despite the use of a multicenter database. First, although we collected data consecutively, it was an observational retrospective study. Second, we cannot ensure the exclusion of unmeasured confounding variables, but we adjusted for several confounders in the statistical analyses. Third, due to the small sample size of subjects with IVT only, the heterogeneity of recanalization therapies  in our study may hinder the generalization of our results. However, the subgroup analysis evaluating subjects treated with only IVT showed that higher GA was also associated with SHT. This finding improves the robustness of our main result. Fourth, we did not consider several medical conditions that increase protein metabolism and affect GA levels. Thyroid function, liver function, renal dysfunction and alcohol consumption were not available in our database. However, GA was measured on a routine basis prospectively at the two stroke centers, and missing GA data were uncommon. This measurement is a strength of our study.In conclusion, the results of our study showed that prestroke glycemic variability estimated by GA is associated with SHT and poor functional outcomes in patients treated with IVT. Because the GA level is measured quickly and is a reliable marker for predicting SHT, monitoring prestroke glycemic variability via GA levels may be helpful to establish strategies for the prevention of SHT and poor functional outcomes in acute stroke patients receiving IVT.We consecutively registered all acute ischemic stroke patients between March 2016 and December 2020 at two university-affiliated institutions . We identified acute ischemic stroke patients treated with IVT. All of the included patients received intravenous recombinant tPA according to current guidelines for acute stroke management. We excluded the following patients: (1) patients with unavailable GA measurements; (2) patients without follow-up brain computed tomography (CT) or magnetic resonance imaging (MRI) within 24 h of stroke onset; (3) patients who were not available for assessment with the National Institutes of Health Stroke Scale (NIHSS) after IVT and the modified Rankin Scale (mRS) at 3 months; and (4) patients with a prestroke mRS score ≥ 2.This retrospective cohort study was conducted in accordance with the Helsinki declaration and approved by the Institutional Review Board (IRB) of the Hallym University Chuncheon Sacred Heart Hospital (IRB number: 2021-03-002) and Hallym University Dongtan Sacred Heart Hospital (IRB number: 2021-03-006). The need for written informed consent was waived and confirmed by the IRB of Hallym University Chuncheon Sacred Heart Hospital (IRB number: 2021-03-002) and Hallym University Dongtan Sacred Heart Hospital (IRB number: 2021-03-006) because this was a retrospective cohort study.27; and (4) laboratory data, such as platelet count, prothrombin time, systolic blood pressure and level of hemoglobin, creatinine, initial random glucose, fasting blood glucose, low-density lipoprotein and HbA1c. The infarct volume confirmed by diffusion weighted imaging was calculated using the ABC/2 method28.The following data were directly obtained from the registry database: (1) demographics, including age and sex; (2) stroke risk factors, medical history, prior stroke, the presence of hypertension, DM, hyperlipidemia, atrial fibrillation, current smoking status, prestroke status, and prior use of statins and antithrombotic drugs; (3) stroke characteristics, acute stroke treatment, initial NIHSS score, ischemic stroke mechanism according to the Trial of Org 10,172 in Acute Stroke Treatment (TOAST) classification, tPA dose and reperfusion therapy , modified Thrombolysis In Cerebral Infarction (mTICI) grade 2b to 3 after IATSITS)29. Two trained neurologists (S–H Lee and MU Jang) reviewed the CT/MR images to confirm the subtypes of HT in a double-blinded manner. The secondary outcome measures were any HT and poor functional outcomes assessed by a mRS score of 3–6 at 3 months30. Any HT was defined as any newly developed bleeding on the follow-up CT or MRI after IVT.The primary outcome measure was the occurrence of SHT. SHT was defined according to the Safe Implementation of Thrombolysis in Stroke-Monitoring Study (SITS-MOST): type 2 parenchymal hemorrhage with deterioration in the NIHSS score of ≥ 4 points or death  and a lower GA group (GA < 16.0%). GA may be a useful marker for monitoring prestroke glycemic variability because GA is not affected by several medical conditions , which account for a large proportion of stroke patients32. All procedures were conducted in accordance with the STROBE guidelines.Recent previous studies established that a GA level ≥ 16.0% reflected the presence of prestroke glycemic variability based on the following equation of HbA1c and GA: HbA1c = 0.216 × GA + 2.978We hypothesized that high glycemic variability prior to stroke, as defined by a higher GA level, would increase the risk of SHT and poor functional outcomes after IVT. Statistical analyses were performed using IBM SPSS version 21.0 software  and R version 4.0.3 . Summary statistics are presented as the number of subjects (percentage) for categorical variables and the means ± SD or median [interquartile range (IQR)] for continuous variables. Group comparisons were made using Pearson’s chi-squared test for categorical variables and Student’s t-test or the Mann–Whitney U test for continuous variables, as appropriate.For the primary and secondary outcome measures, the higher GA group and lower GA group were compared using Pearson’s chi-squared test for categorical variables and Student’s t-test or the Mann–Whitney U test for continuous variables. To evaluate the independent effects of GA level on outcome measures, we performed binary logistic regression analysis. Variables for adjustment in the multivariable analysis were selected when their p values were < 0.1 in comparisons according to the GA level and if their associations with each outcome variable were clinically plausible. Crude and adjusted ORs and 95% CIs were estimated.p value was calculated using Delong’s test. The cutoff values of GA and HbA1c levels for SHT were calculated using the Youden index.To assess the predictive ability of GA and HbA1c levels on SHT, we generated an ROC curve using the ‘pROC’ package of R. The 95% CI for the area under the curve (AUC) and A sensitivity analysis was also conducted to explore the impact of the GA to HbA1c ratio (GA/HbA1c ratio) and raw GA level on outcomes using a logistic regression model because the GA/HbA1c ratio represented a more accurate glycemic control status. For subgroup analyses, we performed the same statistical models to explore the impact of GA on outcomes in patients receiving IVT only and combined IVT and EVT separately and in those with DM. DM patients were defined as patients with high HbA1c levels (≥ 6.5%) at the time of hospitalization or patients with a history of or current use of hypoglycemic agents or insulin.Supplementary Information.


