Under our conditions, borylation was observed only at the sterically least encumbered para‐positions of the acceptor group. As boronate esters are versatile building blocks for organic synthesis  the C−H borylation represents a simple potential method for post‐functionalization by which electronic or other properties of d‐π‐A systems can be fine‐tuned for specific applications. The photophysical and electrochemical properties of the borylated (21‐(Bpin)) and unborylated (1) diphenylamino‐substituted d‐π‐A systems were investigated. Interestingly, the borylated derivative exhibits coordination of THF to the boronate ester moieties, influencing the photophysical properties and exemplifying the non‐innocence of boronate esters.We observed a surprisingly high electronically driven regioselectivity for the iridium‐catalyzed C−H borylation of donor‐π‐acceptor phenyl)boryl (FXyl)2B as the acceptor group. The borylated system exhibits an unexpected hypsochromic shift of the emission from toluene to THF, likely due to coordination to the boronate ester moieties (see figure). Based on this, potential applications of three‐coordinate boron have been investigated, such as for linearz‐orbital. This can be avoided by using bulky substituents or by fixing the boron center in a rigid scaffold.ortho‐trifluoromethylated aryls in triarylboranes has been established as a strategy to improve acceptor strength as well as stability.z‐orbital, as well as a direct interaction of the fluorine lone pairs with the empty pz‐orbital on boron. The introduction of boron into organic systems can be efficiently carried out via a metalation/borylation strategy with different metal‐stabilized organic nucleophiles such as organolithium2  as the precatalytic species, bis(pinacolato)diboron (B2pin2) as the boron source and 4,4’‐di‐tert‐butyl‐2,2’‐bipyridine (dtbpy) as the ligand  systems with bisphenyl)boryl (FXyl)2B as the acceptor group.d‐π‐A systems, exclusively. Based on previous results, we decided to use the iridium‐catalyzed C−H borylation and found it to exhibit a surprisingly high electronically driven regioselectivity for our systems.We have previously investigated donor‐π‐acceptor  compound 2 (Scheme i and ii) was lithiated using nBuLi in hexane. Afterwards, bisphenyl)fluoroborane (FXyl)2FB was added in methyl tert‐butyl ether (MTBE). We found that the solvent mixture is important. When Et2O is used for the lithiation, the lithiation is much faster, but the reaction with FXyl)2FB/2‐(Bpin), 21‐(Bpin)/22‐(Bpin) or unreacted 1/2 were detected. This high degree of regioselectivity was surprising to us, as no borylation at all at the sterically unencumbered arylamino moieties was observed. Note that the selectivity is clearly electronic in nature, as the two arenes have similar steric properties, at least at their para‐positions. It should be noted that, in a competitive borylation experiment of a 1:1 mixture of m‐xylene and 1,3‐bis(trifluoromethyl)benzene, 6 % borylation of m‐xylene and 65 % borylation of 1,3‐bis(trifluoromethyl)benzene were observed,Compound 2pin2 is increased to 2.2 equivalents, borylation at the donor moieties can be observed via 1H NMR spectroscopy. Residual, small amounts of mono‐borylated d‐π‐A can be removed by washing with methanol and recrystallization. No column chromatography was needed, which is beneficial, as aryl‐boronates can be very difficult to purify by column chromatography due to their strong interaction with the solid phase. All compounds exhibit 1H, 11B, 19F and 13C NMR spectra in accordance with their structures. The 19F NMR spectra exhibit strong signal broadening, indicative of a hindered rotation about the B−C bonds, as previously observed for similar boranes with ortho‐trifluoromethyl groups.However, when the amount of B1 and 21‐(Bpin) were obtained from a saturated hexane solution or from a saturated CH2Cl2 solution that was layered with hexane, respectively. The molecular structures are depicted in Figure 22‐(Bpin) was also obtained via single‐crystal X‐ray diffraction . Although the quality of the data does not allow a detailed discussion of the structural parameters, it does provide proof of the connectivity, confirming that borylation took place at the electron‐poor arene rings.Crystals suitable for X‐ray diffraction analysis of 1 and 21‐(Bpin) exhibit trigonal planar geometries with the sum of the angles around the centers of ≈360°. Compounds 1 and 21‐(Bpin) exhibit two longer B−C bonds (1.606(3)–1.617(3) Å) towards the terminal FXyl moieties and one shorter B−C bond (1.525(3) and 1.531(3) Å) towards the phenylene bridge. This can be mainly attributed to the steric demand of the FXyl ortho trifluoromethyl groups. Due to the steric hinderance the FXyl moieties also exhibit larger torsion angles (51.45(7)–53.51(9)°) with respect to the BC3 plane than the phenylene bridge (20.55(18) and 23.31(11)°). The C−N bond lengths of 1 and 21‐(Bpin) show a similar behavior and exhibit two longer bonds (1.430(12)–1.440(3) Å) to the terminal phenyl moieties and one shorter bond (1.395(3) and 1.395(4) Å, respectively) to the phenylene bridge. As the steric demand of all three substituents at the N atom is equal, this can be attributed to a polarized ground state, which is well known for d‐π‐A systems.3 plane (41.86(9)–71.3(3)°), compared to those of the phenylene bridge (16.71(8) and 8.2(2)°). The polarized ground state also results in a quinoidal distortion of the phenylene spacer, as the c and f bonds in 1 and 21‐(Bpin)  show B⋅⋅⋅F contacts (2.769(3)–2.876(3) Å), which are shorter than the sum of their van der Waals radii (3.39 Å),3 fluorine atoms and the empty pz‐orbital of the boron center.The triarylboron and ‐nitrogen centers in 1, 21‐(Bpin), and 22‐(Bpin) were recorded in CH2Cl2 with [nBu4N][PF6] as the electrolyte and a scan rate of 250 mVs−1  and listed in Table d‐π‐A systems 2,N,N‐diphenylaniline (I),N,N‐diphenylaniline (II)Cyclic voltammograms of 1  and 21‐(Bpin)  both exhibit reversible reduction and oxidation waves, that can be attributed to the triarylboron and nitrogen centers, respectively. 22‐(Bpin)  exhibits a reversible reduction corresponding to the triarylboron center and an irreversible oxidation characteristic of the carbazolyl moiety, analogous to what was previously observed for 2 .1 is 150 mV cathodically shifted compared to 2, indicating that diphenylamine is a stronger donor than carbazolyl.para to the boron on the FXyl moieties shifts the reduction potentials anodically by 140 and 100 mV, respectively for 1 and 2. This indicates the electron‐withdrawing nature of the boronate ester moieties. This is in line with our previously published comprehensive comparison of different boron based acceptor groups.1 is anodically shifted by 490 mV compared to its mesityl analogue (I), and cathodically shifted by 450 mV compared to its fluoromesityl analogue (II).para to the boron center as compared to the ortho trifluoromethyl groups.Compounds 1   were also examined in hexane  level of theory suggest that the S1←S0 transition can be attributed to a HOMO to LUMO transition. The HOMO is localized on the donor and π‐bridge, and the LUMO is mainly localized on the acceptor. As such, the transition can be classified as a charge transfer (CT) transition. Similar observations were made for our previously reported d‐π‐A systems with B(FXyl)2, B(FMes)2 or BMes2 as the acceptor.1 shifts bathochromically with increasing solvent polarity . This is expected for dipolar d‐π‐A systems, due to the stabilization of the excited state in solvents of higher polarity. In comparison to our previously investigated analogues of 1 with BMes2 (I) and B(FMes)2 (II), both absorption and emission are bathochromically shifted compared to I  and hypsochromically shifted compared to II . This supports the results from the cyclovoltammetry measurements, in that B(FXyl)2 is right in the middle between BMes2 and B(FMes)2 in terms of acceptor strength. The lowest energy absorption maxima of 21‐(Bpin) are slightly red shifted compared to 1 . The emission maxima of 21‐(Bpin) in hexane  and toluene  are also red‐shifted compared to 1 and also exhibit a bathochromic shift with increasing solvent polarity. In THF, however, the emission maximum  of 21‐(Bpin) is blue shifted compared to those of 1 and 21‐(Bpin) in toluene. This is unexpected, as due to the higher polarity of THF a further bathochromic shift is to be expected. We suspect, that this is due to coordination of THF to the boron centers of the boronate ester moieties, as a result of which the Bpin moieties change from being weak acceptors to weak donors, decreasing the acceptor strength of the B(FXyl)2 moiety, resulting in a hypsochromic shift. This is further supported by the strongly bathochromically shifted and very weak emission of 21‐Bpin in CH2Cl2, a non‐coordinating polar solvent. Hence, the lowest energy transitions of 21‐Bpin can also be classified as CT transitions. Compared to 21‐(Bpin), 22‐(Bpin) exhibits blue‐shifted lowest energy absorption  and emission  maxima in hexane. This can be attributed to carbazolyl being a weaker donor than diphenylamine, resulting in a weaker charge transfer (CT).λmax, abs=400, 398, and 387 nm) and emission  maxima of 21 in hexane, toluene, and THF, respectively. The quantum yield of 1 in hexane is close to unity, which is similar to that previously observed for 2. Similar to 2, the quantum yield of 1 in toluene is still high, but unlike 2, the quantum yield of 1 drops off drastically in THF. This could be due to the energy gap law, as internal conversion processes become more effective, as the energy gap between the excited and ground state becomes smaller, which we previously observed for similar systems.21‐(Bpin) and 22‐(Bpin) in hexane are lower than those of their respective unborylated parent compounds 1 and 2. The same was observed for 21‐(Bpin) in toluene. Interestingly, the quantum yield of 21‐(Bpin) is higher in THF than that of 1. This is likely a result of the blue‐shifted absorption and emission maxima of 21‐(Bpin) as compared with 1.Compound d‐π‐A systems, we found a surprisingly high electronically driven regioselectivity for an iridium‐catalyzed C−H borylation using [Ir(COD)OMe]2 as the precatalytic species, B2pin2 as the boron source and dtbpy as the ligand in hexane.d‐π‐A systems in order to fine tune the electronic properties as suggested by the literature.FXyl)2 is a stronger acceptor than BMes2 and a weaker acceptor than B(FMes)2, and provides the possibility of further functionalization due to the unsubstituted para position. This is also supported by the photophysical data of 1, 21‐(Bpin), and 22‐(Bpin). The borylated derivatives are slightly red shifted compared to their parent compounds, indicating an electron‐withdrawing effect of the Bpin moieties. The compounds show CT transitions with bathochromic shifts of the emission with increasing solvent polarity. A surprising exception is 21‐(Bpin), which exhibits a hypsochromic shift of the emission when increasing the solvent polarity from toluene to THF. This is attributed to a coordination of THF to the boron centers of the boronate ester groups and a subsequent weakening of its acceptor strength.While looking for a methodology to post‐functionalize