Genome-wide identification of nitrate transporter 1/peptide transporter family (NPF) induced by arbuscular mycorrhiza in the maize genome.

The Transporter 1/Peptide Transporter Family (NPF) is essential for the uptake and transport of nitrate nitrogen. Significant increases in nitrogen have been increasingly reported for many mycorrhizal plants, but there are few reports on maize. Here, we have identified the maize NPF family and screened for arbuscular mycorrhiza fungi (AMF) induced NPFs. In this study, a systematic analysis of the maize NPF gene family was performed. A total of 82 NPF genes were identified in maize. ZmNPF4.5 was strongly induced by AMF in both low and high nitrogen. Lotus japonicus hairy root-induced transformation experiments showed that ZmNPF4.5 promoter-driven GUS activity was restricted to cells containing tufts. Yeast backfill experiments indicate that ZmNPF4.5 functions in nitrate uptake. Therefore, we speculate that ZmNPF4.5 is a key gene for nitrate-nitrogen uptake in maize through the mycorrhizal pathway. This is a reference value for further exploring the acquisition of nitrate-nitrogen by maize through AMF pathway. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01464-3.

Metagenomic next-generation sequencing of bronchoalveolar lavage fluid assists in the diagnosis of pathogens associated with lower respiratory tract infections in children.

Worldwide, lower respiratory tract infections (LRTI) are an important cause of hospitalization in children. Due to the relative limitations of traditional pathogen detection methods, new detection methods are needed. The purpose of this study was to evaluate the value of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) samples for diagnosing children with LRTI based on the interpretation of sequencing results. A total of 211 children with LRTI admitted to the First Affiliated Hospital of Guangzhou Medical University from May 2019 to December 2020 were enrolled. The diagnostic performance of mNGS versus traditional methods for detecting pathogens was compared. The positive rate for the BALF mNGS analysis reached 95.48% (95% confidence interval [CI] 92.39% to 98.57%), which was superior to the culture method (44.07%, 95% CI 36.68% to 51.45%). For the detection of specific pathogens, mNGS showed similar diagnostic performance to PCR and antigen detection, except for Streptococcus pneumoniae, for which mNGS performed better than antigen detection. S. pneumoniae, cytomegalovirus and Candida albicans were the most common bacterial, viral and fungal pathogens. Common infections in children with LRTI were bacterial, viral and mixed bacterial-viral infections. Immunocompromised children with LRTI were highly susceptible to mixed and fungal infections. The initial diagnosis was modified based on mNGS in 29.6% (37/125) of patients. Receiver operating characteristic (ROC) curve analysis was performed to predict the relationship between inflammation indicators and the type of pathogen infection. BALF mNGS improves the sensitivity of pathogen detection and provides guidance in clinical practice for diagnosing LRTI in children.

Arbuscular mycorrhizal fungi enhance phosphate uptake and alter bacterial communities in maize rhizosphere soil.

Arbuscular mycorrhizal fungi (AMF) can symbiose with many plants and improve nutrient uptake for their host plant. Rhizosphere microorganisms have been pointed to play important roles in helping AMF to mobilize soil insoluble nutrients, especially phosphorus. Whether the change in phosphate transport under AMF colonization will affect rhizosphere microorganisms is still unknown. Here, we evaluated the links of interactions among AMF and the rhizosphere bacterial community of maize (Zea mays L.) by using a maize mycorrhizal defective mutant. Loss of mycorrhizal symbiosis function reduced the phosphorus concentration, biomass, and shoot length of maize colonized by AMF. Using 16S rRNA gene amplicon high-throughput sequencing, we found that the mutant material shifted the bacterial community in the rhizosphere under AMF colonization. Further functional prediction based on amplicon sequencing indicated that rhizosphere bacteria involved in sulfur reduction were recruited by the AMF colonized mutant but reduced in the AMF- colonized wild type. These bacteria harbored much abundance of sulfur metabolism-related genes and negatively correlated with biomass and phosphorus concentrations of maize. Collectively, this study shows that AMF symbiosis recruited rhizosphere bacterial communities to improve soil phosphate mobilization, which may also play a potential role in regulating sulfur uptake. This study provides a theoretical basis for improving crop adaptation to nutrient deficiency through soil microbial management practices.

