Calmodulin Research Articles

Walnut (Juglans regia L.), an economically important woody oil crop, is widely cultivated in southern Xinjiang. In August 2024, field surveys conducted across five walnut orchards (covering approximately 20 hectares) in Kalatal Town, Aksu Prefecture, Xinjiang (80.4933°E, 40.7665°N) revealed that 30-70% of trees exhibited leaf necrosis symptoms. The initial symptoms appeared as small, irregular brown necrotic lesions along leaf margins, which progressively expanded across the entire leaf surface as the disease developed. A total of 12 symptomatic leaves were collected from three walnut orchards. Leaf segments (4×4 mm) and twig sections (10 mm in length) were excised from lesion margins, surface-sterilized sequentially in 75% ethanol for 30 s and 1% sodium hypochlorite for 3 min, rinsed 7-8 times with sterile distilled water, dried with sterile filter paper towels, and placed on potato dextrose agar (PDA) for 5-7 d at 28°C. Eight single-spore strains were obtained using the single-spore isolation method. After 4 days of incubation, the colonies initially appeared white to light pink, gradually developing a distinct pink pigmentation upon maturation. The macroconidia produced by this strain were falcate or elliptical, measuring 4.6-16.6 µm× 1.6-4.0 µm (n = 50). Genomic DNA was extracted from the representative isolate KY11 using the cetyltrimethylammonium bromide (CTAB) protocol. Four genetic loci were amplified and sequenced: the elongation factor 1-α (EF-1α) gene, internal transcribed spacer (ITS) region, 28S large ribosomal subunit (LSU) rRNA gene, and calmodulin (CAL) gene, along with the second largest subunit of RNA polymerase II (RPB2) gene. Amplification was performed using the following primer pairs: EF1-α: EF1-728F/EF1-986R, ITS: ITS1/ITS4, LSU: LR0R/LR5, CAL: CL1/CL2A, RPB2: fRPB2-5F/fRPB2-7Cr (White et al. 1990; Schoch et al. 2012; Xia et al. 2019). The sequences of isolate KY11 were deposited in GenBank with the following accession numbers: ITS(PV441516), EF-1α(PV524932), LSU(PV441478), CAL (PV536123), RPB2 (PV524934). BLASTn analysis revealed high sequence similarity with F. incarnatum references: 99.8% (ITS: MN871572.1), 99.61% (EF-1α: ON292430.1), 99.22% (LSU: MG274307.1), 100% (CAL: GQ505503.1), and 98.61% (RPB2: MT889350.1). A maximum likelihood phylogenetic tree was constructed based on concatenated sequences of EF-1α, CAL, and RPB2 using RAxML 8.0.0 software. Phylogenetic analysis confirmed that isolate KY11 clusters within the same clade as F. incarnatum, thereby supporting its taxonomic classification. (Garcia-Estrada et al.,2021). Isolate KY11 was identified as F. incarnatum based on morphological and sequence analyses. Pathogenicity tests were conducted on 3-month-old walnut seedlings to fulfill Koch's postulates. The inoculation procedure was as follows: three fully expanded healthy leaves per seedling were surface-sterilized with 75% ethanol, wounded with a sterile needle, and inoculated with a KY11 spore suspension at a concentration of 106 spores/mL. Control plants were mock-inoculated with sterile water. All plants were maintained at 28°C with 70% relative humidity under a 12-h light/dark cycle. After 12 days, inoculated leaves developed symptoms identical to field observations, while controls remained asymptomatic. The experiment consisted of two independent trials with five seedlings each. F. incarnatum was successfully re-isolated from symptomatic leaves and confirmed through both morphological and molecular methods, thereby fulfilling Koch's postulates and verifying its pathogenicity. This pathogen has been reported to cause cucumber fruit rot in Mexico (Garcia-Estrada et al., 2021), maize leaf blight (Xu et al., 2022), and luffa leaf spot (Chen et al., 2025) in China. To our knowledge, this represents the first report of F. incarnatum causing walnut brown spot in China, providing fundamental information for disease diagnosis and the development of control strategies.

