Pancreatic carcinoma (PC) is a highly lethal cancer with an increasing mortality rate, its five‐year survival rate is only approximately 4%. N6‐methyladenosine (m6A) modification is the most common posttranscriptional modification of RNA, it could affect tumor formation by regulating m6A modifications in the mRNA of key oncogenes or tumor suppressor genes. However, its role in PC remains unclear.We combined bioinformatic analysis with in vitro and in vivo experiments to investigate the expression profile of methylation modulators and identify key m6A regulators in the progression of PC. Further study focused on exploring the target genes binding to the regulators through RIP and immunofluorescence staining experiment.TCGA and Gene Expression Omnibus (GEO) analyses revealed an overall increasing trend in the expression of m6A regulators in PC, and consensus clustering analysis of m6A modification showed that the expression of regulators was negatively correlated with the survival rate. LASSO‐Cox regression analysis revealed that IGF2BP2, METTL3, ALKBH5 and KIAA1429 were associated with hazard ratios (HR), but only IGF2BP2 was sufficiently appropriate for the m6A survival prognosis model. The IHC and WB results verified high protein expression of IGF2BP2 in PC, and IGF2BP2 knockdown inhibited the proliferation and migration of PC cells. We predicted and verified B3GNT6 was observably regulated by IGF2BP2 via RIP assays. In addition, IF staining confirmed the co‐expression of IGF2BP2 and B3GNT6. The tumor‐promoting effect of IGF2BP2 and its co‐expression with B3GNT6 were verified in an animal model.Elevated m6A levels promote PC progression. IGF2BP2 is a credible marker and modulates B3GNT6 mRNA stability, indicating that IGF2BP2 is a potential prognostic marker and therapeutic target in PC progression. 1. Bioinformatics reveals that m6A regulators are generally elevated in PC. 2. IGF2BP2 enhances the stability of B3GNT6 mRNA via m6A methylation. 3. IGF2BP2 plays a carcinogenic role, promoting tumor formation in vivo.  Also, RNA sequencing data was downloaded in GEO to measure the expression of regulators in PC. Patients with rational survival data and clinicopathological characteristics were included in the analysis. For group analysis, the median value was used as the cut‐off site.2.2p < 0.05 were used as the cut‐off values for DEGs. KEGG and GO enrichment analyses were performed with the above DEGs. The risk signature was calculated using the LASSO‐Cox regression algorithm according to the minimum criteria. The risk score was calculated using the formula, where Coefi is the coefficient:We employed the R software package ConsensusClusterPlus to divide 176 PC patients into two subtypes (PA1 and PA2) according to the expression of the regulators and used principal component analysis (PCA) to evaluate the difference in the gene expression distribution between the two clusters. Additionally, we used the R software package limma to conduct differential gene expression analysis on the subgroups, where fold change (FC) > 2 and 2.3To predict and verify the possible downstream targets regulated by IGF2BP2, we analyzed and compared the differential genes between the two subgroups (PA1 and PA2), and the differential genes in PC. In addition, the CLIP dataset in m6A2Target database was used to filter the downstream mRNAs of IGF2BP2, the possible targets of IGF2BP2 were then obtained by intersection, and the follow‐up experiments were carried out to verify it.2.4PC together with counterpart adjacent normal tissue samples were obtained from department of General Surgery, the First Affiliated Hospital of Soochow University. The research was ratified by the Ethical Committee of the first affiliated hospital of Soochow University and written informed consent were successively obtained from all participants before the study.2.52 atmosphere at 37°C. All cells were free of bacterial and mycoplasma contamination.Human CFPAC1 and PANC1 PC cells were purchased from GenePharma Technology Co., Ltd.  and used in our research. These cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum (FBS) in an incubator with a 5% CO2.6The target mRNA interference fragments and the NC construct si‐NC were synthesized  and co‐transfected with Lipofectamine 2000 reagent  to knock down gene expression in PC cells; their sequences are shown in Table 2.7We used an EpiQuik m6A RNA Methylation Quantification Kit (colorimetric)  to quantify the overall m6A methylation level in PC cells. First, we extracted total RNA and approximately 200 ng RNA was separated as an initial input. Then, the sample RNA, standard positive/negative control and binding solution were bound to the wells respectively for 1 h to assay and capture RNA according to the manufacturer's instruction. After washing, the detection antibody and enhancer solution were added, and color developer solution and stop solution was added prior to measurement of the absorbance. The values were calculated using linear regression equations.2.8Total RNA was extracted with TRIzol from PC cells and tissues. All primers used for qRT‐PCR were designed and blasted in the National Center for Biotechnology Information database. RNA was reverse transcribed to cDNA and PCR was then performed using SYBR Green qPCR Master Mix  in triplicate. Human β‐actin was used as the internal control for mRNA expression. The primer sequences are shown in Table 2.93 cells per well. After cultured for 0, 24, 48, 72 and 96 h under the same conditions, PC cells were incubated with diluted CCK‐8 reagent following the instructions. Then, the absorbance was measured in a microplate reader at a wavelength of 450 nm.CCK‐8 kit  was used to detect the proliferation ability of PC cells. Transfected cells were seeded in 96‐well plates at a concentration of 5 × 102.10An EdU kit  was used to detect the cell proliferation capability. In brief, after stable transfection for 48 h, cells were incubated with 5 μM EdU reagent diluted with DMEM for 3 h. Then, cells were permeabilized with 0.5% Triton X‐100 for 20 minutes prior to fixation with 4% paraformaldehyde for 30 minutes. Apollo and DAPI dyes were used to stain DNA and then nuclei. Images of EdU‐ and DAPI‐positive cells were acquired under a fluorescence microscope.2.11To verify colony formation ability, transfected cells were seeded into 6‐well plates at a density of 5000 cells per well and distributed evenly by pipetting. After 14 days, the cells were washed with PBS twice, fixed with 4% paraformaldehyde for 30 min and stained with 1% crystal violet for 30 min. Eventually, the cells were imaged and the colonies were counted.2.12To evaluate the cell migration ability, cells were cultured in serum‐free medium in the upper chamber of a Transwell plate , and 700 μl of DMEM containing 10% FBS was added to the lower chamber. After 48 h, the cells were fixed and stained with crystal violet for 20 min, and the cells migrating through the upper chamber membrane were counted. Images were acquired by microscopy.2.13The B3GNT6 fragment containing the possible m6A modification site and the corresponding mutated fragment were synthesized and cloned into the pmirGLO vector  named B3GNT6‐WT and B3GNT6‐Mut. The synthesized vectors were separately co‐transfected with IGF2BP2‐si or si‐NC into 293 T cells. After co‐incubation for 24 h, cells were collected, and luciferase activities were measured with a Dual Luciferase Reporter Assay kit .2.147 treated cells were collected and lysed for 30 min. DNA impurities were removed with DNase, the supernatant was collected after centrifugation, and the supernatant was then incubated with anti‐m6A and anti‐IGF2BP2 primary antibodies overnight at 4°C in a vertical orientation. Then, magnetic beads were added to incubate for 1 h. Finally, RNA was extracted, and target gene expression was detected by qRT‐PCR.Immunoprecipitation assays of m6A and IG2BP2 were performed with an RIP kit . In brief, approximately 1 × 102.15The overall methylation inhibitor 3‐DAA  was used to inhibit RNA methylation levels. After inoculation, cells were treated with 10 ug/ml 3‐DAA for 48 hours, then cells were collected for protein analysis.2.16Paraffin sections of 5 paired clinical pancreatic specimens were prepared and baked in an oven at 65°C for 30 min. After routine dewaxing, hydration and antigen repair, sections were sealed with 5% goat serum for 30 min, placed in a wet box and incubated overnight with the IGF2BP2 primary antibody  at 4°C. The next day, sections were incubated with the secondary antibody for 30 min, incubated with DAB for color development for 3 min and stained with hematoxylin for 3 min. Sections were sealed with neutral gum, and images were acquired with a microscope.2.17After dewaxing, hydration and antigen repair, paraffin sections were blocked with immunostaining blocking solution for 1 h, incubated with IGF2BP2 and B3GNT6  primary antibody at 4°C overnight and secondary antibody for 1 h. After counterstaining with DAPI, sections were sealed with an anti‐fluorescence quenching agent and imaged under a fluorescence microscope. Treated PC cells were prepared as cell slides and subsequent experiments were performed as described above.2.18We used a RIPA mixture containing 1% PMSF lysate to lyse and obtain total protein from transfected cells. A BCA protein detection kit  was used to determine the protein concentration. Protein separation under 80 V, 30 min and 120 V, 1 h electrophoresis conditions, followed by transferring to polyvinylidene difluoride membranes at 200 mA for 1.5 h. Then, membranes were blocked with 5% skim milk and incubated with IGF2BP2 primary antibody  and B3GNT6  primary antibody overnight at 4°C. After washing, immunoreactions were visualized with an electroluminescence detection system.2.196 cells were injected into the axilla of nude mice for subcutaneous tumor formation. After 9 days, modified IGF2BP2‐si or si‐NC was injected into the tumors every 3 days for 3 weeks. Tumor size was measured every 3 days. Finally, the subcutaneous tumors were excised and weighed to compare the tumor sizes, and protein expression was detected by immunohistochemistry and immunofluorescence. The care of laboratory animals was in accordance with the guidelines and ethical requirements of the Laboratory Animal Centre of Soochow University.To evaluate the effect of IGF2BP2 on the tumor formation ability in vivo, 2 × 102.20p < 0.05 (*p < 0.05).We used GraphPad 8.0 for charting. For statistical analysis, two‐tailed Student's t‐test between two groups were performed by SPSS Statistic 26 for windows. The Kaplan–Meier method was used for survival analysis. Data was reported as mean ± SD from three duplicate experiments. Results was considered statistically significant when 33.1Considering that methylation regulators perform different biological roles, we first analyzed the expression of 15 regulators in PC and found that compared with normal pancreatic expression profiles in the GTEx database, the regulators were generally upregulated in TCGA, it was also observed in the GEO database (GSE15471) Figure . Spearma3.2To