Front Cover: Highly hydrophilic cationic gold nanorods stabilized by novel quaternary ammonium surfactant with negligible cytotoxicity (J. Biophotonics 12/2019)

Cationic gold nanorods stabilized by quaternary ammonium salts (QAS) are a promising tool for photothermal destruction of cancer cells. However, cytotoxicity of the alkanethiol‐QAS limits their medical applications. A novel design of cationic surfactant composed of the quaternary ammonium group and ethylene glycol chain significantly reduces the compound cytotoxicity in the free state while allowing the preparation of stable nanorods with high cellular uptake and lysosomal localization. Further details can be found in the article by Sarka Salajkova, Michal Sramek, David Malinak, et al. ( e201900024 ).

     

NITROGEN UPTAKE AND ASSIMILATION KINETICS IN ALEXANDRIUM MINUTUM (DYNOPHYCEAE): EFFECT OF N‐LIMITED GROWTH RATE ON NITRATE AND AMMONIUM INTERACTIONS1

Uptake and assimilation kinetics of nitrate and ammonium were investigated along with inhibition of nitrate uptake by ammonium in the harmful dinoflagellate Alexandrium minutum Halim at different nitrogen (N)–limited growth rates. Alexandrium minutum had a strong affinity for nitrate and ammonium (K_s=0.26±0.03 and 0.31±0.04 μmol·L^?1, respectively) whatever the degree of N deficiency of the cells. Ammonium was always the preferred form of nitrogen taken up (=0.42–0.50). In the presence of both forms, nitrate uptake was inhibited by ammonium, and inhibition was particularly marked in N‐sufficient cells (I_max～0.9 and K_i=0.31–0.56 μmol·L^?1). In the case of N assimilation, ammonium was also the preferred form in N‐deficient cells (=0.54–0.72), whereas in N‐sufficient cells, both N sources were equally preferred (=0.90–1.00). The comparison of uptake and assimilation rates highlighted the ability of A. minutum to significantly store in 1 h nitrate and ammonium in amounts sufficient to supply twice the daily N requirements of the slowest‐growing N‐deficient cells. Nitrogen uptake kinetic parameters of A. minutum and their ecological implications are discussed.     

Combinatorial materials research applied to the development of new surface coatings IX: An investigation of novel antifouling/fouling-release coatings containing quaternary ammonium salt groups

Polysiloxane coatings containing chemically-bound (“tethered”) quaternary ammonium salt (QAS) moieties were investigated for potential application as environmental-friendly coatings to control marine biofouling. A combinatorial/high-throughput approach was applied to the investigation to enable multiple variables to be probed simultaneously and efficiently. The variables investigated for the moisture-curable coatings included QAS composition, ie alkyl chain length, and concentration as well as silanol-terminated polysiloxane molecular weight. A total of 75 compositionally unique coatings were prepared and characterized using surface characterization techniques and biological assays. Biological assays were based on two different marine microorganisms, a bacterium, Cellulophaga lytica and a diatom, Navicula incerta, as well as a macrofouling alga, Ulva. The results of the study showed that all three variables influenced coating surface properties as well as antifouling (AF) and fouling-release (FR) characteristics. The incorporation of QAS moieties into a polysiloxane matrix generally resulted in an increase in coating surface hydrophobicity. Characterization of coating surface morphology revealed a heterogeneous, two-phase morphology for many of the coatings investigated. A correlation was found between water contact angle and coating surface roughness, with the contact angle increasing with increasing surface roughness. Coatings based on the QAS moiety containing the longest alkyl chain (18 carbons) displayed the highest micro-roughness and, thus, the most hydrophobic surfaces. With regard to AF and FR properties, coatings based on the 18 carbon QAS moieties were very effective at inhibiting C. lytica biofilm formation and enabling easy removal of Ulva sporelings (young plants) while coatings based on the 14 carbon QAS moities were very effective at inhibiting biofilm growth of N. incerta.     