Indicator-dependent differences in detection of local intracellular Ca2+ release events evoked by voltage-gated Ca2+ entry in pancreatic ß-cells.

Genetically encoded Ca2+ indicators have become widely used in cell signalling studies as they offer advantages over cell-loaded dye indicators in enabling specific cellular or subcellular targeting. Comparing responses from dye and protein-based indicators may provide information about indicator properties and cell physiology, but side-by-side recordings in cells are scarce. In this study, we compared cytoplasmic Ca2+ concentration ([Ca2+]i) changes in insulin-secreting ß-cells recorded with commonly used dyes and indicators based on circularly permuted fluorescent proteins. Total internal reflection fluorescence (TIRF) imaging of K+ depolarization-triggered submembrane [Ca2+]i increases showed that the dyes Fluo-4 and Fluo-5F mainly reported stable [Ca2+]i elevations, whereas the proteins R-GECO1 and GCaMP5G more often reported distinct [Ca2+]i spikes from an elevated level. [Ca2+]i spiking occurred also in glucose-stimulated cells. The spikes reflected Ca2+ release from the endoplasmic reticulum, triggered by autocrine activation of purinergic receptors after exocytotic release of ATP and/or ADP, and the spikes were consequently prevented by SERCA inhibition or P2Y1-receptor antagonism. Widefield imaging, which monitors the entire cytoplasm, increased the spike detection by the Ca2+ dyes. The indicator-dependent response patterns were unrelated to Ca2+ binding affinity, buffering and mobility, and probably reflects the much slower dissociation kinetics of protein compared to dye indicators. Ca2+ dyes thus report signalling within the submembrane space excited by TIRF illumination, whereas the protein indicators also catch Ca2+ events originating outside this volume. The study highlights that voltage-dependent Ca2+ entry in ß-cells is tightly linked to local intracellular Ca2+ release mediated via an autocrine route that may be more important than previously reported direct Ca2+ effects on phospholipase C or on intracellular channels mediating calcium-induced calcium release.

Construction of a virtual simulation laboratory for gene detection.

OBJECTIVE: The current paper aims to discuss the development of a virtual simulation experiment teaching system and review its effectiveness in improving the teaching of clinical skills to college medical students. METHODS: Collaborators used 3D Studio Max, Unity 3D and Visual Studio to develop four modules: laboratory thinking training, biosafety training, gene testing and experimental assessment. Teaching was conducted and a virtual software program was used for evaluation of the students. RESULTS: The laboratory safety training system, virtual gene experiment system and experimental assessment system were developed. The results of the questionnaire survey show that the software provides good interactivity and guidance. The interest of medical students in study is improved and they received training in clinical experimental thinking. Student evaluation assists their scientific research practice, and can improve the awareness of biosafety. CONCLUSION: The virtual simulation experiment teaching system, when applied in the teaching of undergraduate and postgraduate experiment courses, can bring about rapid improvements in the following areas: biosafety awareness, interest in learning about experiments and experimental skills, clinical experimental thinking, and comprehensive experimental ability.

Genome-wide analysis of 14-3-3 gene family in four gramineae and its response to mycorrhizal symbiosis in maize.

14-3-3 proteins (regulatory protein family) are phosphate serine-binding proteins. A number of transcription factors and signaling proteins have been shown to bind to the 14-3-3 protein in plants, which plays a role in regulating their growth (seed dormancy, cell elongation and division, vegetative and reproduction growth and stress response (salt stress, drought stress, cold stress). Therefore, the 14-3-3 genes are crucial in controlling how plants respond to stress and develop. However, little is known about the function of 14-3-3 gene families in gramineae. In this study, 49 14-3-3 genes were identified from four gramineae, including maize, rice, sorghum and brachypodium, and their phylogeny, structure, collinearity and expression patterns of these genes were systematically analyzed. Genome synchronization analysis showed large-scale replication events of 14-3-3 genes in these gramineae plants. Moreover, gene expression revealed that the 14-3-3 genes respond to biotic and abiotic stresses differently in different tissues. Upon arbuscular mycorrhizal (AM) symbiosis, the expression level of 14-3-3 genes in maize significantly increased, suggesting the important role of 14-3-3 genes in maize-AM symbiosis. Our results provide a better understanding on the occurrence of 14-3-3 genes in Gramineae plants, and several important candidate genes were found for futher study on AMF symbiotic regulation in maize.