Soybean (Glycine max L.) is widely cultivated in northeastern Italy, especially in Friuli-Venezia Giulia and Veneto. Since 2013, during early seedling development, necrotic lesions have appeared on upper leaves, spreading downward and causing up to 25% defoliation in the field. Purplish discoloration was observed on petioles, stems, and pods. Seeds showed pale to dark purple blotches on the seed coat, from small spots to full coverage. These symptoms are typical of Cercospora leaf blight (CLB) and Purple Seed Stain (PSS). In 2018-2019, symptomatic tissues were analyzed. Small fragments of infected stems and pods from five different fields (ten plants/site), were surface-sterilized in 1% sodium hypochlorite for 10 min, rinsed with sterile distilled water, and plated onto Potato Dextrose Agar (BD Difco™) with streptomycin sulphate (300 mg/L). Plates were incubated in the dark at 25°C for 7 days. Seeds were disinfected similarly, but for 1 minute. Single-spore cultures produced deep purple mycelium on the lower surface and grey on the upper. Stromata were absent or poorly developed. Conidiophores were straight, pale to dark brown, 120–180 × 3.0–5.5 µm; conidia were solitary, hyaline, slightly curved, and measured 40–250 × 2.5–4.5 µm (n=25). Eight representative isolates from Friuli-Venezia Giulia, three from pods (CREA-CI24, CREA-CI25, CREA-CI26) and five from seeds (C-N7, C-170-1, C-203-4, C-203-5, C-212), underwent multilocus molecular analysis (Groenewald et al., 2012). DNA was extracted and internal transcribed spacer region (ITS), calmodulin (CAL), and histone H3 (HIS3) were amplified (White et al., 1990; Carbone and Kohn, 1999; Crous et al., 2004) and Sanger sequenced. Sequences were deposited in GenBank (ITS: MK989496–98, PX092092–96; CAL: MK991296–98, PX091946–50; HIS3: MK991293–95, PX091951–55). MegaBLAST analysis showed 99% to 100% identity with reference sequences of C. cf. flagellaris (ITS: JX143614, CAL: JX142867, HIS3: JX142621). Phylogenetic analysis using concatenated sequences and 63 references confirmed clustering within C. cf. flagellaris. Pathogenicity tests (Cai & Schneider, 2005) were conducted with strains CI24, CI25, and CI26, in triplicate. Sterile soybean leaves on living plants were sprayed with conidial suspension (10⁴ conidia/mL) and incubated at 25°C under moist conditions. After 8–10 days, necrosis and blight symptoms developed as in the field. No symptoms were seen in controls. Reisolation from symptomatic inoculated leaves confirmed C. cf. flagellaris as the causal agent, based on morphology and ITS sequences. The symptoms observed in this study align with Cercospora-related diseases (CLB and PSS). Previously attributed to C. kikuchii, recent studies (Soares et al., 2015; Albu et al., 2016) showed that C. cf. flagellaris and C. cf. sigesbeckia are also involved. Fernandes et al. (2025) reported that C. cf. flagellaris causes milder symptoms on young soybean leaves than other Cercospora spp., consistent with our observations. To our knowledge, this is the first report of C. cf. flagellaris on soybean in Italy. Soybean covers about 300,000 ha in the country, mainly in northeastern valleys, and is a key food and feed crop. This finding expands known Cercospora diversity associated with CLB and PSS in Italy and highlights the need for further studies on pathogen distribution, epidemiology, cultivar susceptibility, and management strategies to sustain soybean production.

Calmodulin enhancement of mitochondrial calcium uniporter function in isolated mitochondria.

Deciphering mutational effects on inducible NO synthase conformational dynamics via quantitative cross-linking mass spectrometry and AlphaFold2 subsampling.

Molecular, morphophysiological, and pathogenic characterization of Colletotrichum isolates from strawberry plants in Santa Catarina, Brazil.

Exploiting Anisotropic Orientation in Nanoparticle-Aided Cryo-Electron Microscopy Sampling.