visualize the specific functions of m6A methylation regulators, we removed normal pancreatic samples and PC samples with no suitable clinical data, and used the ConsensusClusterPlus R package to perform consensus clustering. According to the expression similarity of the regulators, the cumulative distribution function (CDF) of our data seemed to be ideal when K = 2, and the PA1 (96 samples) and PA2 (80 samples) subgroups were then defined Figure . We compp ≤ 0.05. Finally, 2681 genes were identified, of which 724 genes were upregulated in PA1, and 1957 genes were upregulated in PA2. KEGG and GO enrichment analyses showed that the DEGs were mainly enriched in homeostasis or regulation of membrane potential  and lymph node metastasis (p < 0.05) were highly significant , IGF2BP3 (HR = 1.53), METTL3 (HR = 0.53), ALKBH5 (HR = 0.6) and KIAA1429 (HR = 1.55) Figure . Survivat Figure . In summ3.4TCGA and IHC analysis revealed high expression of IGF2BP2 in PC Figure . What's 3.5For further investigation of the biological function of IGF2BP2 in PC, we knocked down its expression in CFPAC1 and PANC1 cells Figure  and quan3.6As an RNA binding protein, IGF2BP2 has been proven to bind to a variety of RNAs to regulate their expression. To identify potential target genes of IGF2BP2, we analyzed IGF2BP2 CLIP data and identified 6249 targets with relatively high associativity. Subsequently, PC patients were divided into high IGF2BP2 expression and low IGF2BP2 expression group, and differential gene expression analysis was conducted between the groups. In TCGA, we identified 8335 DEGs between PC and normal tissue, among which nine were common highly reliable genes Figure . qRT‐PCR3.7We first knocked down B3GNT6 in cells, CCK‐8 and EdU incorporation assays indicated that silencing B3GNT6 overtly inhibited the proliferation of PC cells Figure . In addi3.8To further identify the underlying mechanism of IGF2BP2‐mediated B3GNT6 expression, we sought to determine whether and how IGF2BP2 interacts with B3GNT6 to affect its expression. We knockdown IGF2BP2 and RIP showed lower B3GNT6 was enriched Figure . Dual luConsidering that IGF2BPs could regulate RNA stability, we sought to evaluate B3GNT6 mRNA stability. PANC1 cells were transfected with IGF2BP2‐si or the si‐NC, then treated with actinomycin D  at 10 ng/ml at different time point . As shown, B3GNT6 mRNA decay rate in the IGF2BP2‐siRNA group was faster than that in the si‐NC group Figure , IGF2BP23.9Subcutaneous tumorigenesis experiments in mice showed that knockdown of IGF2BP2 contributed to a decreased tumor size Figure  and tumo4We conducted a comprehensive expression profiling analysis of 15 methylation regulators and found overall high expression in PC samples. We divided PC samples into two subgroups and performed differential gene expression analysis. Then, we used LASSO‐Cox regression analysis to construct a methylation model and obtained risk score, finding that the survival rate in the high‐risk group was significantly lower than that in the low‐risk group. Finally, we identified the potential m6A regulator IGF2BP2 and downstream target gene B3GNT6. Subsequent in vivo and in vitro experiments were performed to verify the gene methylation modification. IGF2BP2 was proved to modulate cellular biological function through post‐transcriptional regulation and participate in the development and progression of cancers in an m6A manner, such as head and neck squamous carcinoma,The mortality of PC varies greatly in different regions. In addition, due to late detection and the scarcity of effective treatments, the survival rate is generally low.m6A modifies and regulates different aspects of mRNA, including its structure, stability, splicing, nuclear export, translation, decay, etc., and is also involved in cell fate determination, cell cycle regulation, and cell differentiation. Among the regulators, m6A writers mainly increase the level of methylation on RNA, which is the key step in m6A modification.To date, m6A methylation has been found to function as a carcinogenic or tumor‐suppressive mechanism in glioblastoma,B3GNT6 was identified as IGF2BP2 target in an m6A manner, and our research verified its oncogenic role in PC, considering that B3GNT6 is an essential enzyme for the synthesis of core 3 O‐glycanIn theory, the reader, eraser and writer should have different expression patterns due to their different functions. However, our research found that the expression patterns of most regulators, whether promoting or inhibiting methylation, tended to be the same. We speculated that on one hand, there may be a proportional relationship. For example, the ratio of methylation/demethylation regulators exceeding a certain range will promote cancer or suppress cancer. On the other hand, different regulators have different affinities for the same gene and contributions to abnormal downstream activities caused by m6A methylation.5In summary, we have confirmed the elevated m6A level and revealed the significant effect of RNA methylation in PC. Besides we authenticate a novel gene IGF2BP2 is a credible marker and upregulated in PC, PCR and WB indicate that it promotes the B3GNT6 mRNA stability to contribute to the deterioration of PC, indicating that IGF2BP2 is a potential prognostic marker and therapeutic target in PC progression. Hence, our study provides some evidence for research of RNA methylation in PC.6In this study, we first verified that m6A modification features play an important role in the development of PC through bioinformatics analysis. Combined with experiments, it was verified that IGF2BP2 can regulate B3GNT6 stability through m6A and may regulate the metabolic pathway of PC. However the deficiency is that we only carried out cell function experiments on B3GNT6 and no follow‐up pathway analysis. Clinically, only IHC analysis of specimens was performed. Due to insufficient number of specimens and insufficient follow‐up time, clinical research has not been carried out in depth. we will further expand the sample size for verification.Xiaofeng Xue and Lei Qin contributed to the conception and design of the article. Pei Cao, Ding Sun and Weigang Zhang contributed to the acquisition of the data. Pei Cao and Yufan Wu contributed to the implementation of the experiment. Zuxiong Tang, Junyi Qiu and Ding Sun contributed to the acquisition of specimens. Pei Cao, Yufan Wu and Xiaofeng Xue contributed to the bioinformatics analysis. Xiaofeng Xue, Pei Cao and Junyi Qiu contributed to the analysis and interpretation of the data. Xiaofeng Xue, Lei Qin and Pei Cao contributed to the writing, review, and revision of the paper.This research was supported by the Youth Science Foundation of National Natural Science Foundation of China (No. 81802365).The authors declare no competing financial interests.All animal experiments were performed according to the guide for the Laboratory Animal Centre of Soochow University. The care of laboratory animals was in accordance with the guidelines and ethical requirements of the Laboratory Animal Centre of Soochow University.All authors have read and approved the final manuscript.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Table S1Click here for additional data file.


Heterotrimeric G proteins (αβγ subunits) that are activated by G protein-coupled receptors (GPCRs) mediate the biological responses of eukaryotic cells to extracellular signals. The α subunits and the tightly bound βγ subunit complex of G proteins have been extensively studied and shown to control the activity of effector molecules. In contrast, the potential roles of the large family of γ subunits have been less studied. In this review, we focus on present knowledge about these proteins. Induced loss of individual γ subunit types in animal and plant models result in strikingly distinct phenotypes indicating that γ subtypes play important and specific roles. Consistent with these findings, downregulation or upregulation of particular γ subunit types result in various types of cancers. Clues about the mechanistic basis of γ subunit function have emerged from imaging the dynamic behavior of G protein subunits in living cells. This shows that in the basal state, G proteins are not constrained to the plasma membrane but shuttle between membranes and on receptor activation βγ complexes translocate reversibly to internal membranes. The translocation kinetics of βγ complexes varies widely and is determined by the membrane affinity of the associated γ subtype. On translocating, some βγ complexes act on effectors in internal membranes. The variation in translocation kinetics determines differential sensitivity and adaptation of cells to external signals. Membrane affinity of γ subunits is thus a parsimonious and elegant mechanism that controls information flow to internal cell membranes while modulating signaling responses. Activation of the G proteins results in the α subunit exchanging GDP for GTP and the dissociation of a tightly associated βγ complex. Both the α-GTP and the βγ complex are independently capable of modulating the activity of effectors. The α subunits of G proteins are GTPase switches that are active in the GTP-bound form and deactivated in the GDP bound form . α and βchannels , adenylychannels , phosphochannels , GPCR kichannels , and phochannels .Recent reviews have focused on various aspects of the α and βγ subunits , 10, 11.Arabidopsis thaliana, an atypical γ subunit has been identified with a primary structure that is distinctly different from all other γ subunits .G proteins associate with membranes due to lipid modifications—myristoyl and/or palmitoyl on the α subunit and prenyl on the γ subunit . The shuThe shorter 15-carbon farnesyl lipid has lower hydrophobicity and thus affinity for membranes compared to the 20-carbon geranylgeranyl moiety. Live cell imaging of C-terminal mutants of γ subunits, measuring the dissociation of prenylated fluorescent peptides corresponding to the γ subunit C terminus and mathematical modeling showed that apart from the prenyl moieties, variations in a set of hydrophobic and basic residues at the C terminus of the γ subunits determine differential membrane affinity among γ subunits and consequently translocation properties  92). Th. Th92). A phylogenetic tree of the C-terminal domain residues starting from a conserved AsnPro in the γ subunits shows that 12 γ subunits can be classified into three different subgroups based on the physicochemical properties of amino acids in this sequence. The sequence properties are reflected in the translocation behavior of the γ subunits that are part of a βγ complex as seen in their grouping based on translocation rates .Table 5PWhy do translocated subunits accumulate in internal membranes? Do they translocate to specific organelles? How does a lipidated protein complex traverse through the cytosol?It has been shown using different methods that the shuttling of the heterotrimeric G protein between the plasma membrane and internal membranes is likely diffusive . TransloC). In the case of slow translocating βγ subunits, the proportion found in internal