The double membrane of the nuclear envelope (NE) constitutes a selective compartment barrier that separates nuclear from cytoplasmic processes. Plant viability and responses to a changing environment depend on the spatial communication between both compartments. This communication is based on the bidirectional exchange of proteins and RNAs and is regulated by a sophisticated transport machinery. Macromolecular traffic across the NE depends on nuclear transport receptors (NTRs) that mediate nuclear import (i.e. importins) or export (i.e. exportins), as well as on nuclear pore complexes (NPCs) that are composed of nucleoporin proteins (NUPs) and span the NE. In this review, we provide an overview of plant NPC- and NTR-directed cargo transport and we consider transport independent functions of NPCs and NE-associated proteins in regulating plant developmental processes and responses to environmental stresses. The nuclear envelope (NE) is a key compartment border of eukaryotic cells, partitioning cytoplasmic and nuclear processes. It consists of an outer nuclear membrane (ONM) and an inner nuclear membrane (INM). These two lipid bilayers enclose an intermembrane lumen that is termed the perinuclear space and is continuous with the lumen of the endoplasmic reticulum (ER). The partitioning of essential cellular processes by the NE, such as nuclear transcription of genetic information and the cytoplasmic translation of transcripts into proteins by ribosomes, requires the regulated exchange of molecular information between both compartments. The primary pathways for the bidirectional communication across the NE are nuclear pore complexes (NPCs). NPCs are supramolecular protein conglomerates that consist of multiple copies of approximately 30 nucleoporins (NUPs) and fuse the ONM and INM to form a central transport channel across the NE . Nucleopvia NTRs of the karyopherin family include proteins as well as different RNA species that are generated inside the nucleus, such as transfer-RNAs (tRNAs) and diverse regulatory small RNAs and ribosomal RNAs  and importin-β family IMP-β; . α-imporimportin-α gene family , a gain-of-function mutant of a nucleotide−binding leucine−rich repeat (NLR) immune receptor, IMP-α3 was named MODIFIER OF SNC1, 6 , further suggests a specialization of MOS6/IMP-α3 in immunity-related cargo transport  ELONGATED HYPOCOTYL1 (FHY1) that acts as an NLS-containing facilitator in nuclear translocation of FR light-activated phytochrome A (phyA) termed Pfr  is embryo lethal  mutants (2+- and reactive oxygen species (ROS)-mediated cell death responses, arguing for its involvement in several signaling pathways and physiological responses -SR, further outlines the importance of the nucleocytoplasmic trafficking machinery for plant immune responses. The loss of MOS14 influences the splicing patterns of NLR transcripts, including SNC1, due to reduced nuclear accumulation of serine-arginine rich (SR) proteins, which are required for splice site recognition and spliceosome assembly /EXPORTIN-T and HASTY (HST)/EXPORTIN5 display a delay or acceleration in a range of developmental processes, respectively . ogenesis . XPO1A an1 XIW1; . In addir export . Mutantsectively . While Pf miRNAs . Since Hf miRNAs .XPO4 function enhances autoimmunity of the nucleoporin mutant cpr5 (constitutive expresser of PR genes5), the authors show that the export activity of XPO4 counteracts nuclear accumulation of TPL and TPRs, which are involved in transcriptional co-repression of negative regulators of immunity immunity and PCD upon CPR5 gated nuclear cargo influx. Indeed, The modulation of plant immune responses at the level of protein translocation across the NPC has also been revealed for deletion . NUP88/Mdeletion . The gennup88 mutations in other organisms, null alleles of Arabidopsis mos7 are lethal, suggesting that in addition to its function in regulating a diverse set of plant immune responses, wild-type MOS7/NUP88 is also essential for regular plant growth and development  and has been termed DRACULA2  transcriptional regulator-independent manner . FT induces the expression of genes that contribute to the formation of floral primordia, providing an explanation for the early flowering phenotypes of hos1, nup96 and nup160 plants  and reduced (nup160) – tolerance to cold stress  and nup96  show reduced nuclear accumulation of the transcriptional repressor AXR3/IAA17  complex, which associates with transcripts and aids in the recruitment of mRNA export adaptors and receptors  that is required for nuclear mRNA export  BRI1-ASSOCIATED KINASE1 (BAK1) and its closest homolog BAK1-LIKE1 BKK1; . Du et aA export . In Niconfestans . Intriguaponicus , substanTranscriptional adaption is a key process in initiating cellular responses to developmental as well as biotic and abiotic signals. Apart from functions in protein and RNA transport processes described above, NPCs together with NE-associated proteins are also involved in the spatial chromatin organization and mediate gene positioning at the nuclear periphery to regulate gene expression and promote mRNA export. The idea of NPC-mediated gene regulation was already proposed more than 35 years ago in the “gene gating” hypothesis . Indeed,FLOWERING LOCUS C (FLC) that acts as a flowering repressor  has also been described for nup1/136 as well as nup82 mutant plants, further supporting a role in transcriptional regulation of these nucleoporins  1–4  1–4 . Indeed,RWN) 1–4 , while CRWN) 1–4 . As for l nuclei . A role l nuclei . Signifil nuclei .PR1 gene expression  and the transcriptional repressor SUPPRESSOR OF NPR1-1, INDUCIBLE1 (SNI1), thereby repressing pression . Consequbacteria . Similar pathway . In crwneriphery .crwn1 crwn4 double mutant when compared to wild-type plants and the crwn1 crwn2 mutant, outlining differential and – on global gene expression patterns – partially antagonistic functions for the CRWN proteins on transcription. In another recent study, crwn1 mutants show elevated levels of jasmonic acid (JA), a plant hormone required for defense against necrotrophic pathogens that kill host tissues (crwn1 mutant plants correlates with enhanced resistance toward the necrotrophic fungus B. cinerea (crwn1 plants are more