Molecular interactions of the NaV1.5 C-terminal domain: CaM sequestered the IQ motif from the CTD.

The plant species of Cynanchum rostellatum (Apocynaceae; syn: Metaplexis japonica) is widely distributed in China. Because of its multiple active medicinal components in roots, fruit husks and seeds, and therapeutic properties, it is a common traditional Chinese herb (Li et al., 2022). In September 2023, black punctate lesions were observed on the leaves and fruit husks of the plant in roadside shrubs at Dalian Minzu University (39°2'50"N, 121°46'27"E), Liaoning Province, China. Additionally, extensive black necrotic patches grew on leaves, fruits and stems. Initial infections appeared as small black spots on the host's epidermal tissues. As the disease progressed, the adjacent lesions coalesced, forming larger blackened areas. In severe cases, the infected stems became entirely blackened, with branching nodes exhibiting high susceptibility. Microscopic examination revealed that the pathogen primarily colonized superficial host cells without invading vascular bundles or internal fruit husk tissues. On PDA, the fungal isolates initially produced white aerial hyphae after 3–4 days of incubation. After one week the hyphae proliferated, forming a dense mycelial mat. The colony reverse initially appeared yellow, but gradually became brownish-black. After approximately 40 days, mature black perithecia，with aggregated beak-like ostioles developed within the colony. Conidiomata observed on host tissues were globose to ovoid, unilocular, and embedded in stromata, measuring 95-350 µm in diam.. Conidiogenous cells were phialidic, cylindrical, with an enlarged base and a slightly tapered apex, measuring 6.5–12.3 × 1.3–2.5 µm. Two types of conidia were observed, α-conidia were aseptate, fusiform, hyaline, 5.5‒8.0 × 1.8‒3.0 µm and biguttulate; β-conidia were filiform, curved, smooth, 21.5–25 × 1.0–1.5 mm. Conidiogenous cells subcylindrical, phialidic, terminal, slightly tapering towards the apex, 6.5‒12.5 × 1.3‒2.8 mm. These morphological features align with the descriptions of the anamophic Phomopsis charateristics of Diaporthe compacta (Gao et al., 2016). In order to molecularly identify the fungal isolate LM-0035, a multiple locus DNA sequences of Internal Transcribed Spacer (ITS), Elongation Factor 1-alpha (EF-1α), β-tubulin (TUB), histone H3 (HIS), and calmodulin (CAL) were amplified for the fungus by using the primer pairs of ITS1/ITS4(White et al. 1990), EF-F/TEF-R, TI/BT2b(Carbone et al.,1999), CYLH3F/H3-1b(Glass, 1995), and CAL-228F/CAL-737R(Carbone et al.,1999), respectively. The resulting sequences were deposited in GenBank under the accession numbers PV864833 (ITS), PV768881 (EF-1α), PV768880 (TUB), PV768879 (HIS), and PV768878 (CAL). The phylogenetic analysis revealed that the isolated LM-0035 clustered together with several reference strains of D. compacta (e.g., SKJ1, LC3076, ZJUE0403) with strong bootstrap support (100%), indicating a close evolutionary relationship with D. compacta. The phylogenetic tree also clearly delineated other reference isolates into distinct clades corresponding to D. sojae, D. unshiuensis, D. biguttulata, D. sennae, and D. eres, each supported by high bootstrap values. These results demonstrate that the topology of the phylogenetic tree is robust and the phylogenetic relationships are reliable. The outgroup strain Diaporthella corylina effectively rooted the tree and supported the overall structure of the phylogeny. The phylogenetic analysis revealed that isolate LM-0035 is D. compacta. For pathogenicity assessment, the fungal isolate LM-0035 was inoculated onto healthy stems and leaves of Cynanchum rostellatum in vitro (with three replicates and one control). Chlorosis and yellowing of the leaves were observed obviously after 4 days of inoculation, followed by browning and wilting of the entire leaf after 7 days. two weeks later, black granular structures (conidiomata) began to form along the main vascular bundles on both sides. Inoculation on stems showed visible infection after 3 days, with typical near-elliptical brown lesions and surrounded by water-soaked halos after two weeks. These lesions rapidly extended along the stem, reaching about 1.5 cm in length. The re-isolating procedure confirmed the original pathogenic fungus. This fungus was also reported on Camellia in China (Gao et al., 2016). To our knowledge, this is the first report of D. compacta on Cynanchum rostellatum in China. The identification of this fungal pathogen provides a theoretical guidance for the disease management.