Nitrogen‐Doped Graphene Nanoribbons with Surface Enriched Active Sites and Enhanced Performance for Dye‐Sensitized Solar Cells

Superior electrocatalytic activities and excellent electrochemical stabilities of inexpensive counter electrodes (CEs) are crucial to the large‐scale practical application of dye‐sensitized solar cells (DSSCs). Herein, an efficient strategy for fabricating nitrogen‐doped graphene nanoribbons (N‐GNRs) via chemical unzipping of carbon nanotubes coupled with nitrogen doping process is reported, where abundant edge sites are produced and fully exposed basal planes of GNRs are activated by the N atoms within GNRs backbone. Benefiting from such unique characteristics, when first applied as CEs for DSSCs with triiodide/iodide electrolyte, a power conversion efficiency of 8.57% is delivered, outperforming GNRs (8.01%) and being superb to that of Pt (7.84%), and outstanding electrochemical stabilities of N‐GNRs are also demonstrated. Density functional theory calculations reveal that the N species within GNRs matrix, especially the predominant quaternary ones, could remarkably decrease the ionization energy of GNRs, which is instrumental to transfer electrons rapidly from external circuit to triiodide, and reduce charge‐transfer resistance, thus contributing to the enhanced photovoltaic performance. The present work has an insight into the unique role of N species on GNRs to the triiodide reduction, and provides an efficient strategy for design of high‐efficiency carbon electrodes with fully exposed active sites in energy conversion/storage devices.     

N‐ASSIMILATION IN THE NOXIOUS FLAGELLATE HETEROSIGMA CARTERAE (RAPHIDOPHYCEAE): DEPENDENCE ON LIGHT,N‐SOURCE,AND PHYSIOLOGICAL STATE1

The capabilities of the diel vertically migrating flagellate Heterosigma carterae Hulburt for assimilating ammonium and nitrate into cell‐N in light and in darkness were studied using cells of different N‐status. Ammonium utilization in darkness, except by N‐replete cells, attained>50% of use in the light. However, the capacity to use nitrate was poor in darkness, and less than 20% of nitrate‐N that was taken up in darkness was then actually incorporated into cell‐N. The ability to assimilate N in darkness improved as N‐status (N:C) declined, concurrent with an increasing content of water‐soluble carbohydrate. This carbohydrate was used to support dark N‐assimilation. Cells held in darkness for over a day and that had halted nitrate‐uptake were still capable of taking up ammonium. Furthermore, the act of taking up ammonium appeared to make available a source of C to support nitrate uptake that was previously unavailable. The implications of these results for the ecophysiology of this organism and for the construction of mathematical models of algal growth are considered.     

Dysregulation of inter‐photoreceptor retinoid‐binding protein (IRBP) after induced Müller cell disruption

Reduced expression of a ~150 kDa protein was unexpectedly observed while investigating Norrin protein in a transgenic murine model in which Müller cells can be selectively and inducibly disrupted. Isolation of this unknown protein via ion exchange and hydrophobic interaction chromatography followed by Tandem mass spectrometry identified it as Inter‐photoreceptor retinoid‐binding protein (IRBP). Significantly reduced IRBP mRNA expression was observed at the early and late stages after Müller cell disruption. IRBP protein expression was also consistently reduced to 5.7% of the control level as early as 1 week after Müller cell disruption. This down‐regulation of IRBP was accompanied by focal hyperfluorescent dots and cytotoxic N‐retinylidene‐N‐retinylethanolamine (A2E) accumulation. In vitro treatment of cone photoreceptor cell lines with conditioned medium collected from stressed Müller cells suggested that Müller cells regulated photoreceptors expression of IRBP via secreted factor(s). In vivo studies suggested that one of these secreted factors was tumour necrosis factor alpha (TNFα). These findings suggest that dysregulation of IRBP expression caused by Müller cell dysfunction may be an important early event in photoreceptor degeneration in some retinal diseases.

     

Ectonucleotide pyrophosphatase/phosphodiesterase activity in Neuro‐2a neuroblastoma cells: changes in expression associated with neuronal differentiation