membranes would be less (C). The experimental behavior of the different βγ subtypes does reflect this simple model (When a significant proportion of βγ is released rapidly from the plasma membrane as in the case of rapidly translocating subunits, it can explore the surfaces of internal membranes that have a 10- to 20-fold higher surface area compared to the plasma membrane. At steady state, even if affinities are similar for the plasma membrane and internal membranes, the internal will have bound a higher concentration of fast translocating βγ subunits than the plasma membrane C. In the be less C. The exle model .Figure 4There are some differences between the targeting of the βγ complex to different intracellular organelles dependent on the γ subtype. Most translocated βγ complexes predominantly translocate to the Golgi while γ13 translocates mainly to the ER. A recent report that all γ subunits translocate to multiple internal membranes has been performed with a high affinity bait targeted to these membranes which may bias the distribution toward such ubiquity .Future studies to more clearly establish the target of translocating subunits need to be performed at higher resolution where membrane targeting is solely determined by the intrinsic properties of the membranes and γ subunit types in their native state.1/2 of milliseconds. It also indicates that any additional steps would have slowed down this translocation significantly compared to plain diffusion so the cytosolic transit of the βγ complex is unlikely to be aided by a binding protein that masks the prenyl moiety as in other GTP binding proteins like Ras, Rab, and Rho (A question that remains is how a lipid modified protein like the γ subunit is able to diffuse through the cytosol when translocation occurs. In a HeLa cell, the approximate distance from the plasma membrane to the Golgi is 20 μm. Based on calculations, a fluorescent protein–tagged βγ complex will take 9.5 s for the root mean square of this displacement . The exp and Rho , 101. It and Rho . For thiA cell often encounters stimuli localized to one portion of it. What is the effect of inducing βγ complex translocation in a part of a cell? G protein signaling at the cell surface is known to induce completely different effects compared to that at inner membranes. For instance, β-adrenergic receptor and G protein signaling at the cell surface is cardioprotective, while deeper membrane signaling causes cardiac hypertrophy and cardiomyocyte apoptosis , 104. AtAlthough the conventional view of GPCR-mediated G protein signaling was that it was restricted to the plasma membrane, more recent evidence suggests that G proteins are present and function at intracellular locations such as the ER, nucleus, and Golgi complex , 98, 105via a PKD- and PLCβ-mediated pathway (When the potential role of βγ complex translocation in internal membranes was examined, it was found that rapidly translocating βγ11 subunits were capable of inducing Golgi vesiculation . In cont pathway  and Golg pathway  it had e pathway .Similarly, subtype-dependent βγ complex translocation to the Golgi complex regulates the ERK pathway and cancer metastasis through PI3Kγ activation . KnockouThe differences in the kinetics of βγ complex translocation is retained regardless of the receptor, heterotrimer or cell type. These differences can govern signaling properties such as adaptation and sensitivity. Compared to a slow translocating βγ complex, a fast translocating βγ complex can help a cell adapt to a signal or protect the cell from the deleterious effects of overactivation of a receptor by rapidly depleting βγ subunits at the plasma membrane. Similarly, in contrast to a fast translocating βγ complex, a slow translocating βγ complex will result in a higher concentration of G protein heterotrimer available for receptor activation at the plasma membrane and lead to higher sensitivity to a signal. Disparate evidence now suggest that differential translocation kinetics does indeed play such roles.When mammalian cells expressing a slow translocating γ subunit were compared to cells expressing a fast translocating γ subunit for their PLCβ response to increasing doses of a muscarinic receptor agonist, cells with the slow translocating subunit responded at much lower concentrations . A similWhen adrenergic receptor–induced calcium oscillations in human cells was examined, wide variation in the frequency and the duration of oscillations was observed in a population of cells . When a The fast translocating γ1 subunit similarly helps rod photoreceptors adapt to light by translocating away from rhodopsin . AdaptatWhen migratory macrophage cells and largely sedentary HeLa cells were compared, the macrophage cells were found to express high levels of slow translocating γ subunits while HeLa cells expressed subunits that translocate relatively faster . MacrophTo determine how widely such modulation of signaling occurs in cells, studies that focus on quantitatively measuring the translocation, signal input and output simultaneously in real time will be required. Optogenetic methods and live cell imaging are well suited to explore this question further.Little is known about the potential interaction between α subunits and the translocated βγ complex in internal membranes. Results so far suggest that the translocated βγ complex in internal membranes is free and not bound to an α subunit because it is able to reverse translocate to the plasma membrane as soon as the receptor is deactivated. It would also be of interest to address the following questions. Does free αs.GTP and βγ complex that have translocated to internal membranes act synergistically on the same signaling pathway? Do they form a heterotrimer which is activated by a receptor in internal membranes? Do GPCRs/G proteins in internal membranes modulate the activity of effectors that the translocated βγ complex is regulating?The normal cellular functions in biological systems such as cardiovascular, nervous, and endocrine are maintained by networks of signaling pathways. Signaling activity is modulated by a series of activation and deactivation events. These events result from intrinsic catalytic activity as in the case of the G protein α subunit and Ras family GTPase switches; posttranslational phosphorylation and dephosphorylation in the case of protein kinases and phosphatases; and second messenger generation in the case of enzymes like adenylyl cyclase and PLCβ or conformational changes as in the case of GPCRs. In contrast, the mechanistic basis of the role that the γ subunits play in modulating signaling is unique. Investigations thus far strongly suggest that the βγ complex–mediated regulation of signaling is primarily determined by the differential affinity that various γ subunit types have for cellular membranes. A small number of residues at the C terminus of the γ subunits determine these differences in affinity and are conserved across species, suggesting that evolutionary pressure was exerted in the case of these proteins on lipid–protein rather than protein–protein interaction.The potential roles of γ subunits remained relatively less explored because the more obvious functions of α subunits and β subunits attracted considerable attention. Earlier studies that indicated that the γ subunit interacts with a receptor and that this interaction is essential for G protein activation require support from structural data. In the future as methods to capture the transient states of quaternary complexes like receptor–G protein are refined, it is possible that this question will be resolved.The ability to observe the dynamic properties of signaling proteins in intact living cells has revealed some unexpected roles that this family of small proteins play in signaling. The translocation of βγ complexes allows extracellular signals to act on effectors within the cell. Their differential affinity allows the βγ complex to sustain receptor stimulated effector activity or terminate it rapidly. The γ subunit types can thus determine the sensitivity and intensity of a response as well as adaptation or protection of a cell from an overactivated receptor.Although G proteins have been studied for decades these roles have begun to emerge only recently. As laboratories with varied technical expertise begin to probe the roles of these subunits, advances are likely to be rapid. Methods such as time-resolved crystallography and solid-state NMR can provide structural information about the C-terminal region of the γ subunit and its potential role in receptor activation of a G protein. Time-resolved crystallography can show how lipidated proteins transition through discrete series of conformations with nanosecond or shorter lifetimes . SimilarTo obtain more definitive information about the behavior of G protein subunits in a living cell, live-cell super resolution imaging approaches such as super-resolved structured illumination microscopy  and supeKnock-in incorporation of an 11-reside peptide HiBiT by using a single-stranded oligo template  will allThese methods will help address a number of remaining questions that will help us understand the role of the G protein γ subunits more comprehensively. What are the potential effectors regulated by βγ complexes that translocate to various internal membranes? Does the differential affinity of γ subunit types for membranes play a role in the duration and intensity of signaling at internal membranes? What is the impact of selective expression of γ subunit types on GPCR signaling in various cell types, tissues, and organs? The N-terminal domain of the γ subunits is highly variable within the family but conserved across species . Does thReceptor-driven βγ complex translocation provides an assay that can be used to screen drugs specific to GPCRs. This assay is useful because all receptor types and all G proteins on activation induce βγ complex translocation. It is reversible so it can be used to identify antagonists which are often the drugs of therapeutic value. The development of optogenetic methods to examine GPCR signaling in live cells was made possible only because of this assay , 126. ImOne of the most important areas for future studies is the role of individual γ subtypes in cancer. The evidence thus far for such a role is compelling. Regardless of whether the effects of the misregulated γ subunit types are direct or indirect through a secondary effect on the expression of other components of a signaling pathway, the ability to use the γ subunits as targets for therapy or as markers to predict disease progression are attractive and will need be pursued.This family of small proteins has attracted less attention and its role largely unnoticed because its functions are dependent on dynamic behavior that can be detected only in living cells which requires fairly sophisticated imaging techniques. Findings so far suggest that future studies may establish a central role for these proteins in normal and pathological signaling.The authors declare that they have no conflicts of interest with the contents of this article.


