Functional Characterization of Sesquiterpene Synthases and P450 Enzymes in Flammulina velutipes for Biosynthesis of Spiro [4.5] Decane Terpene.

Flammulina velutipes, a popular edible mushroom, contains sesquiterpenes with potential health benefits. We characterized 12 sesquiterpene synthases and one P450 enzyme in F. velutipes using Aspergillus oryzae as a heterologous expression system, culminating in the biosynthesis of 16 distinct sesquiterpene compounds. An enzyme encoded by the axeB gene responsible for the synthesis of the spiro [4.5] decane compound axenol was discovered, and the mechanism of spirocycle formation was elucidated through quantum mechanical calculations. Furthermore, we delineated the role of a P450 enzyme colocated with AxeB in producing the novel compound 3-oxo-axenol. Our findings highlight the diverse array of sesquiterpene skeletons and functional groups biosynthesized by these enzymes in F. velutipes and underscore the effectiveness of the A. oryzae system as a heterologous host for expressing genes in the Basidiomycota genome. These insights into the biosynthesis of bioactive compounds in F. velutipes have significant implications for functional food and drug development.

[Retracted] The AMPK­mTOR axis requires increased MALAT1 expression for promoting granulosa cell proliferation in endometriosis.

[This retracts the article DOI: 10.3892/etm.2020.9453.].

Biochemical characterization of a multiple prenyltransferase from Tolypocladium inflatum.

Prenylation plays a pivotal role in the diversification and biological activities of natural products. This study presents the functional characterization of TolF, a multiple prenyltransferase from Tolypocladium inflatum. The heterologous expression of tolF in Aspergillus oryzae, coupled with feeding the transformed strain with paxilline, resulted in the production of 20- and 22-prenylpaxilline. Additionally, TolF demonstrated the ability to prenylated the reduced form of paxilline, ß-paxitriol. A related prenyltransferase TerF from Chaunopycnis alba, exhibited similar substrate tolerance and regioselectivity. In vitro enzyme assays using purified recombinant enzymes TolF and TerF confirmed their capacity to catalyze prenylation of paxilline, ß-paxitriol, and terpendole I. Based on previous reports, terpendole I should be considered a native substrate. This work not only enhances our understanding of the molecular basis and product diversity of prenylation reactions in indole diterpene biosynthesis, but also provides insights into the potential of fungal indole diterpene prenyltransferase to alter their position specificities for prenylation. This could be applicable for the synthesis of industrially useful compounds, including bioactive compounds, thereby opening up new avenues for the development of novel biosynthetic strategies and pharmaceuticals. KEY POINTS: • The study characterizes TolF as a multiple prenyltransferase from Tolypocladium inflatum. • TerF from Chaunopycnis alba shows similar substrate tolerance and regioselectivity compared to TolF. • The research offers insights into the potential applications of fungal indole diterpene prenyltransferases.

Enhancing surface sensing performance of cascaded high contrast gratings using bound states in the continuum.

We proposed the cascaded high contrast grating (CHCG) structure to enhance surface sensing capabilities through bound states in the continuum (BICs). Utilizing the finite element method (FEM) and rigorous coupled-wave analysis (RCWA), we studied the dispersion relations, far-field contribution CHCGs, and near-field distributions of BICs corresponding to resonance peaks at different wavelengths. Results demonstrate the ability to precisely control symmetry-protected BIC (SP-BIC) and Friedrich-Wintgen BIC (FW-BIC) resonance peaks by altering incident angles and structural parameters, enhancing structure robustness and tunability. Significantly, modes 1 and 2 have demonstrated substantial enhancement in surface refractive index sensing, achieving highest sensitivities at 51 nm/RIU and the figure of merit reaching 490.8 RIU-1, indicating notable advancement in detecting subtle surface changes. In contrast, mode 3 has shown robust performance in bulk refractive index sensing, achieving a sensitivity of 602 nm/RIU and a figure of merit of 5189.65 RIU-1. These findings underscore the significant potential of the structure as a high-performance integrated sensor, particularly for precise environmental and biological monitoring in surface refractive index sensing.

The impact of erythroblast enucleation efficiency on the severity of anemia in patients with myelodysplastic syndrome.