Adenylyl Cyclase isoform 1 (AC1), responsible for synthesizing the signaling molecule cyclic adenosine monophosphate (cAMP), is key in synaptic plasticity, long-term chronic pain syndromes, osteosarcoma-associated pain, and drug abuse. The protein calmodulin (CaM) and the small molecule forskolin (Fsk) stimulate AC1 to form a catalytic site for ATP catalysis; however, molecular AC1 structural changes triggered by CaM and Fsk remain poorly understood. This study developed a computational model for AC1-cofactor complexes and used all-atom molecular dynamics (MD) simulations to determine how CaM and Fsk individually and jointly affect AC1 dynamics and assess whether they exhibit any synergistic effects. Four systems were investigated: AC1-No Partner, AC1-CaM, AC1-Fsk, and AC1-CaM-Fsk. Simulations revealed that individual and joint cofactor bindings induced unique structural changes within the regulatory C1b subdomain of AC1. CaM and Fsk binding result in a reduced cross section of the catalytic site, implying tighter binding for ATP. Notably, the results showed that the AC1-CaM-Fsk system exhibited unique features distinct from the AC1-CaM and AC1-Fsk systems, demonstrating synergistic effects of CaM and Fsk. Our understanding of AC1-cofactor interactions can guide future research toward modulating AC1 activity, potentially contributing to the development of novel treatments for AC1-associated diseases.

Mammalian cyclic nucleotide-gated (CNG) channels play crucial roles in visual and olfactory signal transduction. In olfactory sensory neurons, the native CNG channel functions as a heterotetramer consisting of CNGA2, CNGA4, and CNGB1b subunits and is activated by cAMP. Calmodulin (CaM) modulates the activity of the olfactory CNG channel, enabling rapid adaptation to odorants. Here we present cryo-EM structures of the native human olfactory CNGA2/A4/B1b channel in both CaM-bound closed and cAMP-bound open states, elucidating the molecular basis of the 2:1:1 subunit stoichiometry in channel assembly and the asymmetrical channel gating upon cAMP activation. Combining structural and functional analyses with AlphaFold prediction, we define two distinct CaM binding sites (CaM1 and CaM2) on the N- and C-terminal regions of CNGB1b, respectively, shedding light on the molecular mechanism of Ca2+/CaM-mediated rapid inhibition of the native olfactory CNG channel.

Abstract All‐protein‐based materials are attractive for their genetic encodability, precise structure, and versatile functions, yet integrating mechanical strength, dynamic adaptability, and functional activity in one system remains challenging. Herein, we report a multi‐stimuli‐responsive, self‐healing, all‐protein‐based network with an interwoven network topology, whose mechanics can be further reinforced by topologically confined micro‐association upon tempering. The network was constructed by polymerizing pseudo[2]catenanes—which employ p53dim for entanglement and SpyTag(DA)‐SpyCatcher complex for physical cyclization—that are opened into a star‐like conformation. Network formation can be triggered by increasing concentration, calmodulin (CaM) binding, or light irradiation (when azoswitch‐modified CaM is used). Subsequent tempering unfolds the SpyTag/SpyCatcher complex, inducing micro‐association that acts as additional crosslinks within the topologically confined network. While the entangled architecture minimizes chain slippage, the micro‐associations enhance crosslinking and stress dissipation, collectively improving mechanical properties and long‐term stability. We further demonstrate its practical utility in controlled release and enzyme immobilization, establishing topological proteins as a versatile platform for designing genetically programmable, mechanically tunable, stimuli‐responsive biomaterials.