The detector radiation tolerance is also assessed up to a total of 1.7 × 1012 protons impinging on the beam spot area, with a maximum variation of the detector's response of 14%.This study reports on a novel, flexible, proton beam detector based on mixed 3D–2D perovskite films deposited by solution onto thin plastic foils. The 3D–2D mixture allows to obtain micrometer‐thick and highly uniform films that constitute the detector's active layer. The devices demonstrate excellent flexibility with stable electric transport properties down to a bending radius of 3.1 mm. The detector is characterized under a 5 MeV proton beam with fluxes in the range [4.5 × 10 The direct detection of 5 MeV protons by flexible devices based on 3D–2D mixed perovskite is reported here. The mixture allows to directly deposit onto polymeric foils micrometer‐thick and highly uniform films that constitute the detector's active layer. This sensor can detect the dose of absorbed protons producing an electrical output signal. MOSFETs, demonstrated their reliability as real‐time dosimeters, but their scaling up to cover useful areas requires complex and expensive fabrication procedures. Plastic scintillators and scintillating fibres are also used for proton beam diagnostics and real‐time dosimetry, but they need to be coupled to photomultiplier tubes or silicon photodiodes with a suitable readout chain to enhance the light sensitivity and obtain a reliable detection. Moreover, complex calibration procedures are generally required and sometimes the simultaneous detection with different scintillators is needed for achieving accurate measurements. Finally, mechanical flexibility is a relevant requirement unsolved by detectors currently available on the market.The monitoring of proton beam's flux, energy and position is of utmost importance in various fields, such as beam control during fundamental physics experiment and for personal dosimetry during hadron therapy treatments, the cutting‐edge medical tool for cancer therapy.1 Sili and alpha particles, and more recently for fast and thermal neutrons. The direct proton beam detection or dose‐monitoring by perovskite based devices has not been explored yet and only one paper has been published so far on direct proton detection, implemented by fully organic flexible devices.Research on innovative materials for the detection of ionizing radiation has rapidly grown in the last ten years, focusing on the classes of materials that allow overcoming the main constraints of traditional detectors, i.e., their mechanical stiffness and difficulty to implement them into large‐area pixelated detector matrixes at limited costs. Organic semiconductors and perovskites share the property of being processable from solution by low‐cost and low‐temperature deposition techniques, addressing the challenge of covering large areas at affordable costs on thin flexible plastic substrates. Both these materials have recently demonstrated excellent direct detection performances for high energy photons5, 6 We recently demonstrated how 2D (PEA)2PbBr4 (PEA = C6H5C2H4NH3+) perovskite films can be employed as active layer for flexible X‐ray direct detectors with high performance. By adding the 3D phase (MAPbBr3) we here aim to enhance the radiation absorption of protons by the perovskite film, thanks to the higher density of MAPbBr3 and to the higher thickness of the active layer for mixed compounds.In this work, we propose a novel flexible proton detector based on mixed 3D and 2D perovskites films deposited from solution. Mixed 3D–2D perovskites are formed by mixing 3D (based on methylammonium (MA) cations) and 2D (based on larger organic ammonium (OA) cations) structure perovskites. Their employment has been reported as an effective strategy to retain the exceptional transport properties of 3D perovskites and the high stability induced by the layered structure of 2D perovskites.14 We The here proposed devices demonstrate an accurate monitoring of proton dose with instant feedback and low limit of detection, and provide a stable response even after hard and long‐lasting proton irradiation. They also show stable transport properties under bending and fatigue tests down to curvature radius of 3.1 mm. The presented results provide an effective solution to the challenge of identifying novel functional materials and portable devices for real‐time accurate monitoring of proton dose, addressing the quest for low‐cost scalability over large areas and mechanical flexibility, still unsolved for a range of application which span from personal dosimetry to large area and lightweight detectors for large accelerators facilities and space missions.22.13 (3D) and (PEA)2PbBr4 (2D) perovskite crystalline structure 2PbBr4 as well as those of the PET substrate. However, due to the presence of (PEA)2PbBr4, the perovskite films show an ordered layer structure in out‐of‐plane direction, with poor long‐range order in the a and b directions , which spontaneously crystallize within the film. The MAPbBr3 microcrystals are embedded in a continuous layer of (PEA)2PbBr4. Thanks to the presence of (PEA)2PbBr4 phase, mixed 3D–2D perovskite films result about twofold thicker than films with MAPbBr3 microcrystals only, i.e., an average thickness of 4 µm is estimated from AFM profile  (PET) foil with prepatterned Cr/Au interdigitated electrodes by lithographic techniques . As known from literature, mixed 3D–2D perovskites have both MAPbBrre Figure1A H+ s−1 cm−2, is tuned by changing the proton beam current between 0.01 pA and 49 pA. A rotating chopper, intercepting the beam between the extraction window and the sample, is used to monitor and measure such low proton current values. The experimental setup used during the proton detection tests is reported in Figure , by passing through several layers between the beam exit point and the perovskite detector, such as 200 nm of Si3N4 for the beam extraction window, 8 mm of mixed air‐He (50–50%) atmosphere in the gap between the extraction window and the metal box containing the sample, 14 µm of Al foil for the entrance window of the box, and 14 mm of air inside the box. The linear energy transfer (LET) released by each proton inside the 4 µm thick mixed 3D–2D perovskite layer and inside the 125 µm PET substrate has been calculated by SRIM simulation, resulting about of 59 keV and 1550 keV respectively, as shown in the graph of LET in function of the penetration depth reported in Figure5 H+ cm−2 s−1 and 1.4 × 109 H+ cm−2 s−1. Even at a bias as low as 1 V, the detector's response to protons is fast and box‐shaped within the wide range of proton fluxes employed for the characterization. The experimental sensitivity per unit volume, calculated as the first derivative of the proton induced current (I) versus dose rate (Dr) (SV=dIdDr1V), resulted up to (290 ± 40) nC Gy−1mm−3. We attribute such a value, lower than the theoretical one, to an incomplete collection of the charges produced in the film, possibly due to trapping effects in the device's channel. This result however confirms that the charge collection of electron‐hole pairs created by ionization constitutes the most significant contribution to the current signal recorded under proton irradiation. It is worth noting that the characteristic proton beam aperture time at LABEC, of about 100 ms, limits a precise evaluation of the response time of the here studied mixed 3D–2D perovskite detector, which clearly follows the timing of the beam aperture and of the rotating chopper employed for the proton beam current estimation , employing a 5 MeV proton beam extracted into atmosphere, provided by the 3 MV Tandetron accelerator.25 The, we observed a different behaviour, i.e., when the beam was switched off, the current had an initial fast drop due to the recombination of the charges, followed by a slower decay. This behaviour led to a gradual shift of the baseline scaling with the total dose received by the detector. We attributed such behaviour to the accumulation of proton‐induced charges in the plastic substrate, which, acting as a virtual bottom‐gate for the organic semiconductor layer, increased its electrical conductivity. We did not observe such an effect in the here reported mixed 3D–2D perovskite detectors, fabricated on plastic substrate of the same thickness, suggesting that the electrically active states responsible of the persistent current observed for organic semiconductors active layer are here totally absent. Therefore, the here proposed mixed 3D–2D perovskite detector can monitor the impinging proton flux in real‐time with a complete recovery of its pristine conditions after few hundreds of milliseconds from a previous irradiation.After each proton irradiation cycle, the current completely recovers to its pristine values. It is noteworthy that, in our previous work on flexible organic thin film‐based direct proton detectors fabricated onto PET foils13, we To estimate the LoD, the SNR was plotted as function of proton flux in Figure 5 H+s−1cm−2, a value near the actual minimum flux measured. From the extrapolation of data collected at higher bias, much lower LoD values resulted, i.e., (3.0±0.4) × 103 H+s−1cm−2 and (6.0±0.6) × 103 H+s−1cm−2 for 5 V and 10 V applied bias voltage respectively  parameter, defined as the minimum intensity of radiation which provides a signal to noise ratio (SNR) equal to 3, following the IUPAC standard.30 To Eabs is the energy absorbed by the active layer calculated with SRIM Monte Carlo simulations, i.e., 59 keV released in 4 µm thick mixed 3D–2D perovskite layer; pN is the proton flux; ρmixed is the density of the mixed 3D–2D perovskite, calculated as the weighted average between the 3D and the 2D perovskite composing the mixture and resulting ρmixed = 3.43 g cm−3, and V is the volume of the active layer, considered as the product of the perovskite layer thickness (4 µm) and the whole area of the detector (1 mm2), which is entirely contained within the beam spot (17 mm2). The as converted minimum detectable dose rate resulted (729.2±0.2) µGy s−1 at 1 V, and (72±3) µGy s−1 and (144±4) µGy s−1 at 5 V and 10 V respectively. These values are from 1 to 2 orders of magnitude lower than the ones we reported for organic film based proton detectors operated in real‐time.The dose rate corresponding to each proton flux can be estimated through the equation:To assess the stability and the reliability of the detector's response we performed 10 repeated irradiation cycles. The signal variation resulted within 12% with respect to the mean value over 10 repetitions, as reported in Figure 12 protons impinging on the beam spot area. Figure The radiation hardness of the detector was also evaluated by measuring its response in pristine condition and after 40 min irradiation tests, corresponding to a total of 1.7 × 1033 (3D) and (PEA)2PbBr4 (2D) and by tuning the 2D:3D ratio with a 35% of 2D precursor component we were able to combine the best features of both materials, targeting their use as direct radiation detectors. The presence of 2D perovskite allows to significantly lower the dark current and to achieve micrometer thick layers homogeneously covering the detector's active area, a very challenging goal to achieve with a pure MAPbBr3 active layer. On the other hand, the 3D perovskite allows to maintain a good sensitivity value, quite low in pure 2D perovskites due to its low density (2.27 g cm−3).In this work we report on the use of novel mixed 3D–2D perovskites films deposited from solution as active layers in flexible proton detectors. We deposited mixed 3D–2D perovskites films based on MAPbBr5 – 109] H+ s−1 cm−2 and under repeated sequential radiation exposures, up to 40 min of irradiation tests, corresponding to a total of 1.7 × 1012 protons impinging on the beam spot area. A maximum variation of 14% was recorded in the output signal, assessing the high radiation tolerance of the detector and its reliability as proton beam dosimeter. The minimum detectable dose rate resulted (72±2) µGy s−1, a value 2 orders of magnitude lower than the one recently reported in the only paper dealing with real‐time direct proton detectors based on organic thin films.The response to 5 MeV protons has been assessed in a wide flux range  H+ cm−2 s−1 and 10 s time window. The spot of the proton beam has an area of 0.17 cm2.During the tests samples were enclosed in a small Faraday cage to lower the electromagnetic noise and for keeping the samples in dark. Samples were irradiated with proton flux in the range [10Electrical response under proton beam irradiation was acquired by a Keithley 2614B precision Source/Measure Unit.