BODIPY-Based Multifunctional Nanoparticles for Dual Mode Imaging-Guided Tumor Photothermal and Photodynamic Therapy.

Multifunctional nanoparticles integrating accurate multi-diagnosis and efficient therapy hold great prospects in tumor theranostics. However, it is still a challenging task to develop multifunctional nanoparticles for imaging-guided effective eradication of tumors. Herein, we developed a near-infrared (NIR) organic agent Aza/I-BDP by coupling 2,6-diiodo-dipyrromethene (2,6-diiodo-BODIPY) with aza-boron-dipyrromethene (Aza-BODIPY). Through encapsulating with an amphiphilic biocompatible copolymer DSPE-mPEG5000, well-distributed Aza/I-BDP nanoparticles (NPs) were developed, which exhibited high 1O2 generation, high photothermal conversion efficiency, and excellent photo-stability. Notably, coassembly of Aza/I-BDP and DSPE-mPEG5000 effectively inhibits H-aggregation of Aza/I-BDP in aqueous solution and enhances the brightness simultaneously up to 31-fold. More importantly, in vivo experiments demonstrated that Aza/I-BDP NPs might be used for NIR fluorescent and photoacoustic imaging-guided photodynamic and photothermal therapy.

The value of folate receptor-positive circulating tumor cells in the diagnosis of lung cancer and its correlation with clinical characteristics.

OBJECTIVE: The aim of this research is to investigate the feasibility of folate receptor-positive circulating tumor cells (FR+CTCs) as a biomarker for the diagnosis of malignant pulmonary nodules and the correlation between clinicopathological factors and FR+CTC levels. METHODS: Patients initially diagnosed with one or more pulmonary nodules from a computed tomography scan were prospectively included. Three milliliters of peripheral blood was collected from each participant for FR+CTC analysis prior to surgery. Clinical and pathological parameters and FR+CTC levels were compared between patients with lung cancer and benign diseases. RESULTS: Six hundred fifty-three patients had lung cancer and the other 124 had benign lung diseases based on pathological examinations of the resected specimens. The median FR+CTC value of the lung cancer group was 12.0 (95% CI 9.6-16.2) FU/3 mL and that of the benign group was 7.2 (95% CI 5.78-11.2) FU/3 mL. The difference was statistically significant (P < 0.0001). In a receiver operating characteristic analysis to distinguish the two groups, the area under curve of FR+CTC was 0.7457 (95% CI 0.6893-0.8021; P < 0.0001) using a cutoff of 8.65 FU/3 mL. The sensitivity was 86.37%, and the specificity was 74.19%. Combined with conventional serum tumor biomarkers, the area under curve was 0.922 (0.499-0.963). The sensitivity was 92.20%, and the specificity was 83.05%. FR+CTC levels were related to tumor staging (P4 < 0.001), the degree of tumor invasion both in single (P = 0.011) and multiple lesions (P = 0.022), pathological subtypes (P = 0.013), and maximum tumor diameter (P = 0.014). CONCLUSIONS: FR+CTC is an effective and reliable biomarker for the diagnosis of lung cancer. Further, FR+CTC level is correlated with tumor staging, degree of invasion, pathological subtypes, and tumor size.

The mechanism of promoting rhizosphere nutrient turnover for arbuscular mycorrhizal fungi attributes to recruited functional bacterial assembly.