Anemia is the most common manifestation in myelodysplastic syndrome (MDS) patients, but the cause of ineffective hematopoiesis is not fully understood. Enucleation is an important event in the maturation process of erythroblasts. According to a series of morphological phenotypes of the pathological development of MDS erythroblasts, we speculate that there may be enucleation disorders. To verify this hypothesis, we cultured MDS bone marrow CD34+ cells in vitro and induced erythroblast development. The results showed that erythroblast enucleation in MDS was significantly lower than that in the normal group, and the rate of enucleation was positively correlated with hemoglobin concentration. Risk stratification of MDS was performed to further analyze the differences in enucleation among the normal group, low-middle risk group and high-risk group. The results showed that the enucleation rate of the high risk group was higher than that of the low-middle risk group but still lower than that of the normal group. Moreover, the expression of pERK and pAKT in MDS erythroblasts in the high risk group was higher than that in the normal group, while the expression of pERK and pAKT in the low-middle risk group was lower than that in the normal group. Furthermore, the enucleation of MDS was positively correlated with the phosphorylation degree of ERK and AKT. In conclusion, this study reveals that the enucleation of erythroblasts is one of the possible causes of anemia in MDS. Video Abstract.

Fuzzy clustering algorithm for university students' psychological fitness and performance detection.

Students' psychological fitness is unavoidable, hindering personal development, social interactions, peer influence, and adolescence. Academic stress may be the most dominant factor affecting college students' mental well-being. Therefore, improving the monitoring of mental health issues among college students is a vital topic for study. However, identifying the student's stress level is challenging, leading to uncertainty. Hence, this paper suggests Heuristic Fuzzy C-means Clustering Algorithm (HFCA) for analyzing college students' stress levels, psychological well-being and academic performance detection. The data are collected from the Kaggle stress dataset for predicting student mental health. This study investigates the psychological factors affecting students' academic performance using the suggested HFCA. Students' performance may be predicted using the Fuzzy Cognitive Map (FCM) in this study. This study used fuzzy clustering algorithms to discover the most crucial aspects of student success, such as student involvement and satisfaction. A better understanding of the risk factors for and protective factors against poor mental health can serve as the basis for developing policies and targeted interventions to prevent mental health problems and guarantee that at-risk students can access the help they need. The experimental analysis shows the proposed method HFCA to achieve a high student performance ratio of 96.7%, cognitive development ratio of 97.2%, student engagement ratio of 97.5% and prediction ratio of 95.1% compared to other methods.

Plasma Exosomes Aggravate Acute Pancreatitis by Promoting M1 Polarization of Adipose Tissue Macrophages in Obesity-Related Severe Acute Pancreatitis.

BACKGROUND AND AIMS: Obesity may be a risk factor for severe acute pancreatitis (SAP). However, its precise mechanism is not yet fully understood. METHODS: We fed rats with a standard laboratory diet (SLD) and a high-fat diet (HFD). SAP model rats were established by retrograde injection of sodium taurocholate. Serum non-esterified fatty acids (NEFAs), lipase (LPS), and myeloperoxidase (MPO) were measured, as were adipose IL-1, IL-6, IL-10, and TNF-α levels. HE staining was performed to determine the severity of pancreatitis. Serum exosomes were extracted from rats with obesity-related SAP, verified by transmission electron microscopy (TEM) and western blot analysis, and co-cultured with THP-1 cells. Flow cytometry was used to analyze the M1 and M2 phenotypes of macrophages in adipose tissues and THP-1 cells. Q-PCR was used to analyze the levels of IL-1, IL-6, IL-10, and TNF-α in each group of cells. RESULTS: The body weight and serum NEFA concentrations of rats in the HFD group were significantly higher than those in the SLD group. Adipose tissue macrophages in the HFD group exhibited a higher percentage of the M1 type than those in the SLD group. The severity of pancreatitis were significantly increased in the HFD + SAP group. Pro-inflammatory macrophages and cytokines were significantly higher in the HFD + SAP group and THP-1 cells co-cultured with serum exosomes extracted from rats with obesity-related SAP. CONCLUSIONS: Obesity might worsen the severity of pancreatitis by amplifying the immune response and activating M1 polarization in adipose tissue macrophages via serum exosomes in rats of obesity-related SAP. In our study, we isolated exosomes from the serum of mice with obesity-related SAP. After inducing THP-1 cells to become M0-typed macrophages, we co-cultured the cells with exosomes and observed that exosomes from obesity-related SAP increased the proportion of M1-typed macrophages and promoted the release of pro-inflammatory factors such as IL-1, IL-6, and TNF. Therefore, obesity might worsen the severity of pancreatitis by amplifying the immune response and activating M1 polarization in adipose tissue macrophages via serum exosomes in rats of obesity-related SAP.