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Coexistence of different ordered adlayer phases with (2×62)R45° and (4×4) unit cells was observed in the double layer potential regime. The adlayer is rather dynamic and undergoes a reversible order‐disorder phase transition at 0 V vs. the reversible hydrogen electrode. Density functional calculations indicate that the adlayer consists of coadsorbed carbonate and water molecules and is strongly stabilized by liquid water in the adjacent electrolyte.(Bi)carbonate adsorption on Cu(100) in 0.1 M KHCO 2 reduction, is clarified by in situ scanning tunneling microscopy, revealing two highly complex coexisting adlayer structures above a critical potential. Ab initio calculations identify these as carbonate/water coadsorbate layers and indicate a strongly stabilizing effect of the adjacent liquid solution.The molecular‐scale structure of the interface between copper electrodes and bicarbonate electrolyte, a key system for electrochemical CO Although bicarbonate is the dominant species in solution, most spectroscopic studies suggest that the adsorbed species on the Cu surface is CO32-.The nature of the molecular adlayer of Cu electrodes has been addressed extensively by in situ surface‐enhanced Raman (SERS) and IR absorption spectroscopy, with the main body of work focusing on bicarbonate solutions.−1 in the double layer range and assigned this to adsorbed CO32-that was replaced by adsorbed CO in the CO2RR regime.−1 with a maximal intensity at −0.3 V, which was assigned to monodentate carbonate. In a very recent work by Morazaman and Gul, also an additional weak band at 1344 cm−1 was found between −0.2 and 0.4 V, which the authors attributed to adsorbed HCO3-on the basis of isotope exchange experiments.Already early FTIRS studies of Hori et al. found a band at ≈1500 cmp(2×2) structure was observed on the atomic scale at −0.5 V, which was tentatively attributed to adsorbed CO. No microscopic data on the carbonate adlayer structure has been reported up to now for Cu or any other electrode surface. Only for Ag(110) under ultrahigh vacuum (UHV) conditions, where the carbonate was formed by reaction of CO2 with the (1×2)‐O reconstructed surface, a carbonate adlayer with only local order was observed in STM studies.Much less is known about the microscopic structure of the adlayer at the Cu ‐ bicarbonate electrolyte interface. While Cu in acidic and alkaline solutions has been extensively studied by in situ scanning tunneling (STM) and atomic force microscopy (AFM),Apart from experimental studies, also ab initio theory has been employed for clarifying the adlayer structure. Density functional theory (DFT) studies of relevant adsorbed species on Cu(100) in CO2RR indicated the presence of carbonate3 by in situ STM and DFT calculations. Coexistence of different, rather complex adlayer structures is observed. On the basis of the DFT results, these can be attributed to the effect of water coadsorption, which stabilizes carbonate on the Cu surface.We here present a combined study on the molecular structure of the carbonate adlayer on Cu(100) electrodes in 0.1 M KHCO3 electrolyte, the most common electrolyte for CO2RR, and under argon or CO2 gas atmosphere. Experimental details are provided in the Supporting Information. All potentials are given vs. RHE.The STM experiments were performed on Cu(100) in 0.1 M KHCOCO32-or HCO3-anions. The stripe‐like structure exhibits a long‐range modulation of (2.1±0.1) nm perpendicular to the stripes and an intermolecular distance of (0.35±0.03) nm along the stripes. The stripes are oriented along the [010] and [001] directions of the Cu substrate. These structural parameters are consistent with a (2×62)R45° superstructure. High‐resolution STM images reveal a complex molecular arrangement within the adlayer unit cell, consisting of double rows of prominent maxima  show only double layer charging without any adsorption peaks between the CO2RR regime and the onset of Cu oxidation above 0.3 V. Nevertheless, we observe in the potential range above 0 V, coexistence of two ordered adlayer phases, one with a stripe‐like R45° phase, i.e., parallel to the Cu[011] direction. This corresponds to a (4×4) superstructure. The appearance of this (4×4) structure in high‐resolution STM images can be quite variable , but the images share certain typically characteristics. Similar to the (2×62)R45° phase, also here prominent molecular rows or double rows are observed, with a spacing of 2dCu between the maxima within each row and an additional weak modulation with a period of 4dCu R45° phase.The second adlayer phase can be described by a square lattice with a spacing of 1.02±0.05) nm, which is oriented at an angle of 45° relative to the stripes in the (.02±0.05 2×62)R45° and the (4×4) structure, can exhibit different appearances in high‐resolution STM images. These may result partly from the state and shape of the STM tip, which is known to strongly affect the imaging of adsorbed anion adlayers, as e.g. reported for sulfate on Au(111).2×62)R45° and (4×4) structure can concurrently exist on the surface. This as well as the parallel presence of adlayer phases with different unit cells suggests some structural flexibility in the adlayer of adsorbed anions that apparently permits formation of a variety of structurally related ordered phases. A coexistence of different adlayer phases, specifically of ordered and disordered structures, was also observed in in situ STM studies sulfate and phosphate adlayers on Cu(111) in neutral solutions.Both the 2×62R45° a2×62)R45° phase, only weak dynamic fluctuations at fixed potential are observed.Before discussing the nature and molecular structure of the carbonate adlayer phases in detail, we address the adlayer's dynamics and potential dependency. For the  phase exhibits strong fluctuations with time. As illustrated in Figure 2×62)R45° phase , supporting that both phases are closely related and consist of the same adsorbate species.At potentials around 0 V, a potential‐dependent order‐disorder transition in the anion adlayer is observed Figure b–d, wher2 saturated 0.1 M KHCO3. Furthermore, no significant changes in the adlayer were observed in STM experiments where the argon gas in the STM chamber was exchanged at about 0.14 V by CO2 gas during the experiment. Apparently, the presence of dissolved CO2 and the resulting slightly different pH does not have a major effect on the adlayer structure. Furthermore, we do not observe changes in the Cu surface morphology  during the order‐disorder transition in the adlayer  at neutral pH.CO32-/HCO3-. However, in this case the ratio of these species should be affected by the pH , for which we found no evidence.Various species may account for such coadsorption. First, carbonate as well as bicarbonate anions exist in this system. Each of those has been proposed as active surface species in the double layer potential regime in previous spectroscopic studies.CO32-orHCO3-may be coadsorbed with a water species. The latter has been proposed for ordered adlayers of other oxyanions, such as sulfate and phosphate, on (111) and (100) surfaces of fcc metals in acidic media, where likewise two type of maxima with different apparent height were reported in STM studies.[Second, Furthermore, our observations resemble those found for other molecular anions, such as formate and acetate, which have a similar bidentate adsorption geometry on metal surfaces as proposed for carbonate.3), bicarbonate (HCO3), water (H2O), and hydroxyl (OH) at different coverages, on Cu(100) slabs with a (4×4) and with a (2×62)R45° unit cell, respectively (details given in the Supporting Information). In total we investigate 192 different initial structures in the two cells (see Table S1–S2). In Figure S8 adlayers of different composition and coverage are analyzed by depicting the lowest energy found for each . We find that structures with rows of carbonate and water are the most energetically stable. The energies of all calculated adlayer configurations are shown in Figure S9–S10. On this basis, we now take a detailed look at the mixed carbonate/ water phases.To gain insight into the nature of the adsorbed species and the adlayer structure, we carried out a DFT study of a series of adlayers, consisting of carbonate (CO3 and 4 H2O in the unit cell (corresponding to a coverage of 1/4 ML CO3 and 1/4 ML H2O). We display the full set of these structures in Figure S11. Here it is observed that multiple simulations  have converged into rather similar low energy structures, which are close to a c(2×2) arrangement of CO3 and H2O molecules that are located in bridge sites. Obviously, these structures differ strongly from those observed in the STM experiments. With increasing adlayer energy, we then find structures where water is detached from the surface , which we discard in this analysis. However, at a slightly higher energy of 0.89 eV  structures are obtained that resemble the experimentally observed adlayers  slabs with the (4×4) unit cell the most stable adlayer phases contain 4 CO2×62)R45°unit cell the most stable compositions have 4 CO3 and 2 H2O molecules in the unit cell . Here, the energies of the calculated 12 structures with different carbonate and water orientations are within an interval of 0.7 eV , i.e., differ by <0.18 eV per CO3. Because of this small energy difference, positional perturbations and rotations of the carbonate and water molecules are possible, which is in good accordance with the experimental observations of several coexisting adlayer phases. In the lowest energy structure (ID=10), the CO3 in the two molecular rows within a CO3 stripe are slightly shifted relative to each other; in the slightly less favorable structure ID=2, stripe with this arrangement alternate with stripes where the two CO3 rows are anti‐phase shifted. These arrangements already strongly resemble those in the STM images. In the ID=2 structure one of the CO3  is adsorbed with its center almost on top of the Cu and its binding O atoms occupy near‐bridge sites. Moving these O atoms close to on‐top positions results in a structure which resembles more closely the STM images  .For Cu(100) slabs with (3+4 H2O the minimum energy structure in solvent is not that found in vacuum (ID=5), but the structure ID=11, which resembles the STM images. Admittedly, the energies are quite close, making both structures possible.In general, the minimum energy structures for the carbonate adlayer obtained in these simulations of Cu(100) in vacuum do not exhibit structural features that correspond well with the experimental data . This might be caused by missing relevant compositions or structures in the simulations, even for the case of extensive screening as performed here. However, we attribute this failure to another reason, namely the absence of solvating water on top of the carbonate adlayer in the simulations. We have previously observed that carbonate species are strongly stabilized by the aqueous environment, as compared to vacuum simulations by employing ab initio molecular dynamics (AIMD).2×62)R45° unit cells and carried out a thermodynamic calculation. The stability is displayed in Figure 2×62)R45° structure with 4 CO3 and 2 H2O is slightly more stable than the (4×4) unit cell with 4 CO3 and 4 H2O, in good agreement with the experimental results. The onset of stability is shifted by about 250 mV towards more positive values in comparison to the experimentally observed order‐disorder transition in the adlayer, which seems acceptable in view of the approximate treatment of the electrolyte solution. According to simulated STM images  and with a  electrode surface. These share certain structural features, especially an arrangement of the CO3‐CO3 and CO3‐H2O interactions are strong enough to stabilize ordered phases while also being sufficiently flexible to allow fluctuations between different structures. It is likely that similar molecular interactions also exist at more negative potentials where the adlayer is disordered. Furthermore, related surface species such as adsorbed CO2 and intermediates of the CO2 reduction reaction may likewise be ruled by these types of interactions, as suggested by AIMD findings of strong stabilization of such species by water.According to our observations of a rich and dynamic phase behavior of the carbonate adlayer, the COThe 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.Supporting InformationClick here for additional data file.