All-protein-based materials are attractive for their genetic encodability, precise structure, and versatile functions, yet integrating mechanical strength, dynamic adaptability, and functional activity in one system remains challenging. Herein, we report a multi-stimuli-responsive, self-healing, all-protein-based network with an interwoven network topology, whose mechanics can be further reinforced by topologically confined micro-association upon tempering. The network was constructed by polymerizing pseudo[2]catenanes-which employ p53dim for entanglement and SpyTag(DA)-SpyCatcher complex for physical cyclization-that are opened into a star-like conformation. Network formation can be triggered by increasing concentration, calmodulin (CaM) binding, or light irradiation (when azoswitch-modified CaM is used). Subsequent tempering unfolds the SpyTag/SpyCatcher complex, inducing micro-association that acts as additional crosslinks within the topologically confined network. While the entangled architecture minimizes chain slippage, the micro-associations enhance crosslinking and stress dissipation, collectively improving mechanical properties and long-term stability. We further demonstrate its practical utility in controlled release and enzyme immobilization, establishing topological proteins as a versatile platform for designing genetically programmable, mechanically tunable, stimuli-responsive biomaterials.

Calcium signaling is essential for coordinating plant responses to diverse stimuli and regulating growth and development. Among calcium sensors, calmodulin (CaM) and CaM-like proteins (CMLs) represent a class that, despite increasing research, remains incompletely characterized in wheat, with many interacting partners and biological functions remaining largely elusive. This study conducted bioinformatics analyses of subgroup II CaM/CMLs, characterizing their phylogenetic relationships, conserved motifs, sequence features, and cis-elements. Expression analysis revealed that TaCML49-B was significantly upregulated in roots under salt stress. Moreover, TaCML49-B was localized to nucleus, cytoplasm, and membrane. Function characterization demonstrated that overexpression of TaCML49-B in Arabidopsis enhanced salt tolerance, whereas the BSMV-VIGS silencing of TaCML49-B reduced salt resistance in wheat. Furthermore, STRING database prediction analysis and bimolecular fluorescence complementation (BiFC) assay confirmed that TaCML49-B can physically interact with TaIQD23, which encodes an IQ67 domain protein, suggesting its potential involvement in the salt stress signaling pathway. Collectively, our findings indicate that TaCML49-B functions as a positive role in wheat salt stress response, thereby providing novel insights into the functions of TaCML genes and calcium signaling in wheat.

Flooding caused by Hurricane María promoted fungal growth in homes across Puerto Rico, raising concerns about indoor air quality and health risks. This study focuses on identifying Aspergillus species from water-impacted homes in San Juan using culture-based and molecular methods. Aspergillus is a common indoor contaminant in moisture-damaged environments, with some species associated with significant health risks. However, species-level identification is often limited. To address this, we collected samples from 14 homes, identifying 28 Aspergillus isolates through morphological examination and gene sequencing of ITS2, beta-tubulin (benA), and calmodulin (CaM) genes. Species-level identifications of 22 isolates revealed species belonging to the subgenera Aspergillus, Nidulantes, and Circumdanti. We highlighted the CaM gene’s importance in molecular identification by phylogenetic analyses, which showed superior resolution in species differentiation. Culture-based methods also played a crucial role in differentiating closely related species, such as A. flavus and A. oryzae, which molecular methods alone could not reliably separate. Our findings underscore the challenges of Aspergillus identification in post-hurricane, water-impacted indoor environments and emphasize the value of integrating phenotypic and genotypic techniques for accurate species identification. These results contribute to a better understanding of fungal composition and its potential public health implications in disaster-affected settings.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-19869-9.