susceptible to non-virulent Pseudomonas bacteria and show defects in early defense responses (It is noteworthy that  tissues . This mo cinerea . In contesponses . These rCRWN genes also affect developmental processes such as seed germination, since crwn1 crwn3 double mutant plants show reduced germination rates and are hypersensitive to ABA (crwn1 crwn4 double mutant displays low copper tolerance (Apart from the effect on immune responses, mutations in e to ABA . The autolerance . Indeed,olerance .via NPCs or plant lamin-like proteins can have suppressive as well as enhancing consequences on gene expression depending on the specific locus or stimulus. Given that some crwn mutant combinations have partially antagonistic effects on global gene expression patterns (The examples summarized in this chapter demonstrate that the tethering of chromatin to the nuclear periphery patterns , it willThe work reviewed here emphasizes the plant NPC and its associated transport machinery as an important hub that actively regulates cellular signaling pathways and gene expression as part of developmental programs and in response to environmental factors. Recent work has made significant progress in defining the NPC/NE proteomes, identifying NTR cargo substrates, and in assigning nucleoporin and NTR functions to multiple plant signaling pathways. However, many of the molecular mechanisms underpinning transport-associated and -independent processes in developmental and environmental stress pathways remain to be determined. In particular, open questions to be addressed include: What are the steady-state and stimulus-specific “transportomes” of NTRs and how are NTR cargo binding specificities and transport kinetics across the NPC regulated? Is the nuclear translocation of small molecules, such as plant hormones, diffusion based or actively regulated by binding to transported carrier proteins? What are the effects of positioning particular genes in proximity to the NPC/NE on their expression and epigenetic regulation? How is the recruitment of a certain chromosomal locus regulated, considering that each nucleus contains a multitude of NPCs? Are there tissue/cell-type specific differences in NPC composition and/or post-translational nucleoporin modifications that may account for the involvement in particular response pathways? And finally, is the protein composition of NPCs within a single nucleus identical or variable, and does variability provide sub-populations of NPCs with specific functions? Elucidating these questions will provide valuable insights into the intricate regulation of cellular gene expression and signal transduction pathways by nucleocytoplasmic transport and transport-independent NPC functions in plants.DL and MW conceived the review. DL, PFWR, and MW wrote the manuscript. PFWR prepared the figures and tables. All authors edited and approved the submitted version of the article.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.


Background: Previous studies in mice indicated that Paneth cells and c-Kit-positive goblet cells represent the stem cell niche of the small intestine and colon, respectively, partly by supporting Wnt and Notch activation. Whether these cell populations play a similar role in human intestinal cancer remains unexplored. Methods: We performed histopathological evaluation and immunohistochemical analysis of early colorectal adenomas and carcinoma adenoma from patients at the Hospital del Mar in Barcelona. We then determined the possible correlation between the different parameters analyzed and with patient outcomes. Results: Paneth cells accumulate in a subset of human colorectal adenomas directly associated with Notch and Wnt/β-catenin activation. Adenoma areas containing Paneth cells display increased vessel density in the lamina propria and higher levels of the stem cell marker EphB2. In an in-house cohort of 200 colorectal adenoma samples, we also observed a significant correlation between the presence of Paneth cells and Wnt activation. Kaplan–Meier analysis indicated that early adenoma patients carrying Paneth cell-positive tumors display reduced disease-free survival compared with patients with Paneth cell-free lesions. Conclusions: Our results indicate that Paneth cells contribute to the initial steps of cancer progression by providing the stem cell niche to adenoma cells, which could be therapeutically exploited. Colorectal cancer (CRC) is the second leading cause of death by cancer in the industrialized countries. However, most CRC patients do not die because of the primary lesions that are generally removed by surgery, but as a consequence of more aggressive invasive or metastatic tumors. Tumor relapse after therapy and tumor metastasis mostly depend on an exclusive population of cells known as “tumor initiating cells” or cancer stem cells (CSCs) that share specific features with normal intestinal stem cells (ISC) including their transcriptional profiles .In the normal mouse intestine, a limited number or Paneth cells constitute the niche of the ISC populations residing at the bottom of the crypts , which aLgr5, Bmi1, EphB2 or Hes1.Multiple evidence indicates that tumor-initiating cells depend on the same signaling pathways that regulate normal ISC function including Notch ,16,17,18Using a mouse model of intestinal cancer that carries mutations in the APC gene , we previously demonstrated that aberrant activation of β-catenin in the adenoma cells induced transcription of the Notch ligand Jagged1 leading to Notch signaling , which wApart from being the source of stem cell signals in the small intestine, Paneth cells have previously been associated with β-catenin pathway activation in human adenomas  and a spHere, we investigate the relationship between Notch and β-catenin signaling and the presence of Paneth cells in human colorectal tumors, and its possible contribution to tumor malignancy.Samples of 38 patients from the Hospital del Mar  with pathologically determined colorectal adenocarcinomas were initially selected for this study based on the presence or absence of Paneth cells. General information about these tumors is shown in In brief, the tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and processed by