The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Exogenously supplied SNP (10 µM) and H2O2 (1 µM) lessen the toxicity of Cd (6 µM) but without NO; H2O2 was unable to release the stress from cyanobacterial cells potentially. The reduced contents of exopolysaccharide, protein content, endogenous NO and enzymatic antioxidants  due to Cd toxicity, were found increased significantly after exogenous application of H2O2 and SNP thereafter, cyanobacterial calls flourished much better after releasing toxic level of Cd. Moreover, increased level of ROS due to Cd stress also normalized under exogenous application of H2O2 and SNP. However, chelation of NO hindered the signaling mechanism of H2O2 that diminished its potential against Cd stress while signaling of NO has not been hindered by chelation of H2O2 and NO potentially released the Cd stress from cyanobacterial cells. In conclusion, current findings demonstrated the synergistic signaling between H2O2 and NO towards the improvement of cyanobacterial tolerance to Cd stress, thereby enhancing the growth and antioxidant defense system of test cyanobacteria that improved fertility and productivity of soil even under the situation of metal contamination.In the present study, defensive strategies of H Cd ions in agricultural soil can also negatively affect the beneficial microbes present in the rice field4. Cyanobacteria are pioneer phototrophic prokaryotic microorganisms that are known to display their potential role in sustainable agricultural development especially for paddy fields. Nostoc muscorum and Anabaena sp. are categorized as heterocystous cyanobacteria and known for their nitrogen-fixing ability. They are mainly present on soil and water bodies and fix about 20–25 kg/ha atmospheric nitrogen. More efficiently, Anabaena sp. can fix about 60 kg/ha of nitrogen in a season5. Unfortunately, Cd ions present in rice soil are major threats to these nitrogen fixers. Previous studies demonstrated that some photosynthetic bacteria can tolerate up to 1 mg Cd kg−1 soil in the association with Hay bacillus (Bacillus subtilis) and some other lactic acid bacteria6. But the release of Cd is much higher (200 mg Cd kg−1 soil) in the soils near the leather and electro-plating factories7 that creates a hazardous environment for paddy field cyanobacteria which ultimately results to the poor quality of rice crop. Cd interrupts the cell functioning and hangovers the regular metabolic processes of cyanobacteria. Thus, Cd ions can enter into the cell through P-type Cd2+ ATPases (e.g. cadA1 and cadA2) and interrupt electron transport chain and produce excessive ROS  excess level of Cd2+ enhances the expression of miR398 that ultimately inhibits the functioning of Cu/Zn/SOD and finally, a drastic rise of ROS; (2) excess Cd2+ in cells can inhibit the regulatory role of second messengers like Ca2+, or other signaling molecules like NO and H2O2; (3) excess accumulation of Cd2+ enhances the activity of NADPH oxidase, that is a major producer of ROS. So to enhance the tolerance against different environmental stresses the external applications of different signaling molecules like H2O2, NO, and H2S, are considered as one of the most beneficial methods10.Cadmium (Cd) contamination in agricultural fields like rice crop fields has become a serious environmental issue. Potentially active Cd ions in the soil can accumulate in rice plants and ultimately reduce the uptake and translocation of vital minerals and nutrients for plants2O2 are the potentially active and key biological signaling molecules known for the plant defense. At a low and very specific concentration, H2O2 and NO work as signal messengers for cell communication during stress situations. Tiwari et al.11 reported that exogenous supplementation of SNP (a donor of NO) reduced the level of ROS which is produced by aluminium stress in Anabaena PCC 7120. Verma et al.12 reported that in stress situation the increased ROS  level promotes the production of reactive nitrogen species  that finally control the balancing of antioxidants to protect the plants from severe damage. Several recent findings show the existence of several signaling topologies between NO and H2O213. It has been reported that H2O2 mediates some other signaling molecules like NO or H2S to regulate oxidative stress14. Hasanuzzaman et al.15 suggested that Cd toxicity has been removed expressively by pretreatment of H2O2 in the seedlings of Brassica nupus. Pretreatment of H2O2 possibly empowers the internal defense system of the organism, but its downstream signal transduction mechanisms and pathways are not effusively clear in cyanobacteria. The defense strategies in organisms are a much complex process in which different signaling molecules are linked together as reported in the study of Li et al.16; it is proven that there is a multifaceted interaction among NO, H2S, and H2O2 promoting thermotolerance in maize seedlings. Christou et al.17 also noticed a faster and stronger response against salt stress by the priming of H2O2 and SNP (donor of NO) in strawberry plants. Gonzalez et al.18 have also revealed the cross linking mechanism of calcium, H2O2, and NO in copper treated Ulva compressa. However, in the case of cyanobacteria to the best of our knowledge the interacting role of signaling molecules H2O2 and NO in Cd stress alleviation is lacking. Therefore, we have hypothesized that synergistically H2O2 and NO could initiate certain protective mechanisms that may be associated with metal stress particularly Cd tolerance in cyanobacteria.NO and H2O2 and NO as defense signaling molecules to cadmium stress tolerance in cyanobacteria Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120. Also a key objective was to explore the probable mechanism and connective pathway between H2O2 and NO.Hence, the prime focus of this study was to evaluate the potential role of HNostoc muscorum ATCC 27893 and 30% in Anabaena sp. PCC 7120 (hereafter referred as Nostoc muscorum and Anabaena sp.) in comparison to control. However, on exogenous supplementation of H2O2, growth was recovered and inhibition was noticed only 9 and 11% respectively for Nostoc muscorum and Anabaena sp. Similar to this, exogenous SNP significantly reduced (P < 0.05) the inhibition exerted by Cd, and found only 11 and 7%, respectively as compared to control. Further, to know the regulatory role of endogenous ROS and NO on growth, Cd treated cells were subjected to NAC, DPI, PTIO and LNAME; therefore, a greater reduction in dry weight was noticed under PTIO and LNAME treatment in comparison to control  and inhibitors  enzymes) with Cd stress. In this case, our results showed a critical decline in growth under treatments of PTIO or LNAME i.e. 41 and 44% respectively in Nostoc muscorum and 44 and 46% respectively in Anabaena sp. even in the presence of H2O2. Contrastingly, growth was found improved moderately under SNP treatment even in the presence of NAC and DPI  by 23 and 25% respectively under Cd treatment in contrast to control values  by 20 and 24% respectively in Nostoc muscorum and 17 and 19% respectively in Anabaena sp. comparative to control values. Contrastingly, under the same stress, the decline in EPS secretion was found more critical under the treatments of PTIO or LNAME i.e. 28 and 43% respectively in Nostoc muscorum and 33 and 51% respectively in Anabaena sp. even in the presence of H2O2. Whereas, treatments of NAC or DPI along with SNP do not hinder the signaling mechanism of NO and an increased EPS content was noticed i.e. 11 and 8% respectively in Nostoc muscorum and 10 and 7% respectively in Anabaena sp. Moreover, combined treatments of H2O2 and SNP along with PTIO and LNAME under the same stress crucially declined the EPS content that showed the incapability of H2O2 without NO. On the other hand, enhanced EPS content was noticed on the combined treatment of H2O2 and SNP along with NAC and DPI under the same stress showed the potential of NO without H2O2 in the removal of the toxic impact of Cd on EPS secretion.Impact of exogenous supplementation of H2O2 and SNP supplementation on the protein content in Cd challenged N. muscorum and Anabaena sp. have been depicted in Fig. N. muscorum, and by 37% in Anabaena sp. respectively, in comparison to control values. However, exogenous supplementation of H2O2 and SNP to Cd stressed cyanobacteria, considerably lowered the inhibitory effect of Cd on protein content but values were still less than control. Contrastingly, all the positive effects of H2O2 and SNP on protein content were reversed on the application of PTIO and LNAME. Whereas application of NAC and DPI does not alter the role of SNP on protein content.The results pertaining to the effect of H2O2 and SNP on the endogenous level of NO have been depicted in Fig. Nostoc muscorum and Anabaena sp. respectively. Contrastingly, exogenously supplied H2O2 significantly (P < 0.05) enhanced the content of endogenous NO by 26 and 25% and more pronouncedly SNP enhanced (P < 0.05) the NO content inside the cell by 28 and 27% in Nostoc muscorum and Anabaena sp. respectively. Under similar stress, a very severe reduction in NO content was noticed on the separate treatments of PTIO and LNAME even in the presence of H2O2. In contrary to this exogenous supplementation of SNP slightly improved NO content even in the presence of NAC and DPI . However, exposure of PTIO enhanced the level of SOR, H2O2 and MDA even in the presence of exogenous H2O2 and this increasing trend was continued when LNAME applied together with exogenous H2O2. While exposure of SNP along with NAC or DPI was significantly able to lessen the toxic levels of SOR, H2O2 and MDA. Furthermore, the combined addition of H2O2 and SNP along with PTIO and LNAME vigorously enhanced the levels of SOR, H2O2 and MDA while combined exposure of H2O2 and SNP along with NAC and DPI under the same stress declined the levels of SOR, H2O2 and MDA in both Cd stressed cyanobacteria.Results pertaining to the contents of SOR, H2O2 and MDA were supported more strongly by in-vivo analysis inside the cells of both tested organisms Anabaena sp. and Nostoc muscorum  shown by the sky blue spots  H2O2 and NO can reduce the intracellular accumulation of Cd  of H2O2  is used and very satisfactory results have been obtained against the Cd toxicity and enhanced growth of test cyanobacteria have been found under H2O2 treatment , that showed a direct relationship between H2O2 and NO  kinase cascade pathway to empower the defensive mechanism of maize leaves. During stress situation H2O2, can regulate NO, and NO itself works as a ROS scavenger that is the point of interaction between these two incredible signaling molecules15.Moreover, Cd can block ETS (electron transport chain) or displace the iron (Fe) molecule with other protein molecules that cause the abundant production of ROS inside the cellss Table  which mi2O2 and SNP alleviated electrolyte leakage  and bands of their isoenzymes were also found less enhanced . Their homogenous cultures were grown in controlled growth conditions; at a temp of 25 ± 2 ºC under the illumination of 75 µmol photons m−2 s−1 of photosynthetically active radiation  with a controlled regime of 14:10 h light/dark cycle.g for 15 min and washed