Arecanut (Areca catechu L.), commonly known as betel nut, is one of the economically important crops in the family Arecaceae, cultivated in Hainan Province, China. This region's warm, humid climate promotes the growth of microorganisms, especially pathogenic fungi. From 2023 to 2024, a leaf spot disease was observed on arecanut seedlings in the research area of the Coconut Research Institute, Chinese Academy of Tropical Agricultural Sciences. The disease affected approximately 8% of seedlings across 6 hectares under natural environmental conditions. The main objective of this study was to identify the causal agent of this unknown disease of arecanut based on pathogenicity tests and morpho-molecular identification using phylogenetic analysis. A total of two isolates were obtained from symptomatic leaf samples collected between 2023 and 2024. The infected leaves exhibited typical disease symptoms, characterized by yellowish to brown spots on the leaf blades, which were regular to irregular in shape and surrounded by a chlorotic yellow halo. The minor spots became more prominent, reducing the chlorophyll content, causing the leaves to split from the center, and giving a broom-like appearance. The color ranged from black to off-white, and the leaf eventually dried and dropped off. We obtained monoconidial isolates by transferring pycnidia onto water agar supplemented with alfalfa rod medium at 25°C and then purified them using the single-spore technique (Shabong and Kayang 2023). The morphological characteristics of the fungal colonies were light grey, with a dense and fluffy appearance, and exhibited a small amount of yellow pigment deposition on the backside of the colony. On the water agar plus alfalfa rod medium, the pile of conidia exuded from the sporangium, appearing as black droplets. α-spores were aseptate, colorless, and transparent, oval to spindle-shaped, 3.9–9.5 × 2.1–3.0 μm; β-spores were colorless, transparent, linear, smooth, curved, base truncated,15.3–28.6 × 1.3–1.9 μm, like those of Diaporthe spp. (Udayanga et al. 2015; Moodispaw et al. 2023). Partial sequences of the internal transcribed spacer (ITS) (White et al. 1990), calmodulin (CAL) (Carbone and Kohn 1999), and translation elongation factor 1-a (TEF) (Carbone and Kohn 1999) were amplified. GenBank accession codes for BLX2 and BLX3 were PV653305 and PV653306 for ITS, PV696108 and PV696109 for CAL, and PV696110 and PV696111 for TEF, respectively. A multilocus phylogenetic analysis based on ITS, CAL, and TEF, using the neighbor-joining method, showed that the two isolates formed a distinct lineage with Diaporthe chamaeropicola CDP460. Arecanut pot plants were artificially inoculated with these isolates using a 1 × 106 conidia/ml suspension, while sterile distilled water mixed with Tween was used as a control. Five plants were used for each replication, and all the experiments were repeated thrice and incubated at 26±2°C, >95% RH, and a 12-h light and dark period. Leaf symptoms with brown leaf spots surrounded by regular to irregular chlorotic yellow halo were observed on the inoculated plants, whereas the control plants did not exhibit any symptoms. Morphological and molecular tests confirmed the identity of the reisolated fungal colonies, fulfilling Koch's postulates. This first report of D. chamaeropicola causing leaf spot on arecanut in China highlights a new threat to arecanut cultivation, emphasizing the need for early detection and effective disease management strategies.

The fruiting stage of soybean (Glycine max L.) is critical for determining both its yield and quality, thereby influencing global production. While some studies have provided partial explanations for the occurrence of Fusarium species on soybean seeds and pods, the fungal diversity affecting soybean pods in Sichuan Province, a major soybean cultivation region in Southwestern China, remains inadequately understood. In this study, 182 infected pods were collected from a maize–soybean relay strip intercropping system. A total of 10 distinct pod-infecting fungal genera (132 isolates) were identified, and their pathogenic potential on soybean seeds and pods was evaluated. Using morphological characteristics and DNA barcode markers, we identified 43 Fusarium isolates belonging to 8 species, including F. verticillioides, F. incarnatum, F. equiseti, F. proliferatum, F. fujikuroi, F. oxysporum, F. chlamydosporum, and F. acutatum through the analysis of the translation elongation factor gene (EF1-α) and RNA polymerases II second largest subunit (RPB2) gene. Multi-locus phylogenetic analysis, incorporating the Internal Transcribed Spacer (rDNA ITS), β-tubulin (β-tubulin), Glyceraldehyde 3-phosphate dehydrogenase (GADPH), Chitin Synthase 1 (CHS-1), Actin (ACT), Beta-tubulin II (TUB2), and Calmodulin (CAL) genes distinguished 37 isolates as 6 Colletotrichum species, including C. truncatum, C. karstii, C. cliviicola, C. plurivorum, C. boninense, and C. fructicola. Among these, F. proliferatum and C. fructicola were the most dominant species, representing 20.93% and 21.62% of the isolation frequency, respectively. Pathogenicity assays revealed significant damage from both Fusarium and Colletotrichum isolates on soybean pods and seeds, with varying isolation frequencies. Of these, F. proliferatum, F. acutatum, and F. verticillioides caused the most severe symptoms. Similarly, within Colletotrichum genus, C. fructicola was the most pathogenic, followed by C. truncatum, C. karstii, C. cliviicola, C. plurivorum, and C. boninense. Notably, F. acutatum, C. cliviicola, C. boninense, and C. fructicola were identified for the first time as pathogens of soybean pods under the maize–soybean strip intercropping system in Southwestern China. These findings highlight emerging virulent pathogens responsible for soybean pod decay and provide a valuable foundation for understanding the pathogen population during the later growth stages of soybean.