standard histological methods. From each selected paraffin block, 4 μm serial sections were cut. Immunohistochemical (IHC) studies were performed with avidin-biotin-peroxidase complex kits according to the manufacturer’s instructions . Primary antibodies  were incubated in a humidified chamber at 4 °C overnight. After that, the samples were developed with liquid DAB + substrate chromogen system  under microscope  and the results were counted by two independent researchers. The method of IHC READING calculation was as follows: Staining intensity: 0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining; percentage of positive cells: 0 = 0%, 1 = 1–10%, 2 = 10–50%, 3 ≥ 50%.Briefly, after the samples were incubated with primary antibody , the samples were washed and then developed with Tyramide Signal Amplification (TSA™) plus the Cyanine 3/Fluorescence system  under the microscope  and then mounted with DAPI ; the positive results were stained green or red and the pictures were taken by confocal laser scanning microscopy .Two researchers without knowledge of the clinical data independently evaluated the IHC results. In all the analyses a minimum of five fields containing tumour areas were randomly selected and evaluated by two independent researchers. Images at 400× magnification were used to determine the percentage of tumor cells that exhibited positive immunoreactivity for the different antibodies. In tissue microarray samples, staining intensity was assessed directly under the microscope by two independent researchers, at 100× magnification, but amplifying tumour areas at 200× when necessary to obtain agreement among researchers. In the case of Notch and β-catenin, nuclear and cytoplasmatic stainings were evaluated separately. Intensity of positive staining was defined as follows: light: 1, moderate 2, intense 3. The value for negative staining was 0. The Histoscore index defined as (0×%) + (1×%) + (2×%) + (3×%) was used to analyze the correlations between IHC results and clinical information including the pathological grade and TNM stage.p value was calculated using a two-tailed confidence interval of 95%. Survival analyses were performed on Graphpad Prism 9, to measure 5-year disease-free survival; X values were measured in months, Y values were coded 1 for death/event, 0 for censored subjects; survival curves were compared using the logrank (Mantel–Cox test) and the Gehan–Breslow–Wilcoxon test.Pearson’s chi-square test with a cross tabulation table of 2 × 2 was performed to determine the statistical association between marker expression (positive or negative) using Excel. R score for correlation (Pearson coefficient) was used to test the correlation between quantitative measurements of marker expression (0-1-2-3 or beta-catenin and 0-1 for lysozyme) using Graphpad Prism 9; Paneth cells are mostly located at the bottom of the small intestinal crypts in both mouse and humans. They are primarily absent from the normal colonic mucosa but their frequency increases in several inflammatory diseases . We havePrevious studies indicated that Paneth cells also express synaptophysin . ConsistTogether these results indicate that Paneth cells accumulate in the pseudostratified adenomas whereas they are absent from adjacent monolayered glands, thus suggesting a role for Paneth cells in early tumor progression.Paneth cells represent the stem cell niche in the small intestine by providing the source for Wnt and Notch activation . Here, wWe then extended this study using a tissue microarray (TMA) containing 250 human samples including normal tissue (54), adenoma (143) and carcinoma areas (53) from 60 different patients. We detected LYZ-positive cells (considering positive samples classified as ++ and +++) in about 40% of the adenoma samples, covering different areas of the tumor tissue. This proportion was highly reduced in the more advanced carcinoma samples and almost negligible in the normal mucosa C. As expThese results indicate that Wnt/β-catenin activation is associated with LYZ expression during early colorectal tumorigenesis.Next, we determined the possible association between Notch1 activation and the presence of Paneth cells and LYZ expression in colorectal tumors. IHC analysis of samples using the ICN1 antibody demonstrated higher Notch1 activity in adenoma and carcinoma samples compared with normal adjacent mucosa of the same patients A. Remarkp < 0.001) between EphB2 levels and nuclear β-catenin that was restricted to Paneth-positive samples  compared with those classified as Paneth-negative   showed reduced overall survival (negative A. Simila = 3.35) B that wa = 3.35) C. Intere = 3.35) D. Analys = 3.35) E.Together our data indicate the Paneth cells may contribute to the activation of essential stem cell signaling pathways at early colorectal adenomas, and suggest the usefulness of using Paneth cell detection for patient prognosis at early stages of tumor progression. However, further studies should be performed, including a higher number of patients, to obtain robust conclusion on the validity of Paneth cell detection in early adenomas as a criterion to refine patient guidance.There are controversial data on the prognosis value of Paneth cell detection in colorectal tumors. Our results indicate that Paneth cell detection and LYZ expression is correlated with Wnt/β-catenin and Notch activation specifically in low-grade adenoma samples. Most importantly, the detection of Paneth cells in this group of tumors is directly associated with poor patient outcomes. The fact that Paneth cell differentiation is significantly reduced in the more advanced carcinoma samples has certainly made difficult the identification of Paneth cells as a poor prognosis factor in the less-advanced lesions. However, we have demonstrated that restricting the analysis to initial tumor stages  is sufficient to confirm the prognosis value of LYZ levels in CRC.We propose that evaluating the presence/absence of mature Paneth cells in the adenoma samples at the time of diagnosis by detection of eosin-positive granules in the H&E-stained samples will provide the clinicians with additional information, which could directly impact patient prognosis with subsequent therapeutic implications.