with sterilized distilled water (DW). Henceforth, cyanobacterial cells were subjected to the growth medium containing concentrations of Cd (6 µM), sodium nitroprusside (SNP: a donor of NO) (10 µM), PTIO ; scavenger of NO (20 µM), LNAME (Nω-Nitro-l-arginine methyl ester hydrochloride); NOS enzyme’s inhibitor (100 µM), H2O2 (1 µM), NAC (N-acetyl-l-cysteine); scavenger of H2O2 (1 mM), DPI (diphenyleneiodonium chloride); an inhibitor of NADPH oxidase enzyme (1 µM). Accordingly, succeeding treatments were designed- Control; C (only in BG-11 medium), Cd (cadmium), Cd + H2O2, Cd + NAC, Cd + DPI, Cd + SNP, Cd + PTIO, Cd + LNAME, Cd + H2O2 + PTIO, Cd + H2O2 + LNAME, Cd + SNP + NAC, Cd + SNP + DPI, Cd + H2O2 + SNP + PTIO + LNAME, Cd + H2O2 + SNP + NAC + DPI.Experimental design used for the present study was completely randomized design (CRD). To perform the experiments, cultures of both tested cyanobacteria were collected at their exponential phase by centrifuging them at 30002O2 is a short-lived photodegradable component so for the experiments of the present study, H2O2 was prepared very carefully in dark and added to the experimental set up at the end wherever it was required. Finally, all cultures were incubated 3 h in dark and after that they were placed for cycle 14:10 h light and dark. All the parameters were analyzed after 24 h of treatment.As Hg for 10 min and then washed twice with distilled water. Further, pellets were dried at 80 ºC and weighed gravimetrically .The growth of test cyanobacteria was measured in terms of dry weight. The 100 ml cultures of both the organisms were collected and centrifuged at 4000.45. For EPS extraction, 100 ml samples were taken from each treatment, centrifuged at 3000g for 15 min supernatants were collected and then dried separately at 40 ºC. The obtained precipitates were washed thrice with isopropanol and dried again at 37 ºC. Further, hydrolysate was analyzed for glucose by Seifter et al.46 and calculation was done by standard curve of glucose.Exopolysaccharides (EPS) content was determined according to Sharma et al47 was adopted. Cells were centrifuged and homogenized with 50 mM potassium phosphate buffer (PPB) (pH 7.8) containing 1 mM EDTA and 2% polyvinyl pyrrolidone at 4 °C. Further, 0.1 ml of obtained homogenates was mixed with 2.5 ml of Coomassie brilliant blue G-250 reagent, then kept for 2 min in dark at 25 °C and absorbance was read at 595 nm. Total soluble protein content was determined by using bovine serum albumin as standard protein solution.For the extraction of protein, the method of Bradford.48 with some modifications. For NO estimation desired volume of cultures was centrifuged and pellets were crushed in 3 ml of 50 mM cool acetic acid buffer . Further, homogenates were centrifuged and supernatant was mixed with 50 mg of charcoal. After vortex and filtration, the filtrate was leached and collected. The 1 ml of filtrate and 1 ml of Greiss reagent was mixed and incubated at room temperature for 30 min. Absorbance was taken at 540 nm. NO content was calculated by the standard curve prepared by graded solution of NaNO2.The NO content was determined by using Griess reagent (Sigma-Aldrich) by following the procedure of Zhou et al49 with some minor changes. To detect the intracellular Cd accumulation; cells were collected by centrifugation then gently washed twice or thrice with double distilled water (DDW) to remove excess Cd. Obtained cells were mixed with 1 ml of dithiozone stock solution containing 6 gm of dithiozone and DW (3:1). Further 1–2 drops of acetic acid glacial were added and after 4 h of incubation; cells were observed under microscope .Histochemical analysis of Cd was conducted through the method of Seregin and Kozhevnikova2•-), H2O2 contents, and lipid peroxidation were quantified by following the procedure of Elstner and Heupel50, Velikova et al.51 and Heath and Packer52 respectively. For SOR content the absorbance was recorded at 530 nm. The determination of SOR content was based on the formation of NO2 from hydroxylamine in presence of O2•- and the content was quantified by the standard curve of nitrite (NaNO2). Absorbance for H2O2 was recorded at 390 nm and content was calculated by the standard curve prepared with graded solution of H2O2. SOR and H2O2 induced oxidative damage to the lipids i.e. lipid peroxidation was calculated in the form of malondialdehyde (MDA) equivalents contents. Absorbance for MDA was recorded at 532 and 600 nm and content was calculated by using an extinction coefficient of 155 mM−1 cm−1.Superoxide radical  and 3, 3 diaminobenzidine (DAB) for in-vivo staining respectively. MDA equivalents contents and intensity of membrane damage were visualized by using Schiff's reagent and Evan’s blue by following the methods of Pompella et al.54 and Yamamoto et al.55 respectively. Images were taken in a high-quality microscope .The Histochemical analysis of the accumulation of O56, Gahagan et al.57, Aebi58 and Habig et al.59 respectively. For SOD activity, the photoinhibition of NBT was recorded at 560 nm after 20 min illumination of light (100 µmol photons m−2 s−1). One unit of SOD activity is demarcated as the required amount of enzyme to cause 50% inhibition in the reduction of NBT. For POD, absorbance of the reaction mixture was recorded at 430 nm for 3 min. The activity of the enzyme was calculated by using an extinction coefficient of 25.5 mM−1 cm−1 and one unit of enzyme activity is defined as 1 nmol pyrogallol oxidized min−1. Activity of CAT was determined by monitoring the decrease in absorbance at 240 nm in the reaction mixture58. The activity of enzyme was calculated by using an extinction coefficient of 39.4 mM−1 cm−1. Here 1 nmol H2O2 dissociated min−1 was equivalent to one unit of enzyme activity. For GST, absorbance of reaction mixture was recorded at 340 nm. Enzyme activity was calculated by an extinction coefficient of 9.6 mM−1 cm−1 and one unit of enzyme activity is equivalent to 1 nmol of CDNB conjugates formed min−1.The activities of superoxide dismutase , peroxidase , catalase  and glutathione-S-transferase  were analyzed by following the methods of Giannopolitis and Ries60. A uniform amount (300 mg) of proteins mixed with sample buffer  was loaded in each well and proteins were electrophoretically separated at 80 V through stacking gel followed by 120 V in the separating gel at 4 ºC. For visualizing SOD isoenzymes, the gels were immersed in PPB  containing NBT (1.125 mM) in darkness for 20 min and followed by incubation in PPB containing TEMED (28 mM) and riboflavin (28 mM) in dark for 15 min. The gels were then placed in PPB containing mM EDTA (0.1 ml) and exposed to light for 20 min at 25 ºC61. For POD, gels were immersed in 1 mg ml−1 benzidine and 1 mM H2O2 in 0.1 M Tris–acetate buffer (pH 5.0) at 25 ºC till the brown colored bands appeared62. For CAT isoenzymes gels were incubated with H2O2 (0.01%) for 15 min, rinsed with water, and shaken in freshly prepared solutions each of 0.1% FeCl3 and K3Fe(CN)6 at 25 ºC until the achromatic bands appeared63. For GST isoenzyme staining a reaction mixture containing 0.1 M PPB (pH 6.5), GSH (5.0 mM), CDNB (1.0 mM) and NBT (1 mM) was used and bands were developed by illuminating the gel under 25 µmol photon m−2 s−1 of photon flux density64. Photographs of isoenzymes were captured with digital camera, photo-plate was prepared in CorelDRAW and image quality was enhanced in Photoshop 7.0.Native–PAGE analysis was carried out on discontinuous polyacrylamide gels (PAGE) with 4.5% polyacrylamide in stacking and with its varying concentrations . The separation of individual isoenzyme was performed in a vertical gel electrophoretic unit  by considering the method of LaemmliStatistical analysis of variance (ANOVA) was performed to test the significance at probability level P < 0.05. Duncan’s multiple range test was applied to compare significant differences among the mean values. Graphical representations are the means of three independent experiments with three replicates in each experiment (n = 9). Lower case letter  shows statistical significance.2O2 that exists. In Fig. 65. But exogenously supplied NO and H2O2 promote the EPS secretion and check the entrance of Cd inside the cells after that they also enhance the antioxidant system and endogenous NO content that's why the level of ROS get minimized. Here, the low amount of exogenous H2O2 works as a signal transmitter to enhance the beneficial amount of RNS species that indirectly respond to the balancing of antioxidants to cope up with cd stress. Afterward PTIO and LNAME with H2O2 block the internal NO that creates a disturbance in the working of signaling mechanism of H2O2 and NO here it is clear that exogenously supplied H2O2 cannot work without NO. However, application of NAC and DPI cannot disturb the signaling of NO. Hence, from this it is clear that NO is a down-stream regulator of H2O2 but the pathway of signaling between both the molecules is very complex because of their multidimensional roles in a stressful environment.From the aforementioned results, it can be concluded that there is an interlinked pathway between NO and HSupplementary Figure S1.


In addition, a mechanism in which Ba0 and BaH2 are both essential is discussed. DFT calculations on benzene hydrogenation with a simple model system (Ba/BaH2) confirm that the presence of metallic Ba has an accelerating effect.Ba metal was activated by evaporation and cocondensation with heptane. This black powder is a highly active hydrogenation catalyst for the reduction of a variety of unactivated (non‐conjugated) mono‐, di‐ and tri‐substituted alkenes, tetraphenylethylene, benzene, a number of polycyclic aromatic hydrocarbons, aldimines, ketimines and various pyridines. The performance of metallic Ba in hydrogenation catalysis tops that of the hitherto most active molecular group 2 metal catalysts. Depending on the substrate, two different catalytic cycles are proposed. A: a classical metal hydride cycle and B: the Ba metal cycle. The latter is proposed for substrates that are easily reduced by Ba 0 and BaH2 are both essential is discussed.Just a pinch of Ba metal: Efficient hydrogenation of a wide range of substrates is achieved with metallic barium previously activated by metal vapor synthesis. A mechanism in which Ba Two major milestones stand at the cradle of heterogeneous metal catalysis.2 and BaH2 are active in ethylene hydrogenation but only after prior thermal treatment under high vacuum, advocating that the presence of the metallic state is of importance.It is hardly known that, following these groundbreaking studies on the catalytic activity of the late transition metals, catalytic ethylene hydrogenation with the much less noble alkaline earth (Ae) metal Ca has been described as early as 1925.3)2]2 3)2 and larger metal hydride clusters of which the surface is decorated with amide ligands (Scheme 2)n salts. It was found that increasing the size of the amide ligand from N(SiMe3)2 to N(SiiPr3)2 led to a considerable boost in catalytic activity and broadened the substrate scope from alkenes to arenes.iPr3)2 form smaller, more active, metal hydride clusters. Although one would expect that the catalyst activity may be increased further using even bulkier ligands, we now report that ligand‐free metallic Ba is a very simple but highly active hydrogenation catalyst.Although these early reports are limited only to ethylene hydrogenation, they become topical again in light of the recent interest in alkene hydrogenation with heavier group 2 metal catalysts.8, 9 Ws Scheme .