Variations in the red blood cell (RBC) lifespan can affect glycosylated hemoglobin (HbA1c) test values, but there is still a lack of evidence regarding how and to what degree the RBC lifespan influences HbA1c in the type 2 diabetes mellitus (T2DM) population owing to the restriction of traditional RBC lifespan–detection means. This study aimed to investigate the influence of RBC lifespan variation on HbA1c values in T2DM patients with a HbA1c detection value lower than 7%.Patients with HbA1c <7% were divided into two groups: RBC lifespan <90 days and RBC lifespan ≥90 days. We collected blood glucose levels at seven time points for three consecutive months, assessed the HbA1c and glycosylated albumin levels, and calculated the hemoglobin glycation index (HGI) for each patient.p < .01). Pearson analysis showed a significant negative correlation between RBC lifespan and the HGI in patients with T2DM .There were no statistical differences in the HbA1c value between two groups, but the estimated glycosylated hemoglobin (eHbA1c) was significantly higher in patients with an RBC lifespan <90 days. The proportion of the eHbA1c ≥7% in the group with an RBC lifespan <90 days was significantly higher than the other group  lifespan variation, is the main cause of variation in glycosylated hemoglobin (HbA1c), independent of blood glucose levels.This study highlighted the important impact of RBC lifespan on HbA1c test value in T2DM with HbA1c test value <7%.We discovered that the HbA1c test value in type 2 diabetes (T2DM) patients with RBC lifespan <90 days significantly underestimated their general blood glucose condition.Our results will provide more useful information to advance management of blood glucose levels for T2DM. Inclusion criteria included adult patients who (1) were >18 years of age and were diagnosed with T2DM based on the 2015 American Diabetes Association diagnostic criteria; (2) received their T2DM diagnosis and at least 3 months of treatment at the Trinity Care clinic; (3) have used a blood glucose monitoring system within the past 3 months; (4) monitor their fasting blood glucose at least twice a week and test their blood glucose via finger stick before each meal and 2 h after each meal, according to the A1C‐derived Average Glucose (ADAG) studyThis study was approved by the Ethics Committee of Zhu Xianyi Memorial Hospital of Tianjin Medical University . All patients provided their written informed consent prior to enrollment in the study.2.2We collected clinical data, including age, sex, duration of diabetes, and medications, using medical records. Blood glucose values using the iHealth software of the Trinity Care patient management system. The weight, height, and brachial artery blood pressure of the right upper limb of each patient were measured by trained personnel. Fasting venous blood was collected and tested at the central laboratory of Chu Hsien‐I Memorial Hospital, Tianjin Medical University. A Beckman Coulter AU5800 automatic biochemical analyzer  was used to detect glycosylated serum albumin, liver and kidney function, blood lipids, and other biochemical indices. Each patient's HbA1c levels were tested using a Sysmex XN instrument . Clinical indices were obtained on the same day as that of gas collection.2.3All of the patients with T2DM enrolled in this study self‐monitored their blood glucose levels using the Trinity Care Outpatient and Diabetes Management System during the previous 3 months. The frequency of blood glucose monitoring followed that of the ADAG study,2.4The RBC detection method was based on a previous study by Ye et al.2.5t test was used to compare data groups with a normal distribution and a chi‐square test was used to compare data groups with a nonnormal distribution. Two‐sided p < .05 values were considered statistically significant.Statistical analysis was conducted using IBM SPSS 25.0  and R v 4.0.3 . A 33.12BG) was 9.20 ± 1.43 mmol/L, and the hemoglobin (Hb) was 142.81 ± 18.6 g/L. There were 183 patients with an RBC lifespan <90 days, and 143 patients with an RBC lifespan ≥90 days. There were no statistical differences in regards to sex, age, systolic blood pressure, diastolic blood pressure, Hb, triglyceride, total cholesterol, low‐density lipoprotein cholesterol, aspartate transaminase, ALT, serum creatinine, urine albumin, blood urea nitrogen, estimated glomerular filtration rate, and therapeutic regimen between the two groups. Metformin was involved in 70.6% of these patients' treatment plans. The duration of T2DM was longer in the RBC lifespan <90 days group than in the RBC lifespan ≥90 days group. The average RBC lifespan of healthy adults is 115 (70 ~ 140) days.Demographic and clinical features of all patients enrolled in this study specifically 326 T2DM patients with HbA1c detection values <7% are shown in Table 3.22BG, AG, and glycosylated albumin (GA) in the group with an RBC lifespan <90 days were significantly higher as compared to the group with an RBC lifespan ≥90 days. We also confirmed that the HbA1c test values were significantly underestimated in patients with an RBC lifespan <90 days. This study calculated the eHbA1c levels of the two groups based on the blood glucose levels of the patients, and as is shown in Figure As shown in Figure 3.3To confirm that the HbA1c detection value led to an underestimation of the blood glucose in patients in the RBC lifespan <90 days group, we further evaluated the target attainment of HbA1c in the two groups according to the patients' eHbA1c level. We set an eHbA1c value <7% as the target for glycated hemoglobin. As shown in Figure 3.4The HGI is an important indicator of whether HbA1c measurement values deviate from a patient's average blood glucose.4HbA1c is an important index not only to diagnose T2DM and evaluate the blood glucose management of T2DM patients but also to guide both the antihyperglycemic therapy and prognosis of T2DM patients. Because the HbA1c detection value is known to be significantly influenced by RBC lifespan, it is critical that the influence of RBC lifespan on blood glucose levels in patients with T2DM be understood. This study was conducted in T2DM patients who were clinically assessed to meet the target of glycemic management  to compare the difference in blood glucose levels among patients with differing RBC lifespans.2BG, mean blood glucose, GA, and eHbA1c were significantly higher in the group with an RBC lifespan <90 days as compared to the group with an RBC lifespan ≥90 days. This study calculated the eHbA1c levels of the two groups based on blood glucose levels of the patients and found that the levels of eHbA1c in the group with the RBC lifespan <90 days was significantly higher than that of the group with the RBC lifespan ≥90 days (Figure In this study, patients with T2DM were divided into two groups according to RBC lifespan: RBC lifespan <90 days and RBC lifespan ≥90 days group. There is growing evidence that HbA1c levels depend not only on average blood glucose levels but also on RBC lifespan.s Figure . AccordiTo analyze the influence of a shortened RBC lifespan on the target attainment of the HbA1c detection value in T2DM patients, this study compared the eHbA1c values between the two groups. As shown in Figure In recent years, the deviation between HbA1c and HbA1c estimated by date‐matched average blood glucose, namely, HGI, has led some diabetes professionals to question the accuracy of HbA1c. Studies have confirmed that HGI is an independent risk factor for microangiopathy, such as diabetic nephropathy, diabetic retinopathy, and macrovascular disease, in patients with T2DM.Limitations of this study include relatively small sample size, average glucose not derived by continuous glucose monitoring, the effect of the background level of CO in the sampling location on RBC lifespan detection values, and the difficulty in correcting the impact of shorter RBC lifespan.In conclusion, the variation in the RBC lifespan of patients with T2DM was an important reason for the deviation of HbA1c detection values from the average blood glucose levels, that is, the HGI. A considerable proportion of patients with T2DM and a shortened RBC lifespan had significantly underestimated HbA1c detection values and blood glucose levels. Thus, accurate evaluation of the blood glucose level in patients with T2DM with shortened RBC lifespan is important, and the effect of RBC lifespan on HbA1c values must be considered. Our results will provide more useful information about blood glucose management for clinical practice and improve patients' therapy compliance.Saijun Zhou conceived the project and designed experiments. Junmei Wang and Li Zhang collected the patients' information and analyzed data. Yu Bai, Junmei Wang, Li Zhang, Xinli Wang, Weilin Wang, and Jing Li collected the patients' information. Junmei Wang and Saijun Zhou had input in writing the paper.None declared.