Symbiosis with arbuscular mycorrhizal (AM) fungi improves plant nutrient capture from the soil, yet there is limited knowledge about the diversity, structure, functioning, and assembly processes of AM fungi-related microbial communities. Here, 16S rRNA gene sequencing and metagenomic sequencing were used to detect bacteria in the rhizosphere of Lotus japonicus inoculated with and without AM fungi, and the L. japonicus mutant ljcbx (defective in symbiosis) inoculated with AM fungi in southern grassland soil. Our results show that AM symbiosis significantly increased bacterial diversity and promoted deterministic processes of bacterial community construction, suggesting that mycorrhizal symbiosis resulted in the directional enrichment of bacterial communities. AM fungi promoted the enrichment of nine bacteria, including Ohtaekwangia, Niastella, Gemmatimonas, Devosia, Sphingomonas, Novosphingobium, Opitutus, Lysobacter, Brevundimonas, which are positively correlated with NPK-related parameters. Through a functional identification experiment, we found that six of these genera, including Brevundimonas, Lysobacter, Ohtaekwangia, Sphingomonas, Devosia, and Gemmatimonas, demonstrated the ability to mineralize organophosphate and dissolve inorganic phosphorus, nitrogen, and potassium. Our study revealed that AM fungi can regulate rhizosphere bacterial community assembly and attract specific rhizosphere bacteria to promote soil nutrient turnover in southern grasslands.

Oxygen self-supplied upconversion nanoplatform loading cerium oxide for amplified photodynamic therapy of hypoxic tumors.

Photodynamic therapy (PDT) has been widely used in preclinical trials for treating various tumors. However, the hypoxic environment of tumors and the limited penetration depth of ultraviolet light severely weaken the PDT effect. To solve the above problems, a near-infrared (NIR) light-triggered oxygen (O2) self-supplied phototherapeutic platform (UCNPs/CeO2/Ce6/BSA) for amplified PDT performance against solid tumors by alleviating tumor hypoxia has been rationally developed. The platform has excellent stability and can continuously decompose H2O2 for sustained O2 supply to synergize 1O2 generation, thus inducing an enhanced mortality rate (59%) of ID8 cells in vitro under hypoxic + H2O2 conditions. The growth of solid tumors was effectively inhibited and the mouse survival rate was dramatically enhanced via a superior PDT therapeutic performance. This reported study facilitated the positive development of multifunctional diagnosis and treatment platforms under long-wavelength excitation for O2 self-supplied tumor treatments.

Tumor-Microenvironment-Activated NIR-II Nanotheranostic Platform for Precise Diagnosis and Treatment of Colon Cancer.

Rational design of tumor-microenvironment (TME)-activated nanoformulation for precisely targeted cancer treatment has recently attracted an enormous attention. However, the all-in-one TME-activated theranostic nanosystems with a simple preparation and high biocompatibility are still rarely reported. Herein, catalase nanocrystals (CatCry) are first introduced as a tumor microenvironment activatable nanoplatform for selective theranostics of colon cancer. They are engaged as (i) a "nanoreactor" for silver nanoparticles (AgNP) synthesis, (ii) a nanovehicle for tumor delivery of anticancer drug doxorubicin (DOX), and (iii) an in situ O2 generator to relief tumor hypoxia. When CatCry-AgNP-DOX nanoformulation is within a tumor, the intratumoral H2S turns AgNP into Ag2S nanoparticles, inducing a photothermal effect and NIR-II emission under 808 nm laser irradiation and also triggering DOX release. Simultaneously, CatCry catalyzes intratumoral H2O2 into O2, relieving hypoxia and enhancing chemotherapy. In contrast, when delivered to healthy tissue without increased concentration of H2S, the developed nanoformulation remains in the "off" state and no theranostic action takes place. Studies with colon cancer cells in vitro and a murine colon cancer model in vivo demonstrated that CatCry-AgNP-DOX delivered a synergistic combination of PTT and enhanced chemotherapy, enabling complete eradication of tumor with minimal side effects. This work not only introduces nanoplatform for theranostics of H2S-rich tumors but also suggests a general strategy for protein-crystal-based nanomedicine.