AtCKX overexpressing centaury plants grown in vitro on graded NaCl concentrations . The levels of endogenous stress hormones including abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) were also detected. The elevated contents of total CKs were found in all analysed centaury shoots. Furthermore, increased amounts of all five CK groups, as well as enhanced total CKs were revealed on graded NaCl concentrations in non-transformed and AtCKX roots. All analysed AtCKX centaury lines exhibited decreased amounts of endogenous IAA in shoots and roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all AtCKX lines. In shoots and roots of both non-transformed and AtCKX transgenic centaury plants, salinity was associated with an increase of ABA and JA and a decrease of SA content.Plant hormones regulate numerous developmental and physiological processes. Abiotic stresses considerably affect production and distribution of phytohormones as the stress signal triggers. The homeostasis of plant hormones is controlled by their de novo synthesis and catabolism. The aim of this work was to analyse the contents of total and individual groups of endogenous cytokinins (CKs) as well as indole-3-acetic acid (IAA) in  Exposure of plants to salt stress results in changes in almost all physiological and biochemical processes leading to a disturbance of normal growth and development. During evolution, plants have developed different mechanisms for successful adaptation on unfavourable environmental conditions. The response of plants to stress conditions depends on multiple factors, among which phytohormones play a crucial role. Thanks to signal transduction and pathway crosstalks, phytohormones defend plants by promoting specific protective mechanisms against abiotic and biotic stresses2.Plants are sedentary organisms that face numerous survival challenges such as abiotic  and biotic stresses . Among numerous environmental hindrances, abiotic stress is known as the significant factor reducing the average yield. Salinization represents one of the major sources of all abiotic stresses damaging at least 20% of the crop production all around the world3. Abiotic stresses often lead to altered production and distribution of endogenous phytohormones making them stress signal triggers4.Plant growth and development are regulated by phytohormones such as cytokinins (CKs), auxins, abscisic acid (ABA), gibberellins, ethylene, salicylic acid (SA), brassinosteroids and jasmonates which control numerous physiological and biochemical procceses5. It has also been reported that CKs are involved in enhancing tolerance against high salinity and temperature stress in plants6. Generally, it is accepted that CKs are predominantly produced in the root tips and developing seeds. After biosynthesis CKs are further translocated from the roots via xylem to the shoots where they regulate plant development and growth processes. The increased activity of the enzyme cytokinin oxidase/dehydrogenase  is directly related to decreased amount of CK. This catabolic enzyme represent a key factor in controlling of CK levels in plant tissues. The free bases, iP and trans-zeatin and their respective ribosides are the preferred CKX substrates7, but cis-zeatin and its riboside have also recently been found to be efficiently degraded by some CKX isoforms8. In different plant species, small gene families encode CKX isoforms with varying number of members. Seven A. thaliana CKX genes (AtCKX1-7) were described almost twenty years ago9. It is also known that individual members of the AtCKX gene family are expressed differentially and have different subcellular localizations10. The AtCKX1 expression is specific for shoot apex of lateral shoot meristems while the AtCKX2 promoter is expressed in the shoot apex and not in Arabidopsis roots. On the other hand, AtCKX1 is a mitochondrial protein with vacuole as final destination while AtCKX2 represent the peptide for targeting to the endoplasmic reticulum and subsequent transport to extracellular region.Various plant growth and developmental processes are regulated by CKs. This group of phytohormones primarily regulates cell division, apical dominance, vascular differentiation, leaf senescence, chloroplast biogenesis, shoot differentiation and anthocyanin production12. Previously it was shown that CKs increase plant tolerance to salinity stress6. These literature data, however, considered exogenous application of CKs. On the other hand, only few literature data exist describing how plants with genetically modified levels of endogenous CKs respond to salt stress15. Considering that some literature data describe both positive and negative influences of CKs on stress tolerance, all of these pieces of information could be crucial for better knowledge of CK involvement in the plant stress tolerance responses.When the main plant organs, roots, are exposed to salt, they are clearly affected by salinity stress. Under salt stress conditions, a reduction of CK biosynthesis in the roots directly affects CK amounts in the shoots16. Literature data indicating involvement of auxin in response to salinity stress are somewhat limited. The root architecture of Arabidopsis thaliana was substantially remodelled by auxin accumulation and its redistribution under salt stress18. Some reports are also describing a significant reduction of IAA level in salt-stressed rice and tomato plants19. Besides, salinity was found to decrease contents of IAA and the non-indole auxin, phenylacetic acid, in both cultivated glycophyte tomato Solanum lycopersicum as well as in its halophyte wild relative S. chilense20.Auxins represent the plant hormones which promote plant growth and development. Indole-3-acetic acid (IAA) as the major auxin representative in plants mainly regulates vascular tissue development, cell elongation and apical dominance21. It is known that under drought or salinity stress conditions, the level of endogenous ABA was significantly increased24. In different plant developmental processes such as stomatal opening in bean leaves and seed germination of Arabidopsis, CKs act as ABA antagonists26. As both these hormones individually play essential roles in stress tolerance, mainly during drought and salinity stress, a crosstalk among CKs and ABA seems to be very important for improving abiotic stress tolerance27. During last ten years, it was shown that CK receptors known as Arabidopsis Histidine Kinases (AHKs) represent crucial regulators during salt, drought and cold stress conditions29. In contrast to AHK1 known as a positive regulator of ABA