Neuro‐2a (N2a) neuroblastoma cells display an ectoenzymatic hydrolytic activity capable of degrading diadenosine polyphosphates. The Ap_nA‐cleaving activity has been analysed with the use of the fluorogenic compound BODIPY^® FL guanosine 5′‐O‐(3‐thiotriphosphate) thioester. Hydrolysis of this dinucleotide analogue showed a hyperbolic kinetic with a K_m value of 4.9 ± 1.3 μM. Diadenosine pentaphosphate, diadenosine tetraphosphate, diadenosine triphosphate, and the nucleoside monophosphate AMP behaved as an inhibitor of BODIPY^® FL guanosine 5′‐O‐(3‐thiotriphosphate) thioester extracellular degradation. Ectoenzymatic activity shared the typical characteristics of the ectonucleotide pyrophosphatase/phosphodiesterase family, as hydrolysis reached maximal activity at alkaline pH and was dependent on the presence of divalent cations, being strongly inhibited by EDTA and activated by Zn²⁺ ions. Both NPP1 and NPP3 isozymes are expressed in N2a cells, their expression levels substantially changing when cells differentiate into a neuronal‐like phenotype. In this sense, it is relevant to point the expression pattern of the NPP3 protein, whose levels were drastically reduced in the differentiated cells, being almost completely absent after 24 h of differentiation. Enzymatic activity assays carried out with differentiated N2a cells showed that NPP1 is the main isozyme involved in the extracellular degradation of dinucleotides in these cells, this enzyme reducing its activity and changing its subcellular location following neuronal differentiation.

     

The physiological and morphological phenotype of a yeast mutant resistant to the quaternary ammonium salt N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride

We investigated the action of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride) on Saccharomyces cerevisiae yeast cells. Changes in the yeast cell ultrastructure were confirmed by electron microscopy. We treated resistant mutant cells with QAS, and confirmed destruction of the mutant cytoplasm, an increase in the thickness of the cell wall, separation of the cell wall from the cytoplasm, and the accumulation of numerous lipid droplets. We also observed a relatively high production of lipids in the cells of the parental wild-type strain Σ1278b and in its IM-resistant (IM^R) mutant in the presence of the QAS. The IM^R mutant showed increased sensitivity to CaCl₂ and SDS, and resistance to ethidium bromide, chloramphenicol, erythromycin and osmotic shock. It also tolerated growth at low pH. We suggest that the resistance to IM could be connected with the level of permeability of the cell membrane because the IM^R mutant was sensitive to this compound in vivo in the presence of SDS and guanidine hydrochloride, which cause increased permeability of the cell plasma membrane.     

Utilization of nitrate,nitrite and ammonium by Chlamydomonas reinhardii

In phototrophically grown Chlamydomonas cells, ammonium strongly inhibited the utilization of nitrate or nitrite. Under darkness, or in the presence of an uncoupler or inhibitor of the non-cyclic photosynthetic electron flow, the utilization of nitrate, nitrite or ammonium was suppressed. l-Methionine-d,l-sulfoximine (MSX) or azaserine, which blocks the assimilation of ammonium, inhibited the consumption of nitrate, but not nitrite, by the cells. Ammonium produced an immediate inhibition of the permease for nitrate in Chlamydomonas growing with nitrate, while ammonium-grown cells lacked this permease. The synthesis of nitrate-reductase activity was dependent on an active permease. In N-starved Chlamydomonas cells, previously treated with MSX, the permease for nitrate was insensitive to inhibition by ammonium, and a significant amount of nitrate reductase was synthetized. These cells photoproduce ammonium by reducing nitrate. Nitrogen-repleted cells, treated with MSX, actively photoproduced ammonium by reducing nitrite, but not nitrate.Abbreviations DCMU N-(3,4-dichlorophenyl)N,N-di-methyl-urea - PCCP Carbonylcyanid-p-trifluoromethoxy-phenylhydrazone - Mops 2-(N-morpholino)propanesulfonic acid - MSX l-Methionine-d,l-sulfoximine     

Synthesis,spectral properties and antimicrobial activity of a new cationic water‐soluble pH‐dependent poly(propylene imine) dendrimer modified with 1,8‐naphthalimides

A three‐step synthesis was implemented to prepare a quaternary ammonium functionalized blue fluorescent poly(propylene imine) dendrimer modified with pyridinium salt of 4‐acylamino‐1,8‐naphthalimide. The new cationic dendrimer absorbs in the ultraviolet light region and emits blue fluorescence. Its spectral characteristics in organic solvents and in an aqueous solution were studied. The influence of pH on the fluorescence intensity of the dendrimer was established with regard to its use as a pH sensor. The effect of hydroxyl ions on the absorption and fluorescence spectra in dry N,N‐dimethylformamide was also investigated. The antimicrobial activity of the dendrimer was assessed against model pathogenic microorganisms in agar, liquid medium, and after its deposition on cotton fabric.     