Hence, the electron‐rich silyl‐substituted complex undergoes cyclometallation or shift of the imine moiety after cooperative activation of the P−H bond across the M=C linkage, depending on the electronics of the applied phosphine. Deuteration experiments and computational studies proved that cyclometallation is initiated by the activation process at the M=C bond and triggered by the high electron density at the metal in the phosphido intermediates. Consistently, replacement of the trimethylsilyl (TMS) group by the electron‐withdrawing 4‐nitrophenyl substituent allowed the selective cooperative P−H activation to form stable activation products.The use of iminophosphoryl‐tethered ruthenium carbene complexes to activate secondary phosphine P−H bonds is reported. Complexes of type [( Metal ligand cooperation. Two different iminophosphoryl‐substituted methandiide‐derived carbene complexes have been applied in the P−H bond activation of secondary phosphines. Surprisingly versatile reaction outcomes were observed depending on the imine substituent at the methandiide ligand and the phosphine used. While more‐electron‐rich combinations led to either cyclometallation or an imine transfer, electron‐poor combinations enabled selective P−H bond activation, thus impressively demonstrating the importance of tuning the M=C in this type of carbene complex for bond activation. In contrast, free phosphines delivered only complex product mixtures. Since bond activation processes across the M=C double bond strongly depend on the nature of the M−C interaction, we turned our attention towards iminophosphoryl‐tethered carbene complexes, which have shown more selective processes due to the less reactive metal carbon linkage. For example, complex 2a  and 13C{1H} NMR spectra , while the H atom bound at the phosphorus atom appears as doublet at 6.95 ppm with a large coupling constant of 1JPH=365.0 Hz. Recording of a 1H coupled 31P NMR spectrum clearly confirmed the P−H unit . Single crystals of 3 a were obtained by diffusion of pentane into a diethyl ether solution of the complex. XRD analysis unambiguously confirmed the nature of the new species  Å and is thus considerably longer than in the starting carbene complex (1.955(2) Å) and within the range of a typical Ru−C single bond.Initially, the reactivity of carbene complex 3 a is revealed to be unstable in solutions of benzene and C6D6. Monitoring the reaction process by 31P{1H} NMR spectroscopy revealed the continuous and selective formation of a single new species with similar NMR features compared to the starting material . Along with this complex, the formation of free cymene was observed, thus suggesting replacement of the cymene ligand by benzene to form complex 5  or naphthalene complexesThe facile replacement of the cymene ligand in 2 a with the less electron rich bisphosphine  the formation of three compounds . One compound showed two doublets in the 31P{1H} NMR spectrum at 32.5 and 47.0 ppm with a coupling constant of 5.0 Hz and revealed itself to be an intermediate of the reaction as the signals completely vanished during prolonged reaction times. The two remaining products exhibited a ratio of approximately 1:0.7, with the main product showing similar signals as the cyclometallated compound 3 a (two doublets at δP=13.9 and 41.7 ppm with a coupling constant of 3JPP=5.1 Hz). Using 1H coupled 31P NMR spectroscopy, it was possible to identify the major product as the cyclometallated complex 3 b . The second product however featured vastly different signals in the 31P{1H} NMR spectrum, namely two doublets at δP=−13.2 and 35.6 ppm with a surprisingly large coupling constant of 47.0 Hz. Unfortunately, we could not identify the second compound at first due to the difficulties in the separation of both products. However, we assumed that the reaction with an even more electron poor phosphine would shift the reaction outcome completely to the new product. Therefore, 2 a was reacted with diphenyl) phosphine (=HPArF2). This led to the selective formation of a single new product 4 b which is characterized in the 31P{1H} NMR spectrum by two doublets at δP=−13.4 and 34.5 ppm with a coupling constant of 2JPP=47.5 Hz and a doublet in the 1H NMR spectrum at 4.93 ppm (2JHP=15.4 Hz) for the methylene bridge. 4 b could be characterized by NMR and XRD  is still rare.κ2‐phosphinomethanide complexes.Next, the reaction of carbene complex 2 a towards the different phosphines we performed computational studies at the PBE0‐D3/def2tzvp level of theory . Calculations revealed that the activation process proceeds via initial coordination of the corresponding phosphine to form the coordination complex Coord, followed by a proton transfer (TS‐Act2) to generate intermediate Act’. Rotation around the P−Ru bond finally gives the activation product Act observed in experiment. Since the P−H activation could also proceed via a concerted 1,2‐addition or an oxidative addition at the metal centre followed by proton transfer, these two pathways have also been investigated exemplarily for the combination 2a′/HPPh2. The energy of the transition state for the concerted 1,2‐addition across the Ru=C linkage , 37.8  and 38.8–38.9 (multiplet) ppm . These findings can be explained by differently deuterated structures . This is at first in contradiction to the assumed P−H activation across the Ru=C bond which suggests the formation of 3 a‐DH. However, the presence of species like 3 a‐HD or 3 a‐DD in the reaction mixture can be explained by the exchange of the phosphine ligand in 3 a‐DH with unreacted DPPh2 which, due to the overall slow reaction process, gives the double deuterated compound 3 a‐DD. This additionally leads to the formation of free HPPh2 which can then be activated by still unreacted carbene complex leading to 3 a. Here, the ruthenium bound phosphine can again be replaced by DPPh2 to afford 3 a‐HD. This also explains the presence of a signal for the proton of the methylene bridge in the 1H NMR spectrum, but with an integral of only 0.5. An additionally performed 2H NMR spectrum  further confirms the presence of deuterium at the phosphine moiety and the carbon bridge. Presumably, all four compounds shown in Figure 3 a was reacted with DPPh2. Indeed, 31P{1H} NMR spectroscopy showed the formation of the aforementioned triplet at δP=37.8 ppm . Furthermore, the signals of 3 a in the 31P{1H} NMR spectrum and the integral of the methylene bound proton in the 1H NMR spectrum remained completely unaffected, suggesting that no H/D exchange at the methanide ligand takes place. This clearly confirms that the deuteration of the carbene centre must be the result of the P−H activation across the Ru−C bond . From this point, the two reaction pathways diverge. For the imine shift to 4’, the iminophosphoryl moiety attacks at the ruthenium bound phosphorous atom (TS2) thus forming a five‐membered Ru−P−N−P−C ring in Int2. Now the original P−N bond is cleaved (TS3) giving the final product of the imine shift (4’).Having confirmed the possible formation of Int1 rotates about the P−C1 bond, so that the ortho C−H bond of one of the phenyl rings coordinates to the unsaturated ruthenium centre. This agostic interaction leads to a pyramidalization of the phosphido moiety in Int3. From this intermediate, a direct σ bond metathesis between the Ru−P and the C−H bond is energetically disfavoured. An oxidative addition of the C−H bond to the ruthenium centre as observed for other orthometallation reactions also does not take place.2 delivered the chloro complex 9 in 87 % yield. Compound 9 is characterized by a singlet in the 31P{1H} NMR spectrum at δP=50.9 ppm and by doublets at δ=4.00 (2JHP=7.4 Hz) and 34.1 ppm (1JCP=64.4 Hz) in the 1H and 13C{1H} NMR spectrum, respectively. In the molecular structure  Å is slightly longer than in the related TMS‐substituted system (1.585(2) Å)),9 to carbene complex 2 b was selectively achieved with potassium tert‐butoxide. Treatment with 1 equiv. of base gave complex 2 b as green solid. The 31P{1H} NMR signal of the carbene complex appears at δP=65.3 ppm and the signal of the carbenic carbon atom at δC=143.6 ppm (1JCP=66.6 Hz), thus being in line with the formation of a carbene species. Unfortunately, separation of the carbene complex from the formed NaCl proved to be difficult, probably due to the coordination of the salt to the carbene complex.In order to enhance the selectivity of the P−H activation process and the stability of the simple activation products we addressed the synthesis of a less electron‐rich carbene complex. We chose complex b Scheme  with the9 with silver tetrafluoroborate was carried out, leading to the formation of the cationic ruthenium complex 10 in nearly quantitative yield. Complex 10 shows a very broad signal in the 31P{1H} NMR spectrum at 41.3 ppm. This is probably due to fluctional behaviour of the weakly coordinating sulfonyl moiety. The proton of the methylene bridge exhibits a doublet in the 1H NMR spectrum at δH=4.50 ppm with a coupling constant of 2JHP=3.7 ppm and the methylene carbon atom appears in the 13C{1H} carbon NMR spectrum at δC=21.8 ppm. Subsequent deprotonation with potassium tert‐butoxide gave the desired carbene complex in 94 % yield and NaBF4 as by‐product which could be successfully removed by filtration. By slowly diffusing pentane in a saturated solution of 2 b in DCM crystals suitable for XRD analysis could be obtained. Carbene complex 2 b crystallizes in monoclinic space group Pn  for 11 a and 19.1 (broad singlet) and 49.8 ppm (doublet with 3JPP=3.8 Hz) for 11 b respectively. Both compounds were characterized by multinuclear NMR spectroscopy and CHN analyses. By slowly diffusing pentane into a saturated solution of 11 b in toluene it was possible to obtain single crystals suitable for XRD analysis which unambiguously confirmed the nature of the activation products  to be 29 kJ/mol lower in energy than the corresponding anti isomer. This high energetic preference suggests that the syn isomer is also thermodynamically favoured und is thus in line with the experimental observation.To our delight, the reactions of s Figure . 11 b cr2 b in combination with the three applied phosphines . The ΔG≠maxvalues reflect the observed reactivities very well. Overall, the activation energies increase for both mechanisms for 2 b in comparison to 2 a. For diphenylphosphine, the barrier in case of the imine shift amounts to 86 kJ/mol and is thus  still viable at room temperature. However, for the two electron poor phosphines HPArCl2 and HPArF2 both pathways show considerably higher values for ΔG≠max(99–117 kJ/mol) which explains why the reactions nearly stop after the P−H activation step which enables the isolation of the “true” activation products. In fact, 31P{1H} NMR experiments of a NMR sample of 11 a after 4 days in solution showed slow decomposition of the substance to  the corresponding orthometallated and imine shift products  thus furthermore validating that both reactions proceed via the activation pathway  should be addressed to the authors.Supporting InformationClick here for additional data file.


Herpetological Conservation and Biology, 17(1), 165–179.” The publication year was corrected from 2021 to 2022.In the recent article by Moreno et al. , the refThe authors apologize for the error.