[9] Th2,2.2 12 bar, 80 °C) cyclohexene was fully reduced within 0.5 hour (Table iPr3)2]2; values for the latter are shown between squared brackets. Attempts to activate bulk Ba metal by dissolution in liquid ammonia and subsequent solvent evaporation gave a Ba0 powder that is essentially non‐active in cyclohexene hydrogenation (Table S5), implying that catalyst synthesis by the metal vapor method is crucial.As a first test case we chose the reduction of cyclohexene, a challenging substrate2C=C(H)Ph could be fully converted at 120 °C (entry 4), the unactivated alkene 1‐Me‐cyclohexene needed a somewhat higher temperature of 150 °C (entry 5). For the first time, also the tetra‐substituted alkene, Ph2C=CPh2, could be quantitatively reduced with a group 2 metal catalyst (entry 6).Encouraged by these preliminary results, hydrogenation of tri‐substituted alkenes was probed. While the activated (conjugated) alkene PhiPr3)2]2, the hitherto most active group 2 metal hydrogenation catalyst. Noticeable, is the unproblematic full conversion of non‐cyclic internal alkenes like cis‐ and trans‐3‐hexene . Also di‐substituted alkynes were fully hydrogenated at rates similar to those reported for Ba[N(SiiPr3)2]2 .Further comparison (entries 7–10) shows that the catalytic performance of Ba metal is at least at par with that of Ba[N(Si3)2]2.For further comparison of Ba metal and Ba amide catalysts, also imine hydrogenation has been investigated (entry 13). The activity of Ba metal is comparable to that of Ba[N(SiMeiPr3)2]2 after three days only 18 % conversion could be reached.2+][C14H102−]}n salts. Indeed, hydrogenation of phenanthrene or more soluble alkylated anthracenes was successful (entries 5–7). While Ba[N(SiiPr3)2]2 was not able to reduce pyrene or acenaphthylene, catalytic quantities of Ba metal reduced up to two out of four of the pyrene rings (entry 8) while also acenaphthylene could be reduced (entry 9).As a last showcase for the full scope of metallic Ba in hydrogenation catalysis, the reduction of aromatic rings was demonstrated Table . Under fiPr3)2]2 is its inability to hydrogenate substrates that contain heteroatoms like O or N. This was attributed to substrate‐metal coordination, blocking potential coordinations sites for H2 activation.One disadvantage of the bulky Ba amide catalyst Ba2 which could be described as two C‐N‐Ba metallacycles connected by bridging nitrogen atoms N(H)Ph was cleanly formed already at room temperature , justifying this route.The feasibility of the Ba metal mechanism B was validated by stoichiometric conversions. Reaction of Phs Figure . The dou0 surface activates the C=C bond for nucleophilic attack by a neighbouring Ba‐H functionality. Adsorption of ethylene on a Ca metal surface was already described as early as 1923d‐orbitals play a role in the chemistry of the heavier Ae metals Ca, Sr and Ba. In metallic form they mimic transition metals and low temperature matrix‐isolation of Ae(CO)8, Ae(N2)8 and Ae(C6H6)3 complexes has been reported.0 and BaH2 which in the presence of H2 are in a temperature dependent equilibrium.2 under high vacuum gave in some cases a catalyst that hydrogenates ethylene already at −78 °C.the catalytic effect occurs at an interface between free metal and metal hydride”.dual sites” and, already at this very early stage, they explain the high activity of Ca and Ba catalysts with “some overlap of s‐, p‐ and d‐bands”.As a third possibility, one could discuss a mechanism that is a hybrid between cycles A and B Scheme : sorptio0/BaH2 pathway needs detailed investigations and advanced calculational studies at surfaces. Although gas phase calculations cannot model complicated heterogeneous systems, they do provide insights in basic steps at an atomic level. Here we present preliminary DFT calculations on very simple model systems. These indicate the activating influence of Ba0 in benzene hydrogenation with BaH2 (Scheme 2. After formation of a benzene⋅⋅⋅BaH2 complex (I1), an activation energy of 8.2 kcal mol−1 is needed to reach the transition state (TS1). The Meisenheimer anion formed during this process is bound to Ba (I2). Hydrogenolysis in ortho‐position (TS2‐o) is slightly preferred over attack in the para‐position (TS2‐p). Note that reduction of the first C=C bond in benzene is endothermic. The exothermic hydrogenation of the two remaining C=C bonds, however, make benzene hydrogenation overall exothermic.It is difficult to verify heterogeneous catalytic pathways. The possibility for a Ba2 Scheme . The nor0 atom (in green). Due to its high reducing power, an interesting starting complex is formed (Ba‐I1) in which the benzene ring is not flat but slightly puckered to a boat form. Different C−C distances in the ring (1.455 Å/1.520 Å) reveal charge transfer from Ba to benzene and the system could be described as having partial Ba2+ character. Similar distortions have been observed in Ae(C6H6)3 complexes.2 unit is close to neutral . This reactivity could be compared to the formation of Mg‐anthracene,2 coordination which acts as a Lewis acid that stabilizes the anti‐aromatic 1,3‐cyclohexadiene2− anion.−1 is nearly halved compared to that for the Ba0‐free route . Partial loss of aromaticity in the benzene ring assists the hydrogenation reaction. Also the activation energy for subsequent hydrogenolysis is lowered by additional interaction with Ba0 . Note that this effect is only noticeable for hydrogenation in ortho‐position. Hydrogenolysis in para‐position leads to a drastic increase of the barrier. The preference for the ortho‐position can be explained by the favorable interaction of Ba0 with conjugated dienes (Ba‐I4‐o) compared to isolated alkenes (Ba‐I4‐p). Complex Ba‐I4‐o should rather be described as the product of 1,4‐addition of Ba0 to a diene.[0 can have an accelerating effect on catalytic hydrogenation.The same mechanism was calculated in presence of a Ba a diene.19, 20Metallic barium is shown to be an all‐round catalyst for effective hydrogenation of challenging alkenes, alkynes, arenes and imines. In strong contrast with our previous approach to increase the activity of Ba amide catalysts by increasing ligand size, use of metallic Ba as a versatile, highly active, early main group metal hydrogenation catalyst not only extends the scope of substrates but is also remarkably simple. Although the metal needs to be activated, the current method represents a welcoming short‐cut in respect to the syntheses of superbulky Ba amide catalysts. Precise understanding of the fundamental mechanisms is at this stage difficult but the idea that the combination of Ba metal and Ba hydride dual sites is key to its activity has been demonstrated by DFT calculations on a simplified model system. This important aspect will be comprehensively investigated and exploited further in future research.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Herein, we propose a conceptually different mechanism for surface electron density modulation based on the model of Au anchored CdS. We firstly manipulate the direction of electron transfer by regulating the vacancy types of CdS. When electrons accumulate on vacancies instead of single Au atoms, the adsorption types of CO2 change from physical adsorption to chemical adsorption. More importantly, the surface electron density is manipulated by controlling the size of Au nanostructures. When Au nanoclusters downsize to single Au atoms, the strong hybridization of Au 5d and S 2p orbits accelerates the photo-electrons transfer onto the surface, resulting in more electrons available for CO2 reduction. As a result, the product generation rate of AuSA/Cd1−xS manifests a remarkable at least 113-fold enhancement compared with pristine Cd1−xS.The surface electron density significantly affects the photocatalytic efficiency, especially the photocatalytic CO 2 photoreduction.The electron density of reactive sites significantly affects catalytic performances. Here, authors demonstrate the electron density of different reactive sites can be modulated by regulating the type of vacancy and the size of Au, leading to effective CO Photocatalytic CO2 conversion technology has attracted extensive interest, since the conversion of CO2 into high value-added chemicals could potentially be achieved under relatively mild conditions, which facilitates its resource utilization4. Generally, photocatalytic CO2 conversion is a typical photoreduction reaction, which needs electrons to participate in7. Different from other photoreduction reactions, such as hydrogen evolution reaction (two electrons), photocatalytic CO2 conversion is an inherently multi-electron reduction process10. To this end, photocatalysts are required to offer abundant electrons for the CO2 molecule to promote its transformation in the reaction process11.As a representative greenhouse gas, carbon dioxide  could affect the electron transfer direction between Pt nanoparticles and BN nanosheets17. Hence, it is speculated that the electrons may be localized at other sites instead of metal nanostructures. Indeed, previous studies suggest that the interaction of adsorbed CO2 molecule and metal site is relatively weak since the formed metal–C or metal–O bonds are weaker than the highly stable C–O bonds in CO2 molecule19. This will lead to the easy cleaving of metal–C or metal–O bonds during the reaction process, hindering the further transformation of CO28. The above considerations suggest that controlling the size of metal nanostructures and/or the type of vacancies might realize the accumulation of electrons at other sites instead of metal nanostructures, which is likely to promote the adsorption and conversion of CO2.Previous studies suggest that metals, especially noble metals, have been commonly introduced to enhance the surface electron density of photocatalysts22. According to the pre-experiments in Supplementary Fig. 25. We have designed and fabricated a series of Au-anchored CdS photocatalysts and reported a conceptually different mechanism for modulating surface electron density to promote the conversion of CO2. As displayed in Fig. 2 could be chemically bonded on the Cd vacancies instead of physical adsorption on single Au atoms to promote its effective activation.For this purpose, CdS is selected to be a model material due to the easy-fabricated vacancies and favorable solar light response1−xS) is first prepared by the hydrothermal method. X-ray diffraction (XRD) pattern and transmission electron microscopy (TEM) images  and Au clusters (AuNC) are successfully introduced in the 1%Au/Cd1−xS and 2%Au/Cd1−xS samples, respectively. To distinguish Au nanostructures in different samples, 1%Au/Cd1−xS and 2%Au/Cd1−xS samples are referred as AuSA/Cd1−xS and AuNC/Cd1−xS for simplicity in the following study.Based on the above considerations, the CdS with Cd vacancies  and extended X-ray absorption fine structure (EXAFS) spectra are presented in Fig. SA/Cd1−xS sample is different from