In myocardial ischemia-reperfusion (I/R) injury, the downregulation of Cx43 is a critical factor influencing intercellular electrical coupling and the incidence of reperfusion arrhythmias (RA). Restoring Cx43 protein levels in cardiomyocytes has been proposed as an effective strategy to reduce RA. However, the impact of I/R on the functionality of Cx43-based gap junction (GJ) remains unclear. In this study, we utilized a 40-minute hypoxia model that did not alter Cx43 expression in cardiomyocytes but impaired GJ function. This model enabled the assessment of GJ functionality via fluorescent dye transfer without the confounding factor of reduced GJ quantity. Further investigation revealed that hypoxia/reoxygenation (H/R) upregulated calmodulin (CaM) expression and enhanced the interaction between CaM and Cx43. SP15, a peptide mimicking the CaM-binding sequence of Cx43, effectively disrupted the CaM-Cx43 interaction, mitigating H/R-induced GJ dysfunction. Additionally, in an ex vivo rat heart I/R model, SP15 improved myocardial electrophysiological parameters and reduced arrhythmia scores by interfering with CaM-Cx43 binding. These findings provide promising evidence for targeting the CaM-Cx43 interaction to improve GJ function and reduce RA.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-18366-3.

Lonicera japonica Thunb. (Caprifoliaceae), a perennial shrub, is widely used in traditional Chinese medicine (Zheng et al. 2022). In June 2023, a severe outbreak of anthracnose was observed in honeysuckle plantations in Changzhou (31.666°N, 119.505°E), Jiangsu Province, China. This disease still exists to this day, affecting the growth of honeysuckle and reducing the yield of flower buds. The planting area was approximately 12 ha, and examination of 120 L. japonica plants revealed an anthracnose leaf infection rate above 30% (n = 120). In the initial stages of the disease, the spots are scattered and appear yellowish-brown. As the disease progresses, the spots gradually expanded into a round or polygonal shape. The edges of the leaves desiccate, leading to extensive necrosis that ultimately results in leaf abscission. Five symptomatic leaves were collected from five different plants, and small tissue fragments (5×5 mm2) were excised from the periphery of the lesions. The tissues were surface sterilized with 75% ethanol for 45 s followed by 1% NaClO for 90 s, and then rinsed three times with sterile water. Subsequently, the leaves were cultured on PDA medium at 25°C in the dark. After 3 days, all samples displayed similar culture morphology. A representative strain (LJ4-1) was obtained through single spore isolation from all samples. The mycelium on PDA medium was white and fluffy at first and turned gray after 7 days. Conidia were unicellular, hyaline, cylindrical, measuring 10.52 to 17.63 μm in length and 4.5 to 5.6 μm in width (n = 50). These isolates were preliminarily identified as Colletotrichum fructicola based on morphological and cultural characteristics (Jiang et al. 2024). To confirm the pathogen's identity, total DNA was extracted and the internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), actin (ACT), and chitin synthase (CHS-1) regions were amplified using the primer pairs ITS1/ITS4, GDF/GDR, CL1C/CL2C, ACT-512F/ACT-783R, and CHS-79F/CHS-345R, respectively (Weir et al. 2012). The sequences obtained were submitted to GenBank (PQ157567 and PQ179695-PQ179698) and showed 99 to 100% identity to C. fructicola strains (OQ429091 for ITS (572/573 bp), MK949020 for GAPDH (280/280 bp), MZ888803 for CAL (773/773 bp), MZ153138 for ACT (289/289 bp), and MW768831 for CHS-1 (299/299 bp)). Phylogenetic analysis of the concatenated sequences from the five genetic loci, using the maximum-likelihood method, indicated that the isolate was classified as C. fructicola. To assess pathogenicity, the conidial suspension (1 × 105 conidia/ml) of LJ4-1 was introduced onto the leaves of one-year-old L. japonica seedlings without causing any injury, while sterile water served as the control. Six duplicates were conducted for each treatment and all plants were covered with plastic bags to maintain high humidity and placed in a greenhouse at 25°C with a photoperiod of 12 h. After 7 days, all leaves inoculated with the pathogen exhibited typical lesions analogous to those observed in field plants, whereas the control group remained asymptomatic. The morphological and molecular characteristics of the pathogen isolated from infected leaves indicated that both infections were identical. Furthermore, no fungi were recovered from the control plants, thereby supporting Koch's postulates. Previous studies indicated that C. fructicola has been shown to cause anthracnose on more than 110 plants (USDA Fungal Databases, https://fungi.ars.usda.gov/). To the best of our knowledge, this is the first report of C. fructicola causing anthracnose on L. japonica in China. The relevance of our findings lies in their potential to inform the development of strategies aimed at controlling anthracnose on L. japonica.