Dual-band refractive index sensor with cascaded asymmetric resonant compound grating based on bound states in the continuum.

We propose a cascaded asymmetric resonant compound grating (ARCG) for high-performance dual-band refractive index sensing. The physical mechanism of the sensor is investigated using a combination of temporal coupled-mode theory (TCMT) and ARCG eigenfrequency information, which is verified by rigorous coupled-wave analysis (RCWA). The reflection spectra can be tailored by changing the key structural parameters. And by altering the grating strip spacing, a dual-band quasi-bound state in the continuum can be achieved. The simulation results show that the highest sensitivity of the dual-band sensor is 480.1 nm/RIU, and its figure of merit is 4.01 × 105. The proposed ARCG has potential application prospects for high-performance integrated sensors.

High-efficient production of mushroom polyketide compounds in a platform host Aspergillus oryzae.

BACKGROUND: Orsellinic acid (2,4-dihydroxy-6-methylbenzoic acid, OA) and its structural analog o-Orsellinaldehyde, have become widely used intermediates in clinical drugs synthesis. Although the research on the biosynthesis of such compounds has made significant progress, due to the lack of suitable hosts, there is still far from the industrial production of such compounds based on synthetic biology. RESULTS: With the help of genome mining, we found a polyketide synthase (PKS, HerA) in the genome of the Hericium erinaceus, which shares 60% amino acid sequence homology with ArmB from Armillaria mellea, an identified PKS capable of synthesizing OA. To characterize the function of HerA, we cloned herA and heterologously expressed it in Aspergillus oryzae, and successfully detected the production of OA. Subsequently, the introduction of an incomplete PKS (Pks5) from Ustilago maydis containing only three domains (AMP-ACP-R), which was into herA-containing A. oryzae, the resulted in the production of o-Orsellinaldehyde. Considering the economic value of OA and o-Orsellinaldehyde, we then optimized the yield of these compounds in A. oryzae. The screening showed that when maltose was used as carbon source, the yields of OA and o-Orsellinaldehyde were 57.68 mg/L and 15.71 mg/L respectively, while the yields were 340.41 mg/Kg and 84.79 mg/Kg respectively in rice medium for 10 days. CONCLUSIONS: Herein, we successfully expressed the genes of basidiomycetes using A. oryzae heterologous host. As a fungus of ascomycetes, which not only correctly splices genes of basidiomycetes containing multiple introns, but also efficiently produces their metabolites. This study highlights that A. oryzae is an excellent host for the heterologous production of fungal natural products, and has the potential to become an efficient chassis for the production of basidiomycete secondary metabolites in synthetic biology.

Progress in pathogenesis research of Ustilago maydis, and the metabolites involved along with their biosynthesis.

Ustilago maydis is a pathogenic fungus that causes corn smut. Because of its easy cultivation and genetic transformation, U. maydis has become an important model organism for plant-pathogenic basidiomycetes. U. maydis is able to infect maize by producing effectors and secreted proteins as well as surfactant-like metabolites. In addition, the production of melanin and iron carriers is also associated with its pathogenicity. Here, advances in our understanding of the pathogenicity of U. maydis, the metabolites involved in the pathogenic process, and the biosynthesis of these metabolites, are reviewed and discussed. This summary will provide new insights into the pathogenicity of U. maydis and the functions of associated metabolites, as well as new clues for deciphering the biosynthesis of metabolites.

Structural and photoelectron spectroscopic study on the heterotrinuclear nickel-titanium dioxide carbonyl complexes Ni2TiO2(CO) n - (n = 2-4).

Herein, the configurations and intrinsic electronic properties of heteronuclear transition metal dioxide carbonyl anions Ni2TiO2(CO) n - (n = 2-4) in the gas phase were investigated using mass spectrometry coupled anionic photoelectron spectroscopy, ab initio calculations, and simulated density-of-state (DOS) spectra. The results clearly show that the binding of electrons is enhanced by the addition of CO. The ground state structures of Ni2TiO2(CO) n - (n = 2-4) are characterized to show that three transition metal atoms (one Ti atom and two Ni atoms) forming a quasi-line is favored. The interaction between Ni and C becomes weaker as the cluster size increases. The natural electron configuration shows that the extra electron is enriched on O atoms attached to Ti, and there is strong interaction between Ti and O atoms. This work gives significant insight into the configuration and electronic structures of nickel-titanium dioxide carbonyl anions, which has potential application in adsorption of carbon monoxide on the surfaces/interfaces of alloys.