that of the reference Au foil. The EXAFS spectrum shows that AuSA/Cd1−xS exhibits a peak near 1.7 Å without any significant Au–Au contribution between 2 and 4 Å, confirming the atomic dispersion of Au on Cd1−xS. The quantitative structural parameters of Au in the AuSA/Cd1−xS (cf. Supplementary Table 1−xS surface with different sites (surface S and Cd vacancy) are constructed by density functional theory (DFT) method (cf. Supplementary Table Ef = −1.89 eV). It is hence suggested that single Au atoms are anchored on the Cd vacancies to form the Au–S bonds in the AuSA/Cd1−xS sample.To investigate the coordination environments of Au in the Au1−x) is synthesized by the hydrothermal method  samples are shown in Supplementary Fig. 1−x is similar to that of the CdS and both of them are different from that of the reference Cd foil. According to the EXAFS spectra, the CdS sample shows bulk crystalline CdS. In the case of the CdS1−x sample, intensity of the first coordination shell is lower than that of CdS, implying fewer S-neighbors than in the bulk CdS. Combined with the element analysis, the ratio of Cd and S are quantitated as 1:0.73  are successfully fabricated and the introduced single Au atoms are anchored on the vacancies. In the case of the Au-anchored Cd2 reduction tests are carried out to evaluate the effect of Au on photoreduction efficiency. Cd1−xS shows low CO and CH4 generation rates of 0.2 and 0.1 μmol g−1 h−1, respectively. After anchoring Au nanoclusters, the product generation rates are enhanced. It is interesting that with the introduction of single Au atoms, the CO, CH4, and H2 generation rates are highly promoted to 32.2, 11.3, and 7.9 μmol g−1 h−1, respectively. It manifests a remarkable 161-fold and 113-fold enhancement over Cd1−xS as well as 5-fold, 4-fold, and 2-fold enhancement compared with AuNC/Cd1−xS. For CdS1−x systems . These first two steps are aimed to activate the C–O bond of CO2 molecule. Finally, these species will be further transformed into the desired products , which need abundant electrons to take part in to promote their effective transformation. Hence, the adsorption behavior of CO2 not only determines the occurrences of reaction, but significantly affects the conversion efficiency.To uncover the main factors of enhanced photocatalytic CO2 adsorption process on the surfaces of Cd1−xS and CdS1−x systems : 1200 and 1166 cm−1), monodentate carbonate : 1140–1030 cm−1) and carboxylate s: 1266 cm−1) detected with the prolonged time of adsorption39. Meanwhile, the band assigned to COOH* is detected at 1634 cm−1,41 suggesting that the adsorbed CO2 is combined with H* and partly converted to the COOH*. After anchoring Au clusters, the intensities of absorption bands assigned to HCO3−, CO2−, and COOH* have decreased. There is a new band appearing at 1043 cm−1, which is assigned to HCO*43. This indicates that part of the formed COOH* is converted to HCO*. Different from it, some of the absorption bands (HCO3− and COOH*) almost disappear over AuSA/Cd1−xS. It should be noted that there is a new band detected at 1110 cm−1 assigned to H3CO*45. The intensity of the absorption bands assigned to HCO* is much lower than that over AuNC/Cd1−xS, revealing that the majority of formed COOH* are quickly converted into HCO* and then transform into H3CO*. The conversion pathway of CO2 over different samples in Au/Cd1−xS systems during the adsorption process could be deduced as follows:2 on Cd1−xS surface. When Au nanoclusters downsize to single Au atoms, the carbon active species can be deeply converted into H3CO*, which is beneficial for the subsequent reaction. Different from the Cd1−xS system, with the introduction of Au nanostructures, there are no absorption bands assigned to new intermediates appearing on CdS1−x surface in the process of CO2 adsorption  is performed to detect the generated intermediates during the COcf. Fig. . For pricf. Fig. , there a2 adsorption process. In previous studies, reaction sites have possibly affected the generation of intermediates46. Based on it, Au nanostructures (Au site) and vacancies (vacancy site) are selected to be the different CO2 adsorption sites. The CO2 adsorption models on Au/Cd1−xS and Au/CdS1−x are constructed by the DFT method . With the introduction of single Au atoms, the adsorption energy has significantly decreased, implying that single Au atoms promotes the adsorption of CO2 on Cd1−xS surface. It should be noted that CO2 is more likely to adsorb on the Cd vacancies instead of single Au atoms due to the more negative adsorption energy. To further identify the adsorption site in AuSA/Cd1−xS, the different surfaces are probed by N2 sorption  has increased after anchoring single Au atoms (33.9 m2/g). It is suggested that the introduction of single Au atoms enhances the amount of adsorption site. However, the CO2 adsorption capacity of Cd1−xS is decreased from 72.9 to 66.2 μmol g−1 : 1621 cm−1; v(S–O): 1209 and 1162 cm−1)51. It is suggested that the CO molecule adsorbs on the Cd vacancy sites and combines with the S atoms to form C=O–S bonds. Meanwhile, there are some absorption bands detected at 1100 and 1044 cm−1, which are assigned to HCO* and H3CO*. The above results indicate that when CO adsorbs on Cd vacancy, it will combine with H* to further convert into HCO* and H3CO*, which is in accordance with the observations in DRIFTS spectra of the CO2 adsorption process.The above discussions suggest that the types of vacancies and the size of Au nanostructures present a different effect on the intermediates in the COcf. Fig.  and 18. 2 and single Au atoms in the AuSA/CdS1−x is weak. Different from it, there is a strong interaction between CO2 and Cd vacancies in the AuSA/Cd1−xS  to promote its activation, resulting in C–O–C bond angle bending and elongation of both C–O bonds. To further prove the effect of different adsorption sites in the CO2 adsorption behavior, isotope labeling is carried out on AuSA/Cd1−xS and AuSA/CdS1−x using 13CO2 instead of 12CO2 for the DRIFTS analysis . In the process of 12CO2 adsorption . In the process of 13CO2 adsorption, there is a small 13C frequency shift (6 cm−1) occurring on the surface of AuSA/CdS1−x. It should be noted that there is a large 13C frequency shift (16 cm−1) of this absorption band detected on the surface of AuSA/Cd1−xS. It is indicated that, compared with AuSA/CdS1−x surface, the adsorbed CO2 obtains more electrons from AuSA/Cd1−xS surface to weaken the C–O bond, leading to a larger 13C frequency shift. Meanwhile, according to the value of 13C frequency shift, more than one C–O bonds might be bonded to the surface52. Based on the above discussions, it is speculated that CO2 molecule is physically adsorbed on single atoms of AuSA/CdS1−x surface. The formation of bonds and effective electron transfer between surface and CO2 indicate that CO2 molecule is chemically adsorbed on Cd vacancies of AuSA/Cd1−xS surface. In brief, when the type of vacancies changes from S vacancies to Cd vacancies, the CO2 adsorption sites change from single Au atoms to the vacancies. Benefiting from this, CO2 could be chemically bonded on the Cd vacancies instead of physical adsorption on single Au atoms. The strong interaction between CO2 and adsorption sites (Cd vacancies) profits the formation of stable electron-transfer channels. As a result, CO2 could obtain more electrons from the adsorption sites to promote its deep transformation.Furthermore, according to the partial density of states (PDOS) cf. Fig. , it is ocf. Fig. . More imcf. Fig. . As a recf. Fig. . It is ocf. Fig. , there i1−xS and CdS1−x surfaces after anchoring Au nanostructures is explored by DFT calculations . To further identify the electron transfer between Au and Cd1−xS or CdS1−x, X-ray photoelectron spectroscopy (XPS) spectra are presented in Fig. 1−xS system  compared with the pristine Cd1−xS, indicating that Cd1−xS receives electrons from the single Au atoms. While for CdS1−x system  than that of AuSA/Cd1−xS. This suggests that the electrons are localized at single Au atoms instead of CdS1−x in AuSA/CdS1−x system. To identify the internal factors affecting the direction of electron transfer between single Au atoms and Cd1−xS or CdS1−x, the potential energy plots are given in Supplementary Fig. SA/CdS1−x, the potential energy of CdS1−x is much lower than that of single Au atoms. Benefiting from it, there is a driving force promoting electron transfer from CdS1−x to single Au atoms. Opposite to that, the potential energy of single Au atoms is lower than that of Cd1−xS, resulting in the reversal of electron transfer direction. Hence, it is observed that the electrons are localized at the single Au atoms when they anchor on S vacancies. After single Au atoms anchored on Cd vacancies, the electrons will conversely transfer from single Au atoms to Cd1−xS and accumulate on Cd vacancies.To identify the internal factors affecting adsorption sites, the charge population of Cdcf. Fig.  and 6b. cf. Fig. , the peacf. Fig. , the pea1−xS and CdS1−x systems. Hence, to further identify the underlying roles of Au content on the surface electron density, the Au/Cd1−xS system is selected as the representative in the following discussion. In general, electron transfer might be related to the local electronic structure. According to PDOS in Supplementary Fig. NC/Cd1−xS, the hybridization between Au 5d and S 2p orbits in Au–S bonds is much stronger for AuSA/Cd1−xS. As a result, more electrons are transferring from Au 5d orbits in single Au atoms to Cd1−xS compared with Au clusters  and the Au 4f spectrum shifts to high binding energy (0.4–0.6 eV) compared with AuNC/Cd1−xS. It is indicated that the interaction between single Au atoms and Cd vacancies is much stronger than that with Au clusters. Benefiting from it, under light illumination, it is observed that the lifetimes of photogenerated electrons in Cd1−xS are decreased from 2.95 to 0.44 ns (AuSA/Cd1−xS) and 2.22 (AuNC/Cd1−xS)  in Fig. 1−xS. The peak at c.a. 400 nm corresponds to the electrons transferring from the top of valence band to the bottom of conduction band. The other one at c.a. 500 nm is assigned to the defect level introduced by Cd vacancy on the surface which is observed in the band structure  on AuSA/Cd1−xS is remarkably decreased by 91.1% relative to that over AuNC/Cd1−xS. This suggests that the accumulated electrons in Cd vacancies on AuSA/Cd1−xS not only promote the activation of CO2 molecule, but also facilitate its further transformations. It is identified that there is no significant absorption band assigned to COOH* observed among in situ DRIFTS test over AuSA/Cd1−xS. Furthermore, extra electron is introduced to create an electron-rich condition in the process of CO2 transition into COOH in Fig. 7d53. Interestingly, the energy barrier on AuSA/Cd1−xS presents a larger decrease (41%) compared with that on AuNC/Cd1−xS (34%). It is suggested that, compared with AuNC/Cd1−xS, the enhancement of surface electron density much more facilitates the occurrence of CO2 reduction catalyzed by AuSA/Cd1−xS.As presented in Fig. 1−x to Au in CdS1−x system. Quite different from it, the direction of electron transfer has been reversed in Cd1−xS system, resulting in the accumulation of electrons in Cd