TRPA1 is an essential calcium (Ca2+)-permeable channel involved in nociception and inflammation. It exhibits complex and mechanistically elusive Ca2+ regulation with initial potentiation then rapid desensitization. We find that the universal Ca2+ sensor Calmodulin (CaM) binds TRPA1 in cells at rest and suppresses channel activity. Combining biochemical, biophysical, modeling, NMR spectroscopy, and functional approaches, we identify an evolutionarily conserved, high-affinity Ca2+/CaM binding element in the TRPA1 distal C-terminus. Genetic or biochemical perturbation of Ca2+/CaM binding to this site yields hyperactive channels that exhibit drastic slowing of desensitization with minor effect on potentiation. Higher extracellular Ca2+ partially rescues slowed desensitization. Our results identify a critical regulatory element in an unstructured TRPA1 region highlighting the importance of these domains, they reveal Ca2+/CaM is an essential TRPA1 auxiliary subunit required for proper channel function, and they suggest that Ca2+/CaM binding at this distal site stabilizes a long-range allosteric mechanism to drive rapid desensitization.

Neurogranin (Ng), a known regulator of neuronal Ca²⁺-calmodulin (CaM) signaling, is linked to Alzheimer's disease. Though well-studied in neurons, Ng is also expressed in brain vasculature, where its function remains unclear. To investigate Ng's role in brain microvascular endothelial cells, we defined its interactome using immunoprecipitation-mass spectrometry (IP-MS) under high- and low-Ca²⁺ conditions. Among 119 Ng-binding proteins, we discovered a novel interaction between Ng and MYH9, a key regulator of cytoskeletal remodeling. Ng-MYH9 binding was prominent in high Ca²⁺ and validated via CaM affinity pulldown and proximity ligation assays. Ng knockdown reduced F-actin levels, while MYH9 knockdown decreased both Ng and F-actin. Loss of Ng-MYH9 also impaired AKT-GSK3β signaling and elevated the endothelial activation marker VCAM1. Ng-null mice exhibited disrupted brain microvascular architecture and reduced MYH9 expression in endothelial cells. These findings reveal a novel Ng pathway promoting MYH9-dependent cytoskeletal remodeling and a potential role in maintaining blood-brain barrier integrity, a previously unrecognized function for Ng in brain health and Alzheimer's disease.