For the latter, variable‐temperature EPR spectroscopy revealed the presence of a thermally accessible triplet state, with a singlet‐triplet separation of 14.1 kJ mol−1.The synthesis and characterisation of a homologous series of quinoid sulfur‐containing imidazolyl‐substituted heteroacenes is described. The optoelectronic and magnetic properties were investigated by UV/vis, fluorescence and EPR spectroscopy as well as quantum‐chemical calculations, and were compared to those of the corresponding benzo congener. The room‐temperature and atmospherically stable quinoids display strong absorption in the NIR region between 678 and 819 nm. The dithieno[3,2‐ A homologous series of quinoid bisimidazolylidene‐derived thioacenes has been synthesised and investigated for their optoelectronic and magnetic properties. All investigated small‐band‐gap quinoids show stability at room temperature in the presence of oxygen and display strong NIR absorption. Three‐ and fourfold annulated quinoids display partially open‐shell singlet diradical character. The latter reaction was accompanied by a dramatic colour change from pale yellow to deep blue . Single crystals suitable for X‐ray diffraction of 12 and 4 were obtained from concentrated THF and ethanol solutions, respectively. The molecular structures  Å compared to the bisimidazole precursor 12 (C3−C4 1.4650(16) Å). The central six‐membered ring of 4 displays alternating bond lengths of 1.359(2) Å (C1−C2) and 1.424(3) Å (C2−C3), whereas the unoxidised entity (12) features bond lengths typical for benzoid structures (C1−C2 1.3889(16) Å; C2−C3 1.4030(16) Å; C3−C4 1.4650(16) Å).12 than in 4, whereas the shorter bond lengths N1−C5 and N2−C6 in 4 account for localised double bonds. The quinoid 6 was synthesised in two steps from 13 according to the method described by Nakamura (Scheme b]thiophene derivative 7 from 15 (Scheme 13 with p‐anisil (11) in the presence of ammonium acetate provided 14 in 43 % yield. The formation of the bisimidazole 14 was supported by the new 1H NMR resonances at δ=12.79 and 7.92 ppm ([D6]DMSO) for the NH and thiophene CH protons, respectively. The poor solubility of 14 in DMSO did not allow the determination of 13C NMR chemical shifts. The oxidation of 14 and 16 to the corresponding quinoid heteroacenes 6 and 7 was achieved by the reaction with alkaline ferrous cyanide in 1,4‐dioxane. The successful formation of the quinoids 6 and 7 was corroborated by the dramatic colour change from yellow to green (for spectroscopic details see below) and both materials were isolated in 88 and 81 % yield, respectively. The corresponding 1H NMR (CDCl3) spectra of 6 and 7 exhibit a singlet at δ=8.29 and 8.40 ppm for the thiophene CH proton. Finally, ESI mass spectrometry stand in line with the successful oxidation of both bisimidazoles to the corresponding quinoids.Despite the significant interest in benzo‐derived quinoids,10, 118 and 9 were constructed through the domino carbon−sulfur cross‐coupling/cyclisation sequence developed in our group (Schemes 8 a–c commences by the reaction of tetrabromo thiophene (17) with trimethylsilyl‐protected (TMS) acetylene (18) under Sonogashira conditions and provided the 2,5‐bisalkyne 19 in 65 % yield as single regioisomer. At this stage, we selected three imidazole derivatives with different para substitution pattern  to study the electronic impact of the imidazole moiety on the optical and electronic properties. In situ alkyne deprotection with potassium fluoride and subsequent Sonogashira coupling with each of the three trimethylsilylethoxymethyl‐protected (SEM) 2‐iodoimidazoles 20 a–c provided the internal alkynes 21 a–c in 68 to 92 % yield. SEM‐protection of the imidazole throughout the synthesis was necessary because otherwise the poor solubility of the imidazoles and – at a later stage – of the bisalkyne derivatives prevented clean reactions with high conversion. The formation of the bisalkyne 21 b was unambiguously supported by X‐ray diffraction analysis (see the Supporting Information). Treatment of the bisalkynes 21 a–c with potassium thioacetate in the presence of [Pd(dba)2]/DiPPF ferrocene) and potassium phosphate as base furnished the dithienothiophene congeners 22 a–c in 41, 58 and 51 % yield, respectively. Since the deprotection of 22 a–c led to insoluble materials, the latter were directly subjected to oxidation using ferrous cyanide/NaOH, again accompanied by a drastic colour change from brown/orange to dark blue. Thus, the quinoid materials 8 a–c were obtained in 83, 74 and 85 % yield as black  or dark brown (8 b) solids. NMR spectroscopic data could not be obtained due to the insolubility of the materials, but ESI mass spectrometry confirmed the two‐electron oxidation by detection of the corresponding [M+H]+ cationic species.The three‐ and fourfold condensed sulfur heteroacenes  Schemes  and 4.[b]thienothiophene derivative 9 was synthesised according to a similar strategy using the thienothiophene bisalkyne 23 as cyclisation precursor 2]/DiPPF induced the C−S cross‐coupling and cyclisation. The tetraheteroacene was deprotected in situ by treatment with HCl in THF to obtain 26 as red solid in 51 % yield. Again, the low solubility of 26 in common polar organic solvents did not allow the acquisition of NMR spectroscopic data but high resolution molecular mass spectrometry of 26 confirmed the successful synthesis of the oxidation precursor. The oxidation of 26 to the quinoid 9 was achieved by aqueous ferrous cyanide under basic conditions. The product was isolated as dark green powder in 72 % yield which evaded NMR spectroscopic analysis due to insolubility.Finally, the thieno2′,3′:4,5]thienothiophene‐derived quinoid 7 is significantly red‐shifted to the NIR region to 737 nm (1.68 eV) with a significantly smaller molar absorption coefficient of log ϵ=4.69 compared to 6 (log ϵ=4.96). Further extension of the heteroacene core to the dithienothiophene‐derivative 8 a causes a red‐shift by 60 to 797 nm . The fourfold annulated system 9 displays the strongest red‐shift to 819 nm (1.51 eV) in comparison to the thiophene derivative 6, with a comparable high molar absorption coefficient of log ϵ=4.84. The two‐electron oxidation to the quinoids results in a significant decrease of ΔEopt by about one‐half. The trend of bigger heteroacene cores leading to smaller ΔEopt is retained  and the fluoro derivative 8 c exhibit both almost identical optical properties . In contrast, the methoxy substituted derivative 8 a displayed an NIR absorption close to 800 nm . This indicates that the substituent has a significant impact on both the position of the absorption maximum and on the extinction coefficients.The electronic structure changes significantly upon oxidation to the corresponding quinoid heteroacenes. The oxidation process is accompanied in all cases by a significant colour change of the reaction mixtures. Furthermore, oxidation leads to a loss of the fluorescence properties. UV/vis spectroscopy clearly shows the presence of an intense absorption band in the NIR region for all seven quinoid molecules Figure c and d. t 688 nm .80 eV wi thieno3,‐bthiophet 688 nm .80 eV wi7, 8 b/c, and 9 in solution and in the solid state by EPR spectroscopy (X‐band). Note that the EPR spectra of molecules such as Chichibabin's hydrocarbon 2 . In THF solution, an additional monoradical impurity with proton hyperfine couplings of a(1H)=32 MHz  and 5 MHz  was detected . For 8 c in THF between 230 and 305 K, a sharp doublet EPR signal was observed with an additional broad signal, which likely corresponds to a thermally populated triplet state . A detailed analysis, however, failed due to strong line broadening. The diradical nature of 9 was unambiguously confirmed by EPR measurements in THF solution. Variable‐temperature experiments between 230 and 305 K show that the EPR signal intensity increases with increasing temperature , indicating a thermally excited triplet state  as implemented in the T6–9, we observe a lowering of the UKS triplet energy relative to the UKS singlet energy, while the closed‐shell state becomes less favourable. Together with the S2 expectation value (0.090 for 6 and 0.864 for 9), this points to an increasing diradical character. The bond lengths of the optimised UKS singlet geometries further support this finding (see the Supporting Information for details).We find that the UKS singlet is energetically most favoured for all investigated compounds. In the series of thioacene quinoids 6, the bond lengths are similar to the ones of the RKS geometry, being in line with a closed‐shell resonance structure. In contrast, the bond lengths of compound 9 are closer to the ones of the UKS triplet geometry and thus more consistent with a diradical resonance structure.For compound 4 and 6, we find that the UKS triplet spin densities look very similar but that the UKS singlet spin density of 6 is smaller in size, suggesting a smaller diradical character in the thiophene than in the benzo quinoid. Overall, the spin densities are mainly localised at the imidazolyl moieties, leading to a larger distance between the unpaired electrons in the series of compounds 6–9.The spin densities of the UKS triplet and UKS singlet geometries are shown in Figure 6 (41 %) is much larger than the one of the corresponding thiophene quinoid 4 (15 %). This can be attributed to the enhanced aromaticity of benzene compared to thiophene, thus favouring a diradical structure over a cross‐conjugated closed‐shell structure. We observe an increasing diradical character in the series of thioacene quinoids 6–9, with the largest value for compound 9 (74 %).In order to quantify the diradical character, complete active space self‐consistent field (CASSCF) calculations were performed on the UKS singlet geometries using the ORCA program.S2 expectation value, the UKS singlet energy suffers from spin contamination errors in the wavefunction. It is therefore not well suited to compute the energy difference to the UKS triplet state. To obtain a better estimate, we corrected the UKS singlet energy using an approximate spin projection method.EST evaluated with this approach are given in Table 6–9, this is consistent with the above‐mentioned increasing distance between the unpaired electrons. Consequently, the smallest value for ΔEST is found for compound 9 (24.9 kJ mol−1). The trends for both the diradical character and the singlet‐triplet gaps match the findings of the EPR spectroscopic investigation. However, the computed value for the singlet‐triplet gap of compound 9 is larger than the experimentally derived one.According to the 12, 4) and thiophene  derivatives are depicted in Figure Furthermore, we investigated the optical properties of the synthesised compounds. In order to understand the experimentally observed red‐shift of the absorption maxima when comparing bisimidazole intermediates to quinoids, the frontier molecular orbitals of the respective benzo . Based on the RKS results, the prominent absorption band in the spectra of 4, 6, 12, and 14 can be assigned to the HOMO‐LUMO transition (H→L). Regarding the series of quinoid derivatives, TDDFT calculations were not only performed for the closed‐shell state (RKS) but also for the open‐shell broken‐symmetry singlet state (UKS singlet). In both cases, we essentially obtain one single excitation with significant oscillator strength  within less than 0.1 eV, perfectly reproducing the experimentally observed trends regarding the extension of the thioacene core  as well as the influence of different substituents (8 a–c).This is the HOMO‐LUMO transition, being responsible for the NIR absorption band of all investigated quinoids. The excitation energies agree with the measured absorption maxima  and computational procedures including atomic coordinates are given in the Supporting Information.20 a), 2127274 (for 4), 2127276 (for 12), 2127275 (for 21 b)2121377  should be addressed to the authors.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.