Synthesis,physiochemical property and antimicrobial activity of novel quaternary ammonium salts

Twenty-four novel 5-phenyl-1,3,4-oxadiazole-2-thiol (POT) analogues, benzo[d]oxazole-2-thiol, benzo[d]thiazole-2-thiol and 5-methyl-1,3,4-thiadiazole-2-thiol-substituted N,N-bis(2-hydroxyethyl) quaternary ammonium salts (QAS) (5a-d, 6a-d, 7a-d, 10a-d, 13a-d, 16a-d) were prepared and characterised by FTIR, NMR and elemental analysis. Part of target compounds (5d, 6d, 7d, 10d, 13d, 16d) displayed potent antimicrobial effect against ten common pathogens (S. aureus, α-H-tococcus, β-H-tococcus, E. coli, P. aeruginosa, Proteus vulgaris, Canidia Albicans, Cytospora mandshurica, Physalospora piricola, Aspergillus niger) and had relatively low cytotoxity against two human cell lines (HaCat and LO2). TEM and SEM images of E. coli and S. aureus morphologies treated with 7d showed that the antibacterial mechanism might be the QAS fixing on cell wall surfaces and puncturing to result in the release of bacterial cytoplasm. This study provides new information of QAS, which could be used to design novel antimicrobial agents applied in clinic or agriculture.     

Soil moisture variations affect short‐term plant‐microbial competition for ammonium,glycine, and glutamate

We tested whether the presence of plant roots would impair the uptake of ammonium (), glycine, and glutamate by microorganisms in a deciduous forest soil exposed to constant or variable moisture in a short‐term (24‐h) experiment. The uptake of ¹⁵NH₄ and dual labeled amino acids by the grass Festuca gigantea L. and soil microorganisms was determined in planted and unplanted soils maintained at 60% WHC (water holding capacity) or subject to drying and rewetting. The experiment used a design by which competition was tested in soils that were primed by plant roots to the same extent in the planted and unplanted treatments. Festuca gigantea had no effect on microbial N uptake in the constant moist soil, but its presence doubled the microbial uptake in the dried and rewetted soil compared with the constant moist. The drying and rewetting reduced by half or more the uptake by F. gigantea, despite more than 60% increase in the soil concentration of . At the same time, the amino acid and ‐ N became equally valued in the plant uptake, suggesting that plants used amino acids to compensate for the lower acquisition. Our results demonstrate the flexibility in plant‐microbial use of different N sources in response to soil moisture fluctuations and emphasize the importance of including transient soil conditions in experiments on resource competition between plants and soil microorganisms. Competition between plants and microorganisms for N is demonstrated by a combination of removal of one of the potential competitors, the plant, and subsequent observations of the uptake of N in the organisms in soils that differ only in the physical presence and absence of the plant during a short assay. Those conditions are necessary to unequivocally test for competition.     

Targeting lung cancer stem‐like cells with TRAIL gene armed oncolytic adenovirus

Lung cancer stem cell (LCSC) is critical in cancer initiation, progression, drug resistance and relapse. Disadvantages showed in conventional lung cancer therapy probably because of its existence. In this study, lung cancer cell line A549 cells propagated as spheroid bodies (named as A549 sphere cells) in growth factors‐defined serum‐free medium. A549 sphere cells displayed CSC properties, including chemo‐resistance, increased proportion of G0/G1 cells, slower proliferation rate, ability of differentiation and enhanced tumour formation ability in vivo. Oncolytic adenovirus ZD55 carrying EGFP gene, ZD55‐EGFP, infected A549 sphere cells and inhibited cell growth. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) armed oncolytic adenovirus, ZD55‐TRAIL, exhibited enhanced cytotoxicity and induced A549 sphere cells apoptosis through mitochondrial pathway. Moreover, small molecules embelin, LY294002 and resveratrol improved the cytotoxicity of ZD55‐TRAIL. In the A549 sphere cells xenograft models, ZD55‐TRAIL significantly inhibited tumour growth and improved survival status of mice. These results suggested that gene armed oncolytic adenovirus is a potential approach for lung cancer therapy through targeting LCSCs.     