signaling, AHK2 and AHK3 were assigned as negative regulators29. Moreover, the expression of maize ckx1 gene was strongly affected by CKs and ABA under the conditions of abiotic stress30. Functional analysis of CK-deficient Arabidopsis plants showed that overexpression of ipt and ckx genes was repressed by stress and ABA treatments, leading to decreased concentrations of biologically active CKs13.ABA, commonly known as the stress hormone, is probably the most studied phytohormone because of response and distinct role in plant adaptation to multiple abiotic stresses31. It also plays an important role in protection against biotic and abiotic stresses including salinity32. The role of SA in plant defense from salt stress conditions is species-specific and has been well documented35. For example, the endogenous SA level was decreased under salt stress in rice seedlings36 and salt-stressed Iris hexagona plants37. Salinity also strongly decreased SA concentration in glycophyte tomato S. lycopersicum but on the other hand, SA content increased in response to salt in the wild halophyte S. chilense20.SA, endogenous plant growth regulator with phenolic nature, is involved in the regulation of physiological processes such as growth, photosynthesis, and nitrogen metabolism37. It is known that JA and its derivatives also strongly respond to salinity stress conditions38. It has been reported that JA levels in various tomato cultivars changed in response to the salt stress39. When exposed to salinisation, the salt-tolerant tomato cultivar considerably increased its JA level whereas the salt-sensitive cultivar decreased it. Concentration of jasmonates was more than two times higher in the halophyte S. chilense than in the glycophyte S. lycopersicum, and salinity increased its concentration by more than 100% in both species compared to control plants20.Jasmonic acid (JA) is an important cellular regulator involved in diverse developmental processes such as seed germination, root growth, fertility, fruit ripening, and senescenceAtCKX transgenic centaury shoots and roots grown in vitro15. It was shown that centaury roots showed higher salinity tolerance compared to shoots. Morphogenic potential of AtCKX1 transgenic line was similar to non-transformed line under increased NaCl concentrations in vitro. On the other hand, there were significant difference in morphogenic response of two investigated AtCKX2 transgenic lines, specially in root culture. Accordingly, in this followed work, we compared the profiles of endogenous of phytohormones in non-transformed and transgenic centaury AtCKX lines grown in vitro under salinity stress conditions. The contents of CKs, auxins, ABA, JA and SA were determined in shoots and roots cultured in vitro for eight weeks on increased NaCl concentrations. This work represents the first report on altered phytohormone levels in centaury, the significant medicinal plant species, during salt stress. The obtained results might be a useful tool for better understanding of salinity tolerance of non-transformed and CK-deficient transgenic centaury plants grown in vitro.Previous work described salinity stress response of non-transformed and AtCKX1  centaury lines  including bioactive forms (CK nucleobases), transport forms (CK ribosides), storage forms (CK O-glucosides), deactivation forms (CK N-glucosides) and immediate biosynthetic precursors (CK phosphates). The abbreviations for CKs presented in this work were adopted and modified according to41. The levels of individual endogenous CK groups were determined and are shown on Figs. Endogenous CKs were detected and quantified in shoots and roots of non-transformed Fig. , one traKX1 line 9, Fig. 2es Figs. , 4 grownAnalysis of endogenous CKs in non-transformed shoots showed that the levels of bioactive CK forms were significantly increased only on one NaCl concentration 150 mM) while decreased on two NaCl doses (50 and 200 mM). In non-transformed roots, the contents of bioactive CK forms were significantly enhanced on all NaCl concentrations . On the other hand, the contents of CK phosphates were elevated in roots. In analogy to AtCKX1-29 transgenic line, AtCKX2-17 shoots and roots showed increased total CKs on all applied NaCl concentrations (with the exception of 200 mM NaCl for roots) in comparison to the control plants grown on NaCl-free medium.In shoots of transgenic ded Fig. . On the AtCKX2 line, AtCKX2-26, showed different pattern compared with AtCKX2-17 . Accordingly, IAA/bioactive CKs ratios were elevated on 100 and 200 mM NaCl  and ckx (responsible for CK degradation), were proposed as possible target genes involved in stress resistance45. Altered endogenous CK levels in Arabidopsis, Nicotiana tabacum and Hordeum vulgare plants, modified either by loss of the ipt genes expression or by overexpression of the ckx encoding genes, showed that CKs played a negative role in response to heat and/or drought stress49. Also, there are literature data describing that reduced CK contents improved drought and salt stress tolerance in Arabidopsis ckx overexpressing lines and ipt1, ipt3, ipt5 and ipt7 mutants50.Cytokinins are present in all plant tissues regulating numerous developmental processes such as cell division, apical dominance, leaf senescence, vascular and shoot differentiation and anthocyanin productionAtCKX1 and AtCKX2 genes in transgenic centaury plants resulted in altered CKs profile leading to a decline of bioactive CK levels and, at the same time, increased contents of storage CK forms, inactive CK forms and/or CK nucleotides51. Previous investigation showed that overexpression of CKX genes caused hyposensitivity to salt stress and drought stress in arabidopsis13 and Physcomitrella patens52. Our current results generally indicated that all groups of endogenous CKs were considerably altered in non-transformed and AtCKX centaury shoots on all applied NaCl concentrations and that the total CK contents were elevated comparing to the corresponding control. Furthermore, increased levels of all five CK groups as well as of total CKs were recorded on used NaCl concentrations in non-transformed and AtCKX centaury roots. Furthermore, analyses of endogenous CKs showed that the levels of bioactive CK forms varied considerably among non-transformed and all analysed AtCKX shoots depending on NaCl concentration. On the other hand, increased levels of bioactive CK forms were found in roots of all analysed centaury lines, on almost all applied NaCl concentration. These observations indicated that salinity triggered different changes in the CK contents in centaury