A light-up fluorescence platform based DNA: RNA hybrid G-quadruplet for detecting single nucleotide variant of ctDNA and miRNA-21.

The nucleic acid assay is an area of great concern in the diagnosis and treatment of breast cancer. Here, we developed a DNA: RNA hybrid G-quadruplet (HQ) detection platform based on strand displacement amplification (SDA) and Baby Spinach RNA aptamer for single nucleotide variant (SNV) of circulating tumor DNA (ctDNA) and miRNA-21. This was the first in vitro construction of HQ for the biosensor. It found that HQ had much stronger ability to switch on fluorescence of DFHBI-1T than Baby Spinach RNA alone. Taking advantage of the platform and the FspI enzyme with high specificity, the biosensor achieved ultra-sensitive detection of SNV of the ctDNA (PIK3CA H1047R gene) and miRNA-21. The light-up biosensor had high anti-interference ability in complex actual samples. Hence, the label-free biosensor provided a sensitive and accurate method for early diagnosis of breast cancer. Moreover, it opened a new application model for RNA aptamers.

Amino-functionalized multilayer Ti3C2Tx enabled electrochemical sensor for simultaneous determination of Cd2+ and Pb2+ in food samples.

Herein, amino-functionalized multilayer titanium carbide (NH2-Ti3C2Tx) was developed and applied to electrochemical sensor for simultaneous determination of Cd2+ and Pb2+. The NH2-Ti3C2Tx was prepared by grafting (3-amino-propyl) triethylsilane (APTES) onto the surface of Ti3C2Tx. Its morphology, crystal structure and chemical composition were characterized by field emission scanning electron microscope (FESEM), X-ray diffractometer (XRD), and X-ray photoelectron spectroscopy (XPS). Benefiting from the unique multilayer structure, large active surface area, strong adsorption capacity and excellent electrical conductivity, the functionalized NH2-Ti3C2Tx presented satisfactory electrochemical performances for Cd2+ and Pb2+ determination, and the detection limits were 0.41 µg L-1 and 0.31 µg L-1, respectively. Based on chemical coordination and electrochemical accumulation, the suggested mechanism for Cd2+ and Pb2+ determination was also deeply clarified. Besides, it was successfully applied to simultaneous determination of Cd2+ and Pb2+ in food samples including rice, wheat, sorghum and corn, demonstrating a good practicability.

Double-layer symmetric gratings with bound states in the continuum for dual-band high-Q optical sensing.

Herein, we theoretically demonstrate that a double-layer symmetric gratings (DLSG) resonator consisting of a low-refractive-index layer sandwiched between two high-contrast gratings (HCG) layers, can host dual-band high-quality (Q) factor resonance. We find that the artificial bound states in the continuum (BIC) and Fabry-Pérot BIC (FP-BIC) can be induced by optimizing structural parameters of DLSG. Interestingly, the artificial BIC is governed by the spacing between the two rectangular dielectric gratings, while the FP-BIC is achieved by controlling the cavity length of the structure. Further, the two types of BIC can be converted into quasi-BIC (QBIC) by either changing the spacing between adjacent gratings or changing the distance between the upper and lower gratings. The simulation results show that the dual-band high-performance sensor is achieved with the highest sensitivity of 453 nm/RIU and a maximum figure of merit (FOM) of 9808. Such dual-band high-Q resonator is expected to have promising applications in multi-wavelength sensing and nonlinear optics.

A novel electrochemical sensor for simultaneous detection of Cd2+ and Pb2+ by MXene aerogel-CuO/carbon cloth flexible electrode based on oxygen vacancy and bismuth film.

Herein, a novel MXene aerogel-CuO/carbon cloth (MXA-CuO/CC) electrochemical sensor was constructed, and the synergistic adsorption of heavy metal ions by oxygen vacancies and Bi (III) was investigated with Cd2+ and Pb2+ as detection targets. The oxygen vacancies of CuO have a strong affinity for heavy metal ions, which promoted the adsorption of Cd2+ and Pb2+ on the electrode surface. In addition, the introduced Bi (III) can form alloys with heavy metal ions, which effectively enhanced the adsorption capacity of sensing electrodes for Cd2+ and Pb2+. Differential pulse anodic stripping voltammetry (DPASV) was used to study the performance of MXA-CuO/CC sensitive electrode for the detection of Cd2+ and Pb2+ separately and simultaneously. The constructed sensing electrode has excellent detection performance, and can detect Cd2+ (4 µg L-1- 800 µg L-1) and Pb2+ (4 µg L-1- 1200 µg L-1) simultaneously with detection limits of 0.3 µg L-1 (Cd2+) and 0.2 µg L-1 (Pb2+), respectively. The proposed sensor electrode also has good anti-interference performance, excellent stability and reproducibility. It is worth mentioning that the proposed method can accurately detect Cd2+ and Pb2+ in food and water samples, which is consistent with the detection results of inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectroscopy (AAS).