Derivatization of peptides as quaternary ammonium salts for sensitive detection by ESI‐MS

A series of model peptides in the form of quaternary ammonium salts at the N‐terminus was efficiently prepared by the solid‐phase synthesis. Tandem mass spectrometric analysis of the peptide quaternary ammonium derivatives was shown to provide sequence confirmation and enhanced detection. We designed the 2‐(1,4‐diazabicyclo[2.2.2] octylammonium)acetyl quaternary ammonium group which does not suffer from neutral losses during MS/MS experiments. The presented quaternization of 1,4‐diazabicyclo[2.2.2]octane (DABCO) by iodoacetylated peptides is relatively easy and compatible with standard solid‐phase peptide synthesis. This methodology offers a novel sensitive approach to analyze peptides and other compounds. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

The fate of assimilated nitrogen in streams: an in situ benthic chamber study

1. Nitrogen (N) processing in streams has been investigated using whole‐stream ¹⁵N addition experiments that, in general, have found that a large proportion of added nitrate removed from the water column appears to be assimilated by the stream benthos. The long‐term fate of this retained N is unknown, and of particular interest is the possibility that it becomes denitrified through coupled mineralisation–nitrification–denitrification processes (indirect denitrification). 2. We used in situ chambers to produce highly ¹⁵N‐enriched benthic biofilms and removed the chambers to allow biofilms to interact with ambient stream conditions. Nitrogen assimilation and direct denitrification were estimated from the first chamber deployment. Chambers were periodically reinstalled over 4 weeks to measure tracer ¹⁵N in ammonium (), nitrate () and dinitrogen (N₂), from which we estimated subsequent rates of biotic N transformations, including N mineralisation (ammonification), nitrification and indirect denitrification. We also estimated rates of depuration of ¹⁵N tracer from benthic biomass compartments. 3. Nitrate uptake was roughly equivalent in the sand and cobble habitats that dominated the stream. Direct denitrification (denitrification of from the water column) was an order of magnitude higher in cobble habitats than in sand habitats, accounting for c. 26 and 2% of total nitrate uptake in cobble and sand, respectively. 4. Mean residence times of actively cycling organic N in stream benthos (algae and microbes) were 16 days in cobble habitats and 9 days in sand habitats. The difference between habitat types was driven by the influence of N residence time in epilithic biofilms (18 days) on cobbles. 5. Release of enriched ¹⁵ was the primary flux of remineralised N, while release of enriched ¹⁵ was an order of magnitude less. We detected slight ¹⁵N enrichment in dissolved nitrogen gas (N₂) in post‐enrichment sampling, indicating that indirect denitrification was taking place. However, indirect denitrification accounted for <0.1% of the assimilated N. 6. These experiments agree with results of whole‐stream ¹⁵N additions, in that most added N was assimilated rather than directly denitrified. Assimilation was primarily a short‐term N retention mechanism in this stream, and indirect denitrification of assimilated N accounted for only a minor proportion of the observed ¹⁵N loss over time. 7. Remaining possible fates include export of N as particulate organic matter, which may lead to additional storage of assimilated N in downstream habitats, and consumption by grazers.     

稳定性铵态氮肥在黑土和褐土中的氮素转化特征

以稳定性氯化铵为氮源,采用室内培养的方法,研究0.20、0.50、1.00 g N·kg^-1干土3种浓度的稳定性铵态氮肥在黑土、褐土中的氮素转化特征.结果表明: 在褐土中,随着氯化铵添加量的增加,土壤中发生硝化作用的时间逐渐推迟,添加0.20、0.50 g N·kg^-1干土处理开始发生明显硝化反应的时间分别为第3、7天,在高浓度氮量(1.00 g N·kg^-1干土)添加下硝化作用受到明显抑制;在黑土中,各浓度氮量添加处理开始发生硝化反应的时间相同,均为第3天,且随着添加量的增加,硝化作用潜势逐渐减弱.只加铵态氮肥的处理中,添加0.20 g N·kg^-1干土的氯化铵氮肥在褐土和黑土中的硝化反应时间分别可维持3周和2周左右;添加0.50 g N·kg^-1干土的氯化铵氮肥在褐土和黑土中的硝化反应时间分别可维持4周和3周左右.与单施氯化铵相比,黑土和褐土在0.20、0.50 g N·kg^-1干土添加浓度下,按纯氮量的1.0%添加3,4-二甲基吡唑磷酸盐(DMPP)、4.0%添加二氰二胺(DCD)均能显著抑制硝化作用,降低硝态氮的含量,抑制硝化作用潜势.综上,在褐土中,随着氯化铵添加浓度增加,土壤硝化作用受到抑制效果大于黑土.在0.20、0.50 g N·kg^-1干土外源铵态氮时,添加抑制剂可以显著抑制铵态氮的硝化作用.因此室内硝化抑制剂培养试验时,建议铵态氮添加量不超过1.00 g N·kg^-1干土,以0.50 g N·kg^-1干土效果最好.     