shoots and roots. Considering that CKs are produced mainly in roots which are directly involved in responses to nutrients it can be concluded that CKs represent an important signal traveling from roots to the shoots. The transport of CK from roots to shoots is considered as the key factor in response to salt stress53. Therefore, the salt-induced changes in CK profiles may disturb homeostasis on the subcellular level and also further alter the CK content in other compartments. Furthermore, it is important to note that altered CK levels in centaury tissues may also be the result of their prolonged exposure to salinity during eight weeks. Any strong conclusions about CK content is very difficult because the complexity of the cytokinin profiles has not been fully explored53.Previous investigation showed that overexpression of the 2O2 production as well as reduced SOD and POX activities in transgenic AtCKX centaury plants under prolonged salinity conditions15. Similarly, the activity of SOD was considerably decreased in leaves and roots of transgenic AtCKX2 tobacco plants during the salt stress24. Previous investigations showed that ectopic expression of ckx genes in barley activated genes involved in biosynthesis of flavonoids49 and also in drought tolerance54. On the other hand, increased level of antioxidants during the stress could protect plant cells from reactive oxygen species (ROS) accumulation and reduce electrolyte leakage and/or rises in malondialdehyde levels56. All of these results indicated the effects of CKs in stress tolerance and involvement of antioxidant systems in response to manipulation of CK homeostasis. It was also reported that AtCKX transgenic centaury roots showed higher salinity tolerance compared to shoots15. These results are in accordance with findings describing that the ckx overexpression enhanced root growth, nutrient uptake and drought tolerance57. Additionally, several literature data indicate that the growth and development of root tissues could be important in CK-regulated responses to water limiting conditions, including the differentiation of vascular tissue58 and lignification48.It is known that CKs affect antioxidant systems and related processes under drought and salt stress. Recent biochemical analyses showed lower free proline, MDA content and HArabidopsis, maize and Prosopis strombulifera60. A cross-talk between auxins and CKs has been widely studied and possible antagonistic roles in plant defense responses were revealed. Specific mechanisms that modulate this cross-talk are, however, still unknown62. Our results showed altered endogenous IAA content in shoots and roots of non-transformed as well as AtCKX-overexpressing centaury plants grown on graded NaCl concentrations in vitro. Interestingly, non-transformed and AtCKX1-29 shoots and roots showed decreased IAA/bioactive ratio in response to salinity whereas this ratio in both AtCKX2 transgenic lines varied considerably. A close connection between IAA levels and CK contents has been previously reported in AtCKX1 and AtCKX2 centaury transformants51 that contained lower amounts of endogenous IAA in shoots as well as in roots. Consequently, the IAA/bioactive CKs ratios showed a significant variation in the shoots and roots of all analysed AtCKX transformed centaury plants. Previously it was shown that auxins affect CK degradation by downregulating ckx genes63. Jones et al.64 showed that elevated CK level increased auxin biosynthesis in young, developing root and leaf tissues while decreased endogenous CK levels reduced auxin biosynthesis in seedlings and root meristem tissues of Arabidopsis. Literature data also demonstrated reduced IAA contents in AtCKX1 and AtCKX2 Arabidopsis seedlings10. On the other hand, overexpression of AtCKX3 transgenes did not affect endogenous IAA content in tobacco leaves65. Varying endogenous IAA and CKs contents and ratios may be explained by the fact that auxin-cytokinin cross-talk pathways are dependent on the developmental stage and tissue specific.Together with CKs, auxins play a crucial role in regulation of plant growth and development. They are also involved in plant tolerance against various environmental stresses like salinity, however, mechanisms of salt stress regulation by these phytohormones are not yet clear enough. Several studies revealed IAA accumulation under salt treatments in AtCKX centaury shoots and roots, ABA and JA levels were elevated on increased NaCl concentrations, reaching the maxima on 200 mM NaCl. ABA is well known to represent an endogenous signal molecule that helps plants to survive unfavorable environmental conditions. Accordingly, the exposure of plants to drought and high salt stress resulted in a proportional enhancement of ABA and JA levels in salt-sensitive and salt-tolerant plant species66. Increased endogenous ABA and JA contents were also observed in Iris hexagona as a response to salinity37. All these reports indicated that higher ABA and JA levels could act as effective protectors against salt stress conditions. Also, there are literature data describing exogenous ABA application in raising the plant salt tolerance69. Likewise, exogenous application of JA was successfully applied in order to recover the salt-induced defects on plants71. Exogenously amended stress hormones during salt treatment may change the balance of endogenous hormones, and thus provide an important trait for understanding the protection mechanisms against the salt stress. All of these results clearly demonstrate that ABA and JA have positive roles not only during wounding and biotic stress but also as effective protection agents against high salinity.Considering that the plant responses to stress conditions involve numerous physiological and biochemical changes, including alterations in concentrations and ratios of endogenous stress phytohormones, it was observed that increasing salinity in centaury plants is associated with an increment of ABA and JA as well as a decrement of SA contents. In non-transformed and transgenic AtCKX lines. Some plant species contain very high basal levels of SA and consequently, SA does not appear to act as an effective signal molecule for defense genes and inducing the disease resistance72. It is known that CKs enhance the SA response which results in increased transcription of defense-related genes such as SID2 for salicylic acid biosynthesis and PR1, a marker gene for salicylic acid responses73. Accoedingly, it was expected that AtCKX centaury plants with altered endogenous CK content show different SA content comparing to non-transformed. Our