A novel SCARECROW-LIKE3 transcription factor LjGRAS36 in Lotus japonicus regulates the development of arbuscular mycorrhizal symbiosis.

The symbiosis with arbuscular mycorrhizal (AM) fungi improves plants' nutrient uptake. During this process, transcription factors have been highlighted to play crucial roles. Members of the GRAS transcription factor gene family have been reported involved in AM symbiosis, but little is known about SCARECROW-LIKE3 (SCL3) genes belonging to this family in Lotus japonicus. In this study, 67 LjGRAS genes were identified from the L. japonicus genome, seven of which were clustered in the SCL3 group. Three of the seven LjGRAS genes expression levels were upregulated by AM fungal inoculation, and our biochemical results showed that the expression of LjGRAS36 was specifically induced by AM colonization. Functional loss of LjGRAS36 in mutant ljgras36 plants exhibited a significantly reduced mycorrhizal colonization rate and arbuscular size. Transcriptome analysis showed a deficiency of LjGRAS36 led to the dysregulation of the gibberellic acid signal pathway associated with AM symbiosis. Together, this study provides important insights for understanding the important potential function of SCL3 genes in regulating AM symbiotic development. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01161-z.

Surface-enhanced Raman scattering from an electromagnetic induced transparency substrate for the determination of hepatocellular carcinoma.

Surface-enhanced Raman scattering (SERS) is a powerful analytical method that is especially suitable for the detection of protein molecules. Detection sensitivity of SERS is directly related to the enhancement factor of the substrate, which is dependent on the strength of a local surface electric field generated by surface plasmonic resonance from substrate. In this study, an electromagnetic induced transparency like (EIT-like) metamaterial was used as the SERS substrate. The corresponding plasmonic resonance structure not only produces stronger optical near field but also reduces the spectral line broadening due to radiation damping. This is very beneficial for SERS process, which is strongly dependent on electric field intensity, to improve the sensitivity of SERS detection. Compared with the single resonance mode substrate, the enhancement factor for SERS with the double-mode substrate was increased by an order of magnitude. The obtained EIT-like substrate was used as a SERS-active substrate to detect Lens culinaris agglutinin (LCA)-reactive fraction of AFP (AFP-L3), a hepatocellular carcinoma (HCC)-specific maker. Experimental results are in good agreement with the clinical diagnosis, which demonstrates the potential application of metamaterials in the SERS-based diagnosis and biosensing.

Fluorescent Translocation Reporters for Sub-plasma Membrane cAMP Imaging.

A wide range of fluorescent sensors with different properties have been developed for imaging of cAMP signals in living cells and tissues. Most cAMP reporters have been designed to undergo changes in fluorescence resonance energy transfer but there are alternative techniques with advantages for certain applications. Here, we describe protocols for cAMP recordings in the sub-plasma membrane space based on detection of translocation of engineered, fluorescent protein-tagged protein kinase A subunits between the plasma membrane and the cytoplasm. Changes in reporter localization can be detected with either confocal or total internal reflection fluorescence microscopy but signal changes are more robust and image analyses less complicated with the latter technique. We show how translocation reporters can be used to study sub-plasma membrane cAMP signals, including oscillations, in insulin-secreting ß-cells stimulated with glucose and G-protein-coupled receptor agonists. We also demonstrate how translocation reporters can be combined with other sensors for simultaneous recordings of the cytosolic Ca2+ concentration, protein kinase A activity or plasma-membrane binding of the cAMP effector protein Epac2. Fluorescent translocation reporters thus provide a versatile complement to the growing cAMP imaging toolkit for elucidating sub-plasma membrane cAMP signals in various types of cells.