Synthesis of Lanostane‐Type Triterpenoid N‐Glycosides and Their Cytotoxicity against Human Cancer Cell Lines

Seventeen lanostane‐type triterpenoid derivatives ( 2 – 18 ), including 11N‐glycosides ( 8 – 18 ), were synthesized from the natural triterpenoid, lanosterol ( 1 ), and were evaluated for their cytotoxicity against the human cancer cell lines, HL‐60, A549, and MKN45, as well as the normal human lung cells, WI‐38. Among them, N‐β‐d ‐2‐acetamido‐2‐deoxyglucoside ( 10 ) showed cytotoxicity against HL‐60, A549, MKN45, and WI‐38 cells (IC₅₀ 0.0078 – 2.8 μm ). However, N‐β‐d ‐galactoside ( 12 ) showed cytotoxicity against HL‐60 and MKN45 cells (IC₅₀ 0.0021 – 4.0 μm ), but not the normal WI‐38 cells. Furthermore, Western blot analysis suggested that 12 induces apoptosis by activation of caspases‐3, 8, and 9. These results will be useful for the synthesis of other tetracyclic triterpenoids or steroid N‐glycosides to increase their cytotoxicity and apoptosis‐inducing activities.     

Heat shock protein 70 induction by valproic acid delays photoreceptor cell death by N‐methyl‐N‐nitrosourea in mice

Retinal degenerative diseases (RDs) are a group of inherited diseases characterized by the loss of photoreceptor cells. Selective photoreceptor loss can be induced in mice by an intraperitoneal injection of N‐methyl‐N‐nitrosourea (MNU) and, because of its selectivity, this model is widely used to study the mechanism of RDs. Although it is known that calcium‐calpain activation and lipid peroxidation are involved in the initiation of cell death, the precise mechanisms of this process remain unknown. Heat shock protein 70 (HSP70) has been shown to function as a chaperone molecule to protect cells against environmental and physiological stresses. In this study, we investigated the role of HSP70 on photoreceptor cell death in mice. HSP70 induction by valproic acid, a histone deacetylase inhibitor, attenuated the photoreceptor cell death by MNU through inhibition of apoptotic caspase signals. Furthermore, HSP70 itself was rapidly and calpain‐dependently cleaved after MNU treatment. Therefore, HSP70 induction by valproic acid was dually effective against MNU‐induced photoreceptor cell loss as a result of its anti‐apoptotic actions and its ability to prevent HSP70 degradation. These findings might help lead us to a better understanding of the pathogenic mechanism of RDs.

     

Photothermal activation of astrocyte cells using localized surface plasmon resonance of gold nanorods

Although it has been revealed that astrocytes, generally known as star‐shaped glial cells, play critical roles in the functions of central nervous system, there have been few efforts to directly modulate their activities and responses. In this study, an optical stimulation strategy for producing intracellular Ca²⁺ transients of astrocytes is demonstrated using near‐infrared (NIR) light and localized surface plasmon resonance. It is presented that NIR stimulation of micro‐second duration combined with gold nanorods (GNRs) efficiently produces stronger Ca²⁺ transients of astrocytes, which seems to be associated with a local heat generation by photothermal effects of GNRs. Since the proposed scheme can directly activate astrocytes with a high reliability, it is expected that GNR‐mediated NIR stimulation could be utilized to facilitate minimally invasive physiological studies on the astrocyte functions.

Photos of intracellular Ca²⁺ transient of astrocytes with membrane‐bound GNRs after optical stimulation at 30 s.