vacancies. Hence, the adsorption of CO2 is enabled in vacancy sites instead of Au sites. More importantly, there is a stable electron-donation channel forming between CO2 and Cd vacancies, accelerating the transfer of accumulated electrons to CO2 for its effective activation. On the other hand, under light illumination, when Au nanostructures change from Au nanoclusters to single Au atoms, the surface electron density is significantly increased and promotes much more electrons accumulating on the surface as well as the Cd vacancies, which is beneficial for the subsequent reaction. As a result, the CO and CH4 generation rates of AuSA/Cd1−xS manifest a remarkable 160-fold and 113-fold enhancement compared with Cd1−xS as well as the H2 generation rate is promoted from 0 to 7.9 μmol g−1 h−1.In summary, we have designed and fabricated a series of Au/CdS photocatalysts. On the one hand, the types of vacancies highly affect the direction of electron transfer. That is the electrons will transfer from CdS1−xS and CdS1−x were synthesized via a modified hydrothermal method54. For Cd1−xS, the synthesis process as follows: first, CdCl2·5H2O (4 mM) was dissolved in 20 mL aqueous solution . Meanwhile, another 20 mL aqueous Na2S·9H2O (8 mM) solution was prepared, marked as solution B. Then, solution B was added dropwise into solution A under continuous stirring. The mixed solution was transferred into a Teflon-lined stainless steel autoclave (inner volume: 50 mL) and held at 140 °C for 18 h in an oven. After cooling to room temperature naturally, the product was centrifuged and washed with water and ethyl alcohol several times. Finally, the obtained product was dried overnight at 60 °C under vacuum condition. The synthesis process of CdS1−x and CdS were the same as that of Cd1−xS except that 1 and 4 mM of Na2S.9H2O were added in solution B.The Cd1−xS and Au/CdS1−x were synthesized by the impregnation method. For 1%Au/Cd1−xS, 100 mg of Cd1−xS was dispersed in 30 mL water and adding 0.2 mL HAuCl4 solution (5 mg/mL). Then, the mixed solution was stirred at 70 °C for 6 h. The product was centrifuged and washed with water and ethyl alcohol several times and dried overnight at 60 °C under vacuum condition. The synthesis procedure of 2%Au/Cd1−xS was the same as that of 1%Au/Cd1−xS, except that 0.4 mL of HAuCl4 solution was used in the experiment. Meanwhile, Au/CdS1−x samples were synthesized by the same method using CdS1−x instead of Cd1−xS.The Au/Cd2 reduction performance test was carried out in a closed gas system (volume: 276 mL) at atmospheric pressure and ambient temperature. First, 30 mg of photocatalyst was dispersed with H2O in a petri dish (ø = 6 cm) and dried at 70 °C under vacuum condition. Then, petri dish was put on a stainless steel breaker with 1 mL of deionized water in the reactor, which was filled with the CO2 . A 300 W Xe lamp was used as the UV-vis light source (intensity: 600 mW/cm2). Every 2 h, 1 mL of gas was taken from the reactor for qualitative analysis by gas chromatography . CO and CH4 were detected by a flame ionization detector (FID) and a thermal conductivity detector (TCD) was used for detecting H2. The isotope test was carried out by using 13CO2 as the carbon source with the same reaction set as mentioned above and the gas products were analyzed by mass spectrometry .Photocatalytic CO12CO2 (20% CO2/Ar) and H2O vapors (humidity: 5%Rh) with a flow rate of 17 mL min−1 was introduced into the Harrick cell. In case of AuSA/Cd1−xS and AuSA/CdS1−x samples, 13CO2 (20% 13CO2/Ar) was used instead of 12CO2 to perform the isotope test. For CO probe test, a mixture of CO (10% CO/Ar) and H2O vapors (humidity: 5%Rh) with a flow rate of 17 mL min−1 was introduced into the Harrick cell. Before the measurement, the samples were pretreated at 100 °C for 10 min under Ar atmosphere.In situ DRIFTS measurements were performed on a TENSOR II FT-IR spectrometer . The spectrometer was equipped with an in situ diffuse reflectance cell (Harrick) and a high-temperature reaction chamber (HVC). A mixture of Supplementary Information


The silver atoms in 1 show an overall brick‐shape arrangement, where structural resemblance to the close‐packed fcc and hcp structures is realized. Within 1 a 58 electron closed shell system is present. The light sensitivity renders 1 as a model compound for the primary seeds of the photo process, whereby this sensitivity, together with the high‐yield synthesis show that 1 is a perfect starting compound for further investigations like silver‐plating processes.The reduction of ( Light up the dark! The reduction of (nBu3P)AgCl with LiBH(sBu)3 in toluene gives the highly unstable metalloid silver cluster Ag64(PnBu3)16Cl6 as dark red, temperature‐ and light‐sensitive single crystals in high yield. This is the largest structurally characterized metalloid silver cluster exhibiting chlorine and phosphine substituents only. The effect of light‐sensitive substances, like silver halides, forming latent images under light exposure on a medium has been well‐known for many years. Even the chemistry of the photographic processing and developing of a negative image and conversion into a positive image has been in use for a long time and has also been commercialized. Although in the last couple of years the replacement of classical silver‐based photography by computer‐assisted digital images proceeds, the nature of those basic processes of forming latent images on photographic plates is still of fundamental interest. A closer look at the reactions leading to the generation of the latent image shows that the formation of micro‐ or nanoscaled silver particles from silver halide under light exposure is assumed. However, the exact nature of these intermediary formed primary silver nanoparticles is unknown.The photographic process has been the bread‐and‐butter work of photographers since the end of the 19nRm 3 or ligand like PPh3), which are actually often called “metal clusters” or “metal nanoclusters”,84(N(SiMe3)2)20]4− a structural relation to α‐Ga and δ‐Ga,12(C13H9)10 a structural relation to δ‐Ga10[Si(SiMe3)3]6 (α‐ to β‐tin phase transition).Metalloid clusters with a metallic core surrounded by some stabilizing ligands and substituents awakened interest in recent years as small, but crystallizable molecules to provide insight into this area of nanoscaled metal particles.ke SiSiMe3 or liga44(p‐MBA)30]4− (p‐MBA=para‐mercaptobenzoic acid) by Bigioni and co‐workers on a gram scale136(S‐TBBT)64Cl3Ag0.45]−,206(S‐Cyc)x70−FxCl2 (x=1–4),374(S‐TBBT)113Br2Cl2tert‐butylbenzene‐thiolate, Cyc=cyclohexyl) really huge clusters were found. All of these metalloid silver clusters exhibit organic thiolate substituents that form a silver thiolate like shell, showing no light sensitivity. Very recently Wang and co‐workers presented an alkynyl‐proctected metalloid silver cluster, [Ag112Cl6R51]3− 2C6H3), which also exhibits halide atoms bound to the silver core;64(PnBu3)16Cl6 (1), obtained from the reduction of nBu3PAgCl, which can be described as a distorted section of face‐centered cubic packing, showing a high sensitivity to light and even temperature to form metallic silver.Many examples of metalloid clusters of the heaviest coinage metal, gold, have been presented in the last years,I precursors is limited due to the very low solubility of AgIX . However, in the literature a simple synthesis of donor‐stabilized silver halides of the general formula [(R3P)xAgCl]y  can be found, which are well soluble, at least in aromatic solvents like benzene and toluene.I compounds thus seemed to be ideal precursors for the synthesis of metalloid silver clusters via reduction. The reduction of the phosphine‐stabilized silver halides , leads in case of nBu3PAgCl, when reduced with LiBH(sec‐Bu)3, to a dark red solution, which yields red crystals of the metalloid cluster Ag64(PnBu3)16Cl61 after a few days. The extreme light‐ and temperature‐sensitive red single crystals of 1 were prepared for the X‐ray diffraction measurement as quickly and with as little exposure to light as possible, selected in cooled mineral oil (approx. −30 °C) under a dimmed light microscope to prevent decomposition to metallic silver.1 could be refined properly 4467(SPhMe2)32(PPh3)8]3+.The metalloid Ag1, large differences in the measured Ag–Ag distances, from 280 pm to 335 pm (bulk: 289 pm) are found, already indicating that the arrangement of the silver atoms cannot be described as a simple section of the fcc packing. Within 1, a central Ag6 octahedron is present to which other Ag6 octahedra are bound. Thereby edge‐ and face‐sharing of the Ag6 octahedra is realized, as highlighted by gray and yellow polyhedra in Figure 1 there are areas that are structurally similar to both close‐packed structures (fcc and hcp), which might be induced by the arrangement of the stabilizing ligand shell that consists of 16 PnBu3 ligands and six chlorine atoms.Taking a closer look at the structure of 1 seems to be distorted with respect to an ideal metallic arrangement, the observed structure seems to be favorable as 1 is obtained in high yield at low temperature in the dark or under red light. The stability of 1 might have electronic grounds as within 1 an electronic shell closure is realized with respect to the jellium model. Hence, within 1, 58 electrons from the 58 Ag0 atoms are available for the silver core. Since 58 is a jellium number (1S2 1P6 1D10 2S2 1F14 2P6 1G18),1 exhibits a stable closed shell, which might be the reason for the high yield of the synthesis. Although 58 is a so‐called “magic number”, the special structure of the Ag core as a brick‐like arrangement leads to a splitting of the superatomic orbitals, since rather than a spherical a lower symmetric brick‐like background potential is present.1 reveal that the highest occupied orbitals can indeed be described as superatomic orbitals, as shown in Figure Although the arrangement of the silver atoms within 1 is obtained in high yield we found during the various syntheses and attempts at further characterization that 1 is not only sensitive to light, but also quite sensitive to temperature, both in solution and in the solid state. Consequently, the observed temperature sensitivity of the single crystals during X‐ray structure analysis is not or maybe not only due to co‐crystallized solvent molecules, as it is often the case for metalloid clusters. Hence, when single crystals are isolated from the mother liquor under red light at low temperature, dark red crystals are obtained. When those crystals are stored in the dark at room temperature for several days, they become darker ; this does not take place at temperatures below −30 °C where the crystals can be stored without decomposition.1 via NMR or UV/Vis spectroscopy was not possible. Nevertheless, we tried variable temperature (VT) NMR measurements as well as UV/Vis measurements knowing that whatever is observed is maybe not the cluster 1 itself but decomposition products or a mixture of both.1 might be the first example of a model compound that demonstrates the primary chemical steps in photographic processing.Nevertheless, although As previously reported,31P NMR spectrum at −17 ppm (molecule A). Since the cluster is formed upon cooling this solution for at least ten days to −28 °C, we assigned this resonance first to cluster 1. However, NMR measurements of isolated crystals dissolved in deuterated benzene show a fundamental different spectrum . Beside this, the NMR tube of A remains orange‐brown and clear. These observations imply that the crude reaction mixture at room temperature does not contain 1 but another phosphorous‐containing substance. Several attempts to isolate A to get more information about this compound failed.A within the reaction system (side product or intermediate en route to 1) and this aspect is still under investigation. Additionally, we conducted 31P VT‐NMR investigations on single crystals of 1 dissolved in [D8]toluene starting from low temperatures around −60 °C, resulting in broad resonances ! Due to the many modes of decomposition and with respect to the cluster size of 64 silver atoms, this is an extraordinary value.The differences between s Figure . The obt64(PnBu3)16Cl6 (1), which is to our knowledge the first phosphine‐stabilized silver cluster without further protection from other organic or Group 16 based ligands. Within 1, not a spherical but a brick‐shaped arrangement of the Ag atoms is observed, whereby structural relations to cubic (fcc) and hexagonal (hcp) close packing is observed. Additionally, 1 can be considered to be a 58 electron closed shell system, showing low thermal and photochemical stability. Due to the easy synthetic procedure starting from nBu3PAgCl, the high yield, and the good solubility in organic solvents, this cluster is a fruitful starting point for silver deposition, especially regarding its thermal and light sensitivity, which exceeds even that of silver halides.To summarize, we were able to synthesize a rare example of a metalloid Ag cluster, namely AgThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. 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Upon treatment with cytokines, reduced cell viability was observed in the presence of TNF-α and IFN-γ. A significant reduction in insulin secretion was also noted after treatment with TNF-α and IFN-γ and IL1-β. The findings of the present study have shown for the first time that the inhibition of the excitatory effects of cholinergic pathways on glucose-induced insulin secretion may cause β cell injury and dysfunction of insulin secretion in response to cytokine-induced NO production.Dysfunction of the pancreatic β cells leads to several chronic disorders including diabetes mellitus. Several mediators and mechanisms are known to be involved in the regulation of β cell secretory function. In this study, we propose that cytokine-induced nitric oxide (NO) production interacts with cholinergic mechanisms to modulate insulin secretion from pancreatic β cells. Using a rat insulinoma cell line INS-1, we demonstrated that β cell viability decreases significantly in the presence of SNAP (NO donor) in a concentration- and time-dependent manner. Cell viability was also found to be decreased in the presence of a combined treatment of SNAP with SMN (muscarinic receptor antagonist). We then investigated the impact of these findings on insulin secretion and found a significant reduction in glucose uptake by INS-1 cells in the presence of SNAP and SMN as compared with control. Nitric oxide synthase 3 gene expression was found to be significantly reduced in response to combined treatment with SNAP and SMN suggesting an interaction between the cholinergic and nitrergic systems. The analysis of gene and protein expression further pin-pointed the involvement of M Pathophysiology of diabetes mellitus (DM) is highly complex, and multiple factors and pathways are involved. β cell dysfunction is considered to be the main pathological feature of DM, and previous studies have identified several mediators of β cell injury that are associated with DM. However, the mechanisms by which these mediators influence insulin secretion are yet to be identified , 29, 41.For decades, type 1 DM was considered as the sole type of DM that is associated with β cell destruction and subsequent reduction in insulin secretion, whereas type 2 DM was mainly characterized by increased insulin resistance. Nevertheless, several lines of recent evidence have confirmed that β cell loss also plays an important role in the pathogenesis of type 2 DM in the long term. β cell failure and disturbance of insulin secretion affects many organs and leads to dramatic complications including cardiovascular disease, stroke, renal failure, and neuropathy , 34, 44.l-arginine by a family of enzymes known as nitric oxide synthases (NOS). Three distinct isoforms of NOS are known: (i) two isoforms that are constitutively expressed in neurons (nNOS), (ii) endothelial (eNOS) cells, as well as (iii) an inducible isoform (iNOS) which is expressed primarily by immune cells  [It is noteworthy that the mechanisms that underpin β cell failure in the progression of type 1 and type 2 DM differ considerably. In the former, the main process that leads to destruction of the β cell is based on autoimmune-mediated apoptosis, whereas in type 2 DM, the dysfunction of β cells mainly involves circulating cytokines  which indirectly promote apoptosis through excessive production of nitric oxide (NO) . NO is aophages) , 34. Theophages) , 43.2 as well as by reducing cellular levels of ATP [It has been strongly suggested that NO mediates the inhibitory actions of cytokines on β cell function by inhibiting glucose oxidation to COs of ATP , 42. AltInterestingly, our previous findings have speculated that an interaction between NO and cholinergic mediators influences insulin secretion . We haveTaking all these previous findings into account, the present study was designed to investigate the involvement of NO and muscarinic receptors on survival of β cells and the regulation of glucose-induced insulin secretion.l-glutamine, 1 mM sodium pyruvate, and 50 μM β-mercaptoethanol [Rat insulinoma cell line INS-1 832/13, a subclone producing both rat and human insulin, transfected with a CMV promoter carrying a neomycin resistance marker was obtained from Lund University, Sweden, by Dr. Jalal Taneera and was used as our research model. INS-1 832/13 pancreatic cells have been used widely in the field of insulin secretion research. These cells were cultured in RPMI 1640 supplemented with 10% heat inactivated FBS , 100 IU/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 2 mM oethanol .4 cells were seeded per well in 96-well plates  with 200 μl RPMI 1640 culture medium and maintained in the incubator at 37 °C with 5% CO2. Upon reaching > 90% confluency, cells were treated in triplicates with 50 μM, 100 μM, and 200 μM of SNAP (S-nitroso-N-acetylpenicillamine) or LNNA (L-NG-nitro-l-arginine) along with their vehicles 200 μM dimethyl sulfoxide (DMSO) and 200 μM hydrochloric acid (HCl), respectively. Cell viability was also measured in the presence of scopolamine methyl (SMN) at a concentration of 0.2 nM.Measurement of cell viability was as follows: 1 × 104 cells were seeded in 0.1 ml/well culture media in 96-well plate and allowed to adhere overnight. Cells were then incubated for 24 h in the presence of pro-inflammatory cytokines: 125 ng/ml of tumor necrosis factor-alpha , 100 ng/ml of interleukin 1-beta , and 125 ng/ml of interferon-gamma . Cells without any treatment were regarded as controls. After treatment, viability was checked using a colorimetric assay with the tetrazolium dye MTT . Culture medium was discarded and replaced with a mixture containing 20 μl of MTT (5 mg/ml) dissolved in 100 μl of PBS . Cells were then incubated at 37 °C for 2 h in the dark. Post incubation, 100 μl of DMSO was added to each well to dissolve the formazan crystals formed by MTT and absorbance was recorded at 570 nm on a microplate reader with 630 nm wavelength as reference. The percentage of cell viability was calculated from the average 570 nm absorbance value as per the following equation: % cell viability = (OD of sample at 570 nm/OD of control at 570 nm) × 100.Viability of the cells in the presence of cytokines was measured as follows: 2 × 10The concentration of nitrite released by the cells was measured using 50 μl of spent media which was collected from each well after 24 h of treatment with the cytokines—TNF-α (125 ng/ml), IL1-β (10 ng/ml), and IFN-γ (125 ng/ml), respectively. Nitrite concentration was measured using the Greiss Reagent System  as per the manufacturer’s protocol. The absorbance was recorded at 595 nm in a microplate reader using 630 nm wavelength as reference. The concentration of nitrite in each sample was determined using a standard reference curve which was generated to compare the average absorbance value for each sample.5 in 1 ml/well culture media in a 24-well plate  and were allowed to adhere overnight. Cells were then exposed to cytokines—IL-1β (100 ng/ml), TNF-α (125 ng/ml), IFNγ (125 ng/ml), or the nitric oxide donor SNAP (100 μM and 200 μM) or muscarinic receptor antagonist (SMN 0.2 nM) for 24 h. The cells were then washed twice with 1 ml of pre-warmed secretion assay buffer (SAB), pH 7.2  containing 2.8 mM glucose. The cells were then incubated in 2 ml SAB containing 2.8 mM glucose for 2 h. Subsequently, cells were incubated for 1 h in 1 ml SAB containing either 2.8 mM or 16.7 mM glucose. The supernatants were collected and secreted insulin was measured using rat insulin ELISA kit  according to the manufacturer’s instructions. Secreted insulin was normalized to the protein content as determined using Bradford Assay  [Briefly, cells were seeded at density of 3 × 10INS-1 cells were harvested for the isolation of RNA using the PureLink RNA Mini Kit . RNA was isolated following the manufacturer’s protocol with final elution volume of 30 μl. The isolated RNA was then quantified using the Nanodrop2000 spectrophotometer , and purity was determined by the A260/A280 ratio. From this, 0.5 μg RNA was used to reverse transcribe to cDNA with 10 mM dNTP mix and 5 μM random primers  and incubated at 70 °C for 10 min, followed by addition of 200 units of M-MLV reverse transcriptase in a reverse transcriptase buffer and incubated at 37 °C for 50 min and 90 °C for 10 min in a Veriti thermal cycler . The final reaction volume was 20 μl.3), muscarinic acetylcholine receptor 3 (M3), and muscarinic acetylcholine receptor 5 (M5) are listed in Table ΔΔCt method, and rat hypoxanthine guanine phosphoribosyltransferase (HPRT) was used as the house keeping gene. Samples implemented with nuclease free water instead of cDNA were regarded as negative controls. All primer sequences were synthesized using the Primer3 software.The analysis of gene expression was checked, and quantitative real-time PCR was performed using the StepOne Real Time PCR system  in a total reaction volume of 10 μl containing 5 μl of 1× Power SYBR green master mix , 0.5 μl of 10 μM primers  and grown at 37 °C with 5% CO2 till > 90% confluency was reached. Cells were then treated with the desired concentrations of SNAP  and SMN (0.2 nM) depending on the experiments. After 24 h of treatment, cells were harvested for protein extraction using M-PER mammalian protein lysis buffer  containing a protease inhibitor cocktail  in a 1:1000 dilution. The concentration of total protein was determined using the Bradford