Determination of human COVID-19 total antibodies in serum using a time-resolved fluorescence immunoassay.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading rapidly around the world. Antibody detection plays an important role in the diagnosis of COVID-19. Here, we established a new time-resolved fluorescence immunoassay (TRFIA) to determine COVID-19 total antibodies. A double-antigen sandwich TRFIA was optimized and established: recombinant nucleocapsid phosphoprotein (N protein) and spike protein (S protein) of COVID-19 immobilized on 96-well plates captured human COVID-19 antibodies and then banded together with the N/S proteins labeled with europium(III) (Eu3+ ) chelates, and finally, time-resolved fluorometry was used to measure the fluorescence values. We successfully established a TRFIA method for the detection of human COVID-19 total antibodies, and the cutoff value was 2.02. There was no cross-reactivity with the negative reference of the National Reference Panel for IgM and IgG antibodies to COVID-19. The CV of the precision assay was 3.19%, and the assay could be stored stably for 15 days at 37°C. Compared with that of the colloidal gold method and chemiluminescence method, the sensitivity of the TRFIA method was higher, and the false positive/negative rate was lower. This established TRFIA has high sensitivity, accuracy, and specificity, which indicates that this method provides a new detection method for the high-throughput routine diagnosis of COVID-19.

Nitric oxide release activated near-Infrared photothermal agent for synergistic tumor treatment.

Activatable phototherapeutic agents (PTA) in one system with synergistic gas therapy (GT) and photothermal therapy (PTT) hold great promise for highly efficient tumor treatments. In this study, an activatable multifunctional platform with photothermal conversion "turn on" features via nitric oxide (NO) release for synergistic GT and PTT was rationally designed using an aryl N-nitrosamine (NO-donating unit) functionalized aza-BODIPY framework (S-NO). As expected, after NO release from S-NO, the product (Red-S) showed obviously enhanced heat production performance under a longer excited wavelength via improved near-infrared light absorption and decreased fluorescence emission. Furthermore, water-soluble and biocompatible S-NO nanoparticles (S-NO NPs) with negligible dark cytotoxicity successfully suppressed tumor growth and enhanced the survival rate of mice via synergistic GT and PTT under the guidance of multimode imaging. The study offered rational guidance to design better platforms for synergistic tumor treatments and validated that S-NO NPs can act as potential PTAs in biological applications.

Human serum albumin gradient in serous ovarian cancer cryosections measured by fluorescence lifetime.

Human serum albumin (HSA) is a depot and carrier for many endogenous and exogenous molecules in blood. Many studies have demonstrated that the transport of HSA in tumor microenvironments contributes to tumor development and progression. In this report, we set up a multimodal nonlinear optical microscope system, combining two-photon excitation fluorescence, second harmonic generation, and two-photon fluorescence lifetime imaging microscopy. The fluorescence lifetime of a small squaraine dye (SD) is used to evaluate HSA concentrations in tumor tissue based on specific binding between SD and HSA. We used SD to stain the cryosections from serous ovarian cancer patients in high-grade (HGSOC) and low-grade (LGSOC), respectively, and found a gradient descent of HSA concentration from normal connective tissue to extracellular matrix to tumor masses from 13 to 2 µM for LGSOC patients and from 36 to 12 µM for HGSOC patients. We demonstrated that multimodal nonlinear optical microscopy can obtain similar results as those from traditional histologic staining, thus it is expected to move to clinical applications.

Highly stable organic photothermal agent based on near-infrared-II fluorophores for tumor treatment.

BACKGROUND: The aim to develop a highly stable near-infrared (NIR) photoinduced tumor therapy agent stems from its considerable potential for biological application. Due to its long wavelength, biological imaging exhibits a high signal-to-background ratio, deep tissue penetration and maximum permissible light power, which can minimize damage to an organism during photoinduced tumor therapy. RESULTS: A class of stable NIR-II fluorophores (NIR998, NIR1028, NIR980, NIR1030, and NIR1028-S) based on aza-boron-dipyrromethene (aza-BODIPY) dyes with donor-acceptor-donor structures have been rationally designed and synthesized by harnessing the steric relaxation effect and intramolecular photoinduced electron transfer (IPET). These fluorophores exhibit an intense range of NIR-II emission, large Stokes shift (≥ 100 nm), excellent photothermal conversion performance, and superior stability against photobleaching. Among the NIR-II fluorophores, NIR998 possesses better NIR-II emission and photothermal conversion performance. NIR998 nanoparticles (NIR998 NPs) can be encapsulated by liposomes. NIR998 NPs show superior stability in the presence of light, heat, and reactive oxygen nitrogen species than that of indocyanine green NPs, as well as a higher photothermal conversion ability (η = 50.5%) compared to other photothermal agents. Finally, under the guidance of photothermal imaging, NIR998 NPs have been proven to effectively eliminate tumors via their excellent photothermal conversion performance while presenting negligible cytotoxicity. CONCLUSIONS: Utilizing IPET and the steric relaxation effect can effectively induce NIR-II emission of aza-BODIPY dyes. Stable NIR998 NPs have excellent photothermal conversion performance and negligible dark cytotoxicity, so they have the potential to act as photothermal agents in biological applications.