Efficient production of a cyclic dipeptide (cyclo-TA) using heterologous expression system of filamentous fungus Aspergillus oryzae.

BACKGROUND: Cyclic dipeptides are an important class of natural products owing to their structural diversity and biological activities. In fungi, the cyclo-ring system is formed through the condensation of two α-amino acids via non-ribosomal peptide synthetase (NRPS). However, there are few investigations on the functional identification of this enzyme. Additionally, information on how to increase the production of cyclic dipeptide molecules is relatively scarce. RESULTS: We isolated the Eurotium cristatum NWAFU-1 fungus from Jing-Wei Fu brick tea, whose fermentation metabolites contain echinulin-related cyclic dipeptide molecules. We cloned the cirC gene, encoding an NRPS, from E. Cristatum NWAFU-1 and transferred it into the heterologous host Aspergillus oryzae. This transformant produced a novel metabolite possessing an L-tryptophan-L-alanine cyclic dipeptide backbone (Cyclo-TA). Based on the results of heterologous expression and microsomal catalysis, CriC is the first NRPS characterized in fungi that catalyzes the formation of a cyclic dipeptide from L-tryptophan and L-alanine. After substrate feeding, the final yield reached 34 mg/L. In this study, we have characterized a novel NRPS and developed a new method for cyclic dipeptide production. CONCLUSIONS: In this study we successfully expressed the E. Cristatum NWAFU-1 criC gene in A. oryzae to efficiently produce cyclic dipeptide compounds. Our findings indicate that the A. oryzae heterologous expression system constitutes an efficient method for the biosynthesis of fungal Cyclic dipeptides.

Genome sequencing of Inonotus obliquus reveals insights into candidate genes involved in secondary metabolite biosynthesis.

BACKGROUND: Inonotus obliquus is an important edible and medicinal mushroom that was shown to have many pharmacological activities in preclinical trials, including anti-inflammatory, antitumor, immunomodulatory, and antioxidant effects. However, the biosynthesis of these pharmacological components has rarely been reported. The lack of genomic information has hindered further molecular characterization of this mushroom. RESULTS: In this study, we report the genome of I. obliquus using a combined high-throughput Illumina NovaSeq with Oxford Nanopore PromethION sequencing platform. The de novo assembled 38.18 Mb I. obliquus genome was determined to harbor 12,525 predicted protein-coding genes, with 81.83% of them having detectable sequence similarities to others available in public databases. Phylogenetic analysis revealed the close evolutionary relationship of I. obliquus with Fomitiporia mediterranea and Sanghuangporus baumii in the Hymenochaetales clade. According to the distribution of reproduction-related genes, we predict that this mushroom possesses a tetrapolar heterothallic reproductive system. The I. obliquus genome was found to encode a repertoire of enzymes involved in carbohydrate metabolism, along with 135 cytochrome P450 proteins. The genome annotation revealed genes encoding key enzymes responsible for secondary metabolite biosynthesis, such as polysaccharides, polyketides, and terpenoids. Among them, we found four polyketide synthases and 20 sesquiterpenoid synthases belonging to four more types of cyclization mechanism, as well as 13 putative biosynthesis gene clusters involved in terpenoid synthesis in I. obliquus. CONCLUSIONS: To the best of our knowledge, this is the first reported genome of I. obliquus; we discussed its genome characteristics and functional annotations in detail and predicted secondary metabolic biosynthesis-related genes, which provides genomic information for future studies on its associated molecular mechanism.

Enhanced optical sensing performance in stacked resonant compound gratings.

We proposed a high-performance integrated optical sensor based on a stacked resonant compound grating (SRCG). The transmission spectrum of a SRCG is investigated by the theoretical model that combines the coupled mode theory with the eigenmode information of the grating structures. It is found that the spectral width of the SRCG is controlled by changing its structural parameters such as the strip depth, the period of the grating, and cavity length. The simulation results, which are verified by finite element method (FEM), show that the sensitivity of the sensor is 401.8 nm/RIU with its figure of merit (FOM) as high as 57404. The presented sensor is a promising application for high-performance biosensing.