reagent  and quantified by the Bradford Method [3 , M5 , and β-actin (1:5000 dilution). The membranes were washed with TBST and incubated with horseradish peroxidase–linked secondary antibody  at room temperature for 1 h. After incubation, the membranes were washed with TBST and visualized using clarity western ECL substrate  according to the manufacturer’s protocol. The intensity of the observed bands was quantified using ImageJ software.The following protocol was used for protein analysis: 1.5 × 10d Method . Fifty mAll reagents and solutions were obtained from Sigma-Aldrich  unless mentioned otherwise.P < 0.05 were considered as statistically significant.All data are expressed as mean ± standard error, and differences between individual means were assessed using one-way analysis of variance (ANOVA) followed by the Tukey multiple comparison tests. Probability values of Two separate sets of experiments were conducted to investigate the effects of a NO donor (SNAP) and a NOS inhibitor (LNNA) on the survival rate of INS-1 pancreatic cells. In the first group of experiments, three different doses of the NO donor  were used to test the effects of the NO donor on the survival of INS-1 pancreatic cells. As shown in Fig. On the other hand, the results of the second group of experiments have shown differential responses in the presence of the NO inhibitor, LNNA Fig. . A signiAnother group of experiments was conducted in order to investigate the effects of combined treatment with SNAP and a muscarinic acetylcholine receptor antagonist  on the survival rate of the INS-1 pancreatic cells Fig. . As showAs shown in Fig. The effects of SMN, a mAChR antagonist, on glucose-induced insulin secretion were investigated. Two concentrations of glucose (2.8 mM and 16.7 mM) were used to stimulate insulin secretion, and, as shown in Fig. The effects of three cytokines  on the survival rate of INS-1 pancreatic cells were examined. A significant reduction in the survival of these cells in response to the presence of TNF-α, IFN-γ, and IL1-β for a duration of 6 and 24 h was noted compared with controls Fig. . As show3 expression was noted in the presence of IL1-β and IFN-γ. However, the presence of TNF-α did not change NOS3 expression  and M5 . These effects were attenuated significantly in response to combined treatment with SNAP and SMN.Additional experiments examined the expression of cholinergic receptors in the presence of a NO donor and a muscarinic receptor antagonist. SMN alone produced a significant reduction in the expression of M3 and M5 receptors was assessed in the presence of SNAP alone, SMN alone, and in combination of both. The expression of the two receptors was sensitive to SMN; however, more significant outcomes were reported in the expression of M3. As shown in Fig. 3 significantly and the combination of SNAP and SMN attenuated the expression of M3 receptor in a concentration-dependent manner. Figure 5 receptor; however, the combination of SNAP  and SMN produced significant reduction in the expression of the M5 receptor and no significant changes were observed in response to the highest concentration of SNAP (200 μM). Representative western blot of M3 and M5in response to the presense of SMN and different concentrations of SNAP were shown in Fig. Protein expression of MIt is well-documented that progressive impairment of pancreatic β cells and associated dysfunction of insulin secretion are the original signatures of DM. Although previous studies have attempted to determine the participating elements in β cell pathogenesis, the precise mechanisms are still poorly understood .Previously, we and others have shown that insulin secretion seems to be modulated by brainstem cholinergic pathways and NO. These in vivo experiments have confirmed that the blockade of cholinergic pathways and the inhibition of NO synthesis had inhibitory and excitatory effects on insulin secretion, respectively .Cytokines have been implicated as strong candidates for dysfunction of β cells and, subsequently, the secretion of insulin. It is believed that cytokines, including IL-1β, TNF-α, and IFN-γ, mediate this effect via induction of NO formation. It seems that the latter is involved as a causative agent of most of the inhibitory and cytotoxic effect of the cytokines on the pancreatic islets , 36.The present study is the first to find that there is an interaction between the nitrergic and cholinergic pathways to modulate insulin secretion from the β cells. Interestingly, it was also found that the source of NO is cytokine-dependent and the muscarinic acetylcholine receptors are the mediators of the cholinergic involvement in regulation of insulin secretion.In agreement with the present results, previous reports have shown that SNAP produced adverse effects on the survival of pancreatic β cells . SNAP prIn addition, the present study investigated the effects of NOS inhibitors, using the same range of concentrations and exposures, on the viability of the pancreatic β cells. It was evident that the NO donor produced adverse effects on the survival rate of the pancreatic β cells, whereas the viability of these cells was decreased in response to the presence of NOS inhibitors. Although the effect of the latter was not the focus of the present study, an interesting finding was noted; the positive impact of LNNA on the survival rate of the β cells was only observed after a long duration of exposure. Given that the RPMI media used in the present study contains arginine, a NO inhibitor, may compete with it to produce an increase in the survival of β cells. Therefore, LNNA took a longer time to exhibit a positive impact. In addition, it has been found that β cells constitutively express nNOS and eNOS and this may explain the observed effects of LNNA on the survival of the β cells . The basInterestingly, the inhibitory effects of SNAP on the survival rate were enhanced by the presence of a muscarinic antagonist suggesting an interaction between these two agents in modulating the survival of β cells. A recent study by Gupta et al. has shown that ACh receptors play a vital role in maintaining the mass and survival of pancreatic β cells. Similar to the present study, Gupta and his group used the INS-1 832/13 pancreatic β cell line. Therefore, it provides strong support and validation to our present results .It is well established that ACh is a neurotransmitter that is involved in the regulation of secretory function of the insulin secreting pancreatic β cell . However3 were both increased in the presence of cytokines, mainly IL1β, suggesting that the adverse effects of IL1β are mediated via induction of NO production. It is noteworthy that the production of NO and expression of NOS3 were not significant in response to TNF-α. Previous studies have demonstrated that the latter alone does not affect NO production significantly. However, in the presence of IFN-γ, large amounts of NO were produced by IL1-β [The negative impact of cytokines on the function and the survival of pancreatic β cells have been demonstrated, and this further supported by our current findings , 31. We by IL1-β . In agreby IL1-β . In agreby IL1-β .Recently, several biochemical and molecular studies have proposed various mechanisms to explain the inhibitory effects of NO on the survival and secretory function of β cells. This includes targeting iron-sulfur–containing enzymes , reducti3 was increased in response to the presence of a NO donor, and this effect was concentration-dependent suggesting that the inhibitory effects of NO donor on survival and secretory function of β cells may be partially mediated by increased levels of NOS3. Although the involvement of different NOS isoforms in regulation of insulin secretion is controversial, several reports have demonstrated that NOS3 and NOS1 are constitutively expressed in pancreatic vasculature and neurons, respectively [3 seems to play a major role in modulation of pancreatic secretion and has been detected in rat and human pancreatic islets; hence, the present study focused on the involvement of NOS3 in this process [The expression of NOSectively . Compare process .3 and the excitatory effects of the NO donor on expression of NOS3 suggesting that the inhibitory effects of ACh receptor blockade can override the excitatory effects of NO on expression of NOS3. Therefore, further experimentation was conducted to investigate the nature of this interaction.In contrast, mAChR blockade alone attenuated the expression of NOS3 and M5 mAChRs significantly, and this may explain the mechanism of action by which these agents modulate insulin secretion. It is noteworthy that protein expression analysis showed that M3 mAChR protein expression was more sensitive to the mAChR blockade compared with M5 expression. The involvement of M3 and M5 mAChRs is supported by previous reports that stimulation of insulin secretion is mediated by activation of M3 and M5 mAChRs in different β cell lines including rodents and human [Not surprisingly, a mAChR antagonist decreased the expression of Mnd human , 28.3 and M5 mAChRs, and this effect was attenuated in the presence of mAChR antagonist. Although the coupling relationship between NOS and mAChR has been reported previously for regulation of amylase secretion from the salivary gland, the present study is the first to demonstrate this phenomenon in modulation of insulin secretion [2+ mobilization which is activated by muscarinic receptors plays an important role in NO signaling pathways [NO donor seems to increase the expression of Mecretion . Severalecretion . In addipathways . Furtherpathways .3 and M5 mAChRs was reported in the presence of the NO donor. However, two key factors were considered to explain this controversy: (i) NO has a dual role in regulation of β cell survival and insulin secretion; both negative and positive effects and anti- and pro-apoptotic activities have been reported based on NOS isoforms and NO concentrations, and (ii) the interaction between the cholinergic and nitrergic systems seems to be dependent on the nature of the pancreatic tissues, and some of the coupling mechanisms between these two systems are confined to neuronal pancreatic tissue [On the other hand, one can argue that the NO donor had adverse effects on insulin secretion and the survival of β cells and yet an increase in the expression of Mc tissue , 20.3 in response to cytokines. In addition, this study has demonstrated an interaction between the mAChRs and NO in modulation of the survival of β cells and insulin secretion. Although the present findings are interesting, further studies need to be conducted to investigate the involvement of different muscarinic receptor subtypes, and therefore, selective cholinergic blockers will be used. Moreover, future studies will test the effects of muscarinic agonists and, if they are able to reverse the impairments that are caused by SNAP.The findings of the present study have shown that a NO donor, muscarinic acetylcholine receptor blockade, and cytokines produced adverse effects on survival rate of β cells and insulin secretion. The effect of cytokines seems to be mediated via production of NO, and this was evident by the increase in NO production and gene expression of NOS