results also showed declined SA contents in shoots and roots of non-transformed as well as transgenic AtCKX centaury plants grown on increased NaCl concentrations in vitro. The lowest endogenous SA content was observed on 200 mM NaCl. These results correlate with a reported decline of endogenous SA levels in Iris hexagona37 and rice seedlings36 under salt stress. However, some studies showed that SA accumulation is a signal of injury of plant tissues under adverse environmental conditions75. Just because some studies have reported the improved stress tolerance by SA accumulation whereas others have demonstrated less stress tolerance when the endogenous SA level remain high, the role of SA in plant responses to abiotic stress is not clear yet. It is also reported that SA considerable cross-talk with other plant hormones is implicated in drought and salinity tolerance mechanisms. In general, antagonistic interactions between SA and ABA are well known77. Beside, numerous literature data are describing that exogenous application of SA elevated salt stress resistance. The ameliorative effects of SA have been well documented including salt tolerance in crops such as bean78 and mustard79. All of these described studies demonstrated that endogenous SA level has a plastic response that is modified depending on the plant organ, particular species or exposed type of stress. Considering that plants are continuously subjected to exposure to multiple stresses under natural conditions, endogenous hormones need to be interconnected in order to allow them to coordinate the reactions leading to survival using often limited resources. Exploring the interaction of endogenous phytohormones still remains a quite issue considering that all phytohormones excessively involved in cross-talk with each other and also affect and alter each other biosynthesis.SA is generally associated with plant biotic stress. However, numerous reports regarding SA participation in plant responses to abiotic stresses, salinity especially, have been documented recently. Our results showed enhanced SA content in non-transformed shoots and roots in comparison to centaury AtCKX transgenic lines indicated their involvement in responses of centaury plants to salinity. Considering that AtCKX transgenic centaury plants grow on medium supplemented with graded NaCl concentrations but without exogenous application of plant growth regulators, it can be concluded that endogenous phytohormones act as a main determinant for the salt tolerance. The majority of plant hormonal investigations are based on their exogenous applications but the knowledge of changing endogenous phytohormone profiles in response to salt stress during plant growth still remains limited. Although it is known that different plant hormones cross-talk mutually, the mechanisms underlying in these complex signal pathways have not yet been described thoroughly. Unravelling the mechanisms of different phytohormone actions undoubtedly represents a promising tool for future investigations.Altered contents and ratio of endogenous phytohormones such as CKs, auxin IAA, ABA, JA and SA in non-transformed and C. erythraea Rafn. seeds obtained from Jelitto Staudensamen GmbH, Schwarmstedt, Germany. Transgenic AtCKX centaury (Centaurium erythraea Rafn.) plants were obtained as previously described80. Shoots and roots of non-transformed, one transgenic AtCKX1 (AtCKX1-29) and two AtCKX2 (AtCKX2-17 and AtCKX2-26) centaury lines (≈ 10 mm long) were separately cultured in vitro, on half-strength MS medium (½MS81). Shoot explants (10) were cultured in 1 l bottles with 100 ml of culture media, while root explants (20–25) were grown in petri dishes (∅ 10 cm) filled with 25 ml of culture media. The selection of three transgenic lines was based on overexpression of AtCKX genes in centaury shoots and roots which resulted in an altered CKs profile leading to a decline of bioactive CK levels and, at the same time, increased contents of storage CK forms, inactive CK forms and/or CK nucleotides51. The medium was solidified with 0.7% agar and supplemented with 3% sucrose and 100 mg l−1myo-inositol. All explants were cultured on ½MS hormone free or ½MS medium supplemented with graded NaCl concentrations . The medium was adjusted to pH 5.8 with NaOH/HCl and autoclaved at 121 °C for 25 min. All in vitro cultured plants were grown at of 25 ± 2 °C and a 16 h/8 h photoperiod . All methods were performed in accordance with the relevant guidelines and regulations.All experiments were performed with plant material originating from AtCKX transgenic centaury lines. The plant explants were extracted, purified and quantified according to Dobrev and Kamínek82 and Dobrev et al.83. Briefly, samples (≈100 mg FW) were homogenised with 1.3 mm silica beads using a FastPrep-24 instrument  with extraction buffer methanol/H2O/formic acid  supplemented with stable isotope-labeled internal standards, each at 10 pmol per sample. The extracts were subjected to solid phase extraction (SPE) using Oasis-MCX cartridges , with two resulting fractions: (1) fraction A (eluted with methanol) containing hormones of acidic and neutral character ; and (2) fraction B (eluted with 0.35 M NH4OH in 70% methanol) containing hormones of basic character (CKs). The SPE eluates were evaporated to dryness and pellets reconstituted in 30 μl of 5% methanol in water. An aliquot (10 µL) of the extract was injected on an Ultimate 3000 high-performance liquid chromatograph  coupled to a 3200 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer  set in selected reaction monitoring mode. Quantitative analysis was done using isotope dilution method with multilevel calibration curves (r2 > 0.99). Data processing was carried out with Analyst 1.5 software (Applied Biosystems). The concentration of all analysed phytohormones are presented as pmol/g of sample fresh weight.Endogenous CKs, auxins, ABA, JA and SA were analysed in extracts of 8-week-old in vitro-grown shoot and root explants of non-transformed and AtCKX shoots and roots were evaluated using standard two-factor analysis of variance (ANOVA). Determination of endogenous phytohormones in three biological samples (for shoots and roots) per non-transformed and each transgenic line was repeated twice. Results are presented as mean ± SE. The comparisons between the mean values were made using a Fisher LSD (the least significant difference) post-hoc test calculated at a confidence level of p ≤ 0.05.The effect of graded NaCl concentrations (0–200 mM) on endogenous phytohormones content of 8-week-old non-transformed and transgenic