The Vancomycin Resistance-Associated Regulatory System VraSR Modulates Biofilm Formation of Staphylococcus epidermidis in an ica-Dependent Manner.

The two-component system VraSR responds to the cell wall-active antibiotic stress in Staphylococcus epidermidis. To study its regulatory function in biofilm formation, a vraSR deletion mutant (ΔvraSR) was constructed using S. epidermidis strain 1457 (SE1457) as the parent strain. Compared to SE1457, the ΔvraSR mutant showed impaired biofilm formation both in vitro and in vivo with a higher ratio of dead cells within the biofilm. Consistently, the ΔvraSR mutant produced much less polysaccharide intercellular adhesin (PIA). The ΔvraSR mutant also showed increased susceptibility to the cell wall inhibitor and SDS, and its cell wall observed under a transmission electron microscope (TEM) appeared to be thinner and interrupted, which is in accordance with higher susceptibility to the stress. Complementation of vraSR in the ΔvraSR mutant restored the biofilm formation and the cell wall thickness to wild-type levels. Transcriptome sequencing (RNA-Seq) showed that the vraSR deletion affected the transcription levels of 73 genes, including genes involved in biofilm formation, bacterial programmed cell death (CidA-LrgAB system), glycolysis/gluconeogenesis, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle, etc. The results of RNA-Seq were confirmed by quantitative real-time reverse transcription-PCR (qRT-PCR). In the ΔvraSR mutant, the expression of icaA and lrgAB was downregulated and the expression of icaR and cidA was upregulated, in comparison to that of SE1457. The transcriptional levels of antibiotic-resistant genes (pbp2, serp1412, murAA, etc.) had no significant changes. An electrophoretic mobility shift assay further revealed that phosphorylated VraR bound to the promoter regions of the ica operon, as well as its own promoter region. This study demonstrates that in S. epidermidis, VraSR is an autoregulator and directly regulates biofilm formation in an ica-dependent manner. Upon cell wall stress, it indirectly regulates cell death and drug resistance in association with alterations to multiple metabolism pathways. IMPORTANCE S. epidermidis is a leading cause of hospital-acquired catheter-related infections, and its pathogenicity depends mostly on its ability to form biofilms on implants. The biofilm formation is a complex procedure that involves multiple regulating factors. Here, we show that a vancomycin resistance-associated two-component regulatory system, VraSR, plays an important role in modulating S. epidermidis biofilm formation and tolerance to stress. We demonstrate that S. epidermidis VraSR is an autoregulated system that selectively responds to stress targeting cell wall synthesis. Besides, phosphorylated VraR can bind to the promoter region of the ica operon and directly regulates polysaccharide intercellular adhesin production and biofilm formation in S. epidermidis. Furthermore, VraSR may indirectly modulate bacterial cell death and extracellular DNA (eDNA) release in biofilms through the CidA-LrgAB system. This work provides a new molecular insight into the mechanisms of VraSR-mediated modulation of the biofilm formation and cell death of S. epidermidis.

Ground-State Structures of Hydrated Calcium Ion Clusters From Comprehensive Genetic Algorithm Search.

We searched the lowest-energy structures of hydrated calcium ion clusters Ca2+(H2O)n (n = 10-18) in the whole potential energy surface by the comprehensive genetic algorithm (CGA). The lowest-energy structures of Ca2+(H2O)10-12 clusters show that Ca2+ is always surrounded by six H2O molecules in the first shell. The number of first-shell water molecules changes from six to eight at n = 12. In the range of n = 12-18, the number of first-shell water molecules fluctuates between seven and eight, meaning that the cluster could pack the water molecules in the outer shell even though the inner shell is not full. Meanwhile, the number of water molecules in the second shell and the total hydrogen bonds increase with an increase in the cluster size. The distance between Ca2+ and the adjacent water molecules increases, while the average adjacent O-O distance decreases as the cluster size increases, indicating that the interaction between Ca2+ and the adjacent water molecules becomes weaker and the interaction between water molecules becomes stronger. The interaction energy and natural bond orbital results show that the interaction between Ca2+ and the water molecules is mainly derived from the interaction between Ca2+ and the adjacent water molecules. The charge transfer from the lone pair electron orbital of adjacent oxygen atoms to the empty orbital of Ca2+ plays a leading role in the interaction between Ca2+ and water molecules.