Glucose-induced cAMP elevation in ß-cells involves amplification of constitutive and glucagon-activated GLP-1 receptor signalling.

AIM: cAMP typically signals downstream of Gs -coupled receptors and regulates numerous cell functions. In ß-cells, cAMP amplifies Ca2+ -triggered exocytosis of insulin granules. Glucose-induced insulin secretion is associated with Ca2+ - and metabolism-dependent increases of the sub-plasma-membrane cAMP concentration ([cAMP]pm ) in ß-cells, but potential links to canonical receptor signalling are unclear. The aim of this study was to clarify the role of glucagon-like peptide-1 receptors (GLP1Rs) for glucose-induced cAMP signalling in ß-cells. METHODS: Total internal reflection microscopy and fluorescent reporters were used to monitor changes in cAMP, Ca2+ and ATP concentrations as well as insulin secretion in MIN6 cells and mouse and human ß-cells. Insulin release from mouse and human islets was also measured with ELISA. RESULTS: The GLP1R antagonist exendin-(9-39) (ex-9) prevented both GLP1- and glucagon-induced elevations of [cAMP]pm , consistent with GLP1Rs being involved in the action of glucagon. This conclusion was supported by lack of unspecific effects of the antagonist in a reporter cell-line. Ex-9 also suppressed IBMX- and glucose-induced [cAMP]pm elevations. Depolarization with K+ triggered Ca2+ -dependent [cAMP]pm elevation, an effect that was amplified by high glucose. Ex-9 inhibited both the Ca2+ and glucose-metabolism-dependent actions on [cAMP]pm . The drug remained effective after minimizing paracrine signalling by dispersing the islets and it reduced basal [cAMP]pm in a cell-line heterologously expressing GLP1Rs, indicating that there is constitutive GLP1R signalling. The ex-9-induced reduction of [cAMP]pm in glucose-stimulated ß-cells was paralleled by suppression of insulin secretion. CONCLUSION: Agonist-independent and glucagon-stimulated GLP1R signalling in ß-cells contributes to basal and glucose-induced cAMP production and insulin secretion.

Overexpression of a phosphate transporter gene ZmPt9 from maize influences growth of transgenic Arabidopsis thaliana.

Phosphate transporters (PHTs) are well-known for their roles in phosphate uptake in plants. However, their actions in imparting plant growth in plants are still not so clear. In our previous study, we observed that maize PHT1 gene ZmPt9 plays a significant role in phosphate uptake. In this study, we further characterized ZmPt9 in response to low phosphate condition through ZmPt9 promoter inductive analysis by GUS staining and quantification. To elucidate the function of ZmPt9, we generated overexpression plant in Arabidopsis. ZmPt9 overexpressing Arabidopsis plants conferred small leaves and early flowering compared with the wild-type plants. In addition, ZmPt9 can complement the late flowering phenotype of Arabidopsis mutant pht1;2. The qRT-PCR analysis revealed that overexpression of ZmPt9 in Arabidopsis changed expression levels of some flowering-related genes. Further expressed detection of hormone related genes revealed that GA and auxin maybe the main determinant for growth influences of ZmPt9. In conclusion, these results suggest that apart from phosphate transport activity, ZmPt9 can be further exploited for improving crops growth.