Emerging insights into the application of metal-organic framework (MOF)-based materials for electrochemical heavy metal ion detection.

Heavy metal ions are one of the main sources of water pollution, which has become a major global problem. Given the growing need for heavy metal ion detection, electrochemical sensor stands out for its high sensitivity and efficiency. Metal-organic frameworks (MOFs) have garnered much interest as electrode modifiers for electrochemical detection of heavy metal ions owing to their significant specific surface area, tailored pore size, and catalytic activity. This review summarizes the progress of MOF-based materials, including pristine MOFs and MOF composites, in the electrochemical detection of various heavy metal ions. The synthetic methods of pristine MOFs, the detection mechanisms of heavy metal ions and the modification strategies of MOFs are introduced. Besides, the diverse applications of MOF-based materials in detecting both single and multiple heavy metal ions are presented. Furthermore, we present the current challenges and prospects for MOF-based materials in electrochemical heavy metal ion detection.

First Report of Rosa roxburghii Rot Disease Caused by Fusarium annulatum in China.

Rosa roxburghii Tratt is a rosaceous shrub originating from southwest China (Fu et al. 2020). From September to October 2022, R. roxburghii rot occurred in Guizhou Province, China, within a major R. roxburghii production area covering from 5 ha to 50 ha, with an incidence rate of 10 to 15%. Symptoms manifested as black and brown lesions on the fruit surface, which were concave, soft, foul-smelling, and surrounded by grayish-brown tissue. Three infected R. roxburghii shrubs were randomly collected from each household, placed in transparent plastic bags, and pathogen isolation was conducted in a laboratory. Infected R. roxburghii fruits were surface-sterilized with 0.5% NaOCl for 2 min, rinsed five times with sterile water, and dried. Symptomatic tissues from the margin between necrotic and healthy tissues were cut into 5 × 5 mm pieces, placed onto potato dextrose agar (PDA), and incubated at 28ºC in the dark for 5 days. Hyphal tips of fungi growing from the samples were transferred onto new PDA plates and incubated until they produced conidia. A total of five fungal isolates with similar morphological characteristics were obtained. The colony obtained by single-spore purification was light purple to dark purple with abundant aerial mycelium. Macroconidia were relatively slender with a curve and zero to three septate. Microconidia appeared obovoid to pyriform, with sizes of 5.2 to 17.2 × 2.1 to 3.3 µm (n = 50). The morphological characteristics were consistent with Fusarium annulatum (Yilmaz et al. 2021). Genomic DNA of two representative isolates (Zhaochanglin 1621 and 1622) was extracted using the DN14 cetyltrimethylammonium bromide rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing). The TEF1 and RPB2 gene were amplified by Polymerase Chain Reaction using primers EF1-983F/EF1-2218R (Rehner et al, 2005), bRPB2-6F/bRPB2-7.1R (Matheny et al, 2002), respectively. All sequences were deposited in GenBank (TEF1, PP236860, PP236861; RPB2, PP767864, PP767865). BLAST searches of the TEF1, and RPB2 sequences revealed the TEF1 sequences had 99.89% (937/938 bp) identity with F. annulatum isolate CBS 258.54; and the RPB2 sequences had 99.86% (737/738 bp) identity to isolate CBS 267.93. In the phylogenetic tree, the isolates (Zhaochanglin 1621 and 1622) clustered with the representative strains of F. annulatum. The morphology and multi-gene phylogenetic analysis indicated that is the isolates were F. annulatum. To complete Koch's postulates, five mature, healthy R. roxburghii fruits were surface disinfected with 1% NaClO solution for 1 min, rinsed with sterile water, and dried at 25℃ for 30 min. A conidial suspension (106 spores/ml) collected from two isolates (Zhaochanglin 1621 and 1622) was sprayed onto R. roxburghii fruits, and the control treatments were sprayed with sterile distilled water. All R. roxburghii fruits were incubated at 25 ºC with 80% relative humidity. The experiment had five replicates. After 7 days of incubation, all the inoculated fruits showed similar symptoms to those initially observed on the originally infected plants. The same pathogen was reisolated and identified by morphological character ization and molecular analysis, fulfilling Koch's postulates. Thus, the pathogen causing rot of R. roxburghii was determined to be F. annulatum (H. Zhang et al, 2024). To our knowledge, this is the first report of F. annulatum causing R. roxburghii rot disease in China. F.annulatum has a wide range of hosts and has been reported to infect a wide range of crops, fruits, and vegetables (Bacon and Nelson 1994). This study lays a foundation for further study and developing disease control methods and the improvement of the economic benefits of R. roxburghii.

Caffeic Acid Phenethyl Ester Encapsulated in Self-Assemble Rice Peptides Nanoparticles: Storage Stability, In Vitro Release, and Their Interaction Mechanisms.

Caffeic acid phenethyl ester (CAPE) is an important active component of propolis with many bioactivities. However, its efficiency and practical application are restricted due to its poor aqueous solubility and storage stability. In this study, a nanocarrier was fabricated to encapsulate CAPE using self-assembled rice peptides obtained by controllable enzymolysis. The physicochemical properties, encapsulation efficiency, and loading capacity of rice peptides nanoparticles (RPNs) were characterized. The storage stability, in vitro release, and interaction mechanisms between CAPE and RPNs were investigated. The results showed that RPNs, mainly assembled by disulfide bonds and hydrogen bonds, possessed an effective diameter of around 210 nm and a high encapsulation efficiency (77.77%) and loading capacity (3.89%). Importantly, the water solubility of CAPE was increased by 45 times after RPNs encapsulation. Moreover, RPNs encapsulation also significantly increased CAPE stability, about 1.4-fold higher than that of unencapsulated CAPE after 18-day storage. An in vitro release study demonstrated that RPNs could delay the release of CAPE, implying a better CAPE protection against extreme environments during digestion. Hydrogen bond and van der Waals force are the predominant interaction forces between RPNs and CAPE. Therefore, the newly developed nanoparticle is a potential delivery system that could effectively improve the aqueous solubility and stability of CAPE.

CircHIPK3 relieves vascular calcification via mediating SIRT1/PGC-1α/MFN2 pathway by interacting with FUS.

BACKGROUND: Circular RNAs (circRNAs) have been reported to regulate the biological processes of human diseases. CircHIPK3 has been implicated in vascular calcification, but the downstream regulatory mechanisms remain unclear. Our study aimed to understand the regulatory function of circHIPK3 in vascular calcification. METHODS: CircHIPK3 expression in atherosclerosis (AS) serum samples and vascular smooth muscle cells (VSMCs) calcification model was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The binding relationships between fused in sarcoma (FUS) and circHIPK3 or sirtuin 1 (SIRT1) were verified by RNA immunoprecipitation (RIP) assay and RNA pull-down assays. Alkaline phosphatase (ALP) activity and alizarin red staining assays were performed to evaluate the biological effect of ß-glycerophosphate (ß-GP) and circHIPK3 on calcium deposition. qRT-PCR and western blot assays were used to examine the effect of ß-GP, circHIPK3, SIRT1, mitofusin 2 (MFN2), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) on VSMCs calcification and the expression of calcification-related proteins. RESULTS: In AS serum samples and VSMCs calcification model, the expression of circHIPK3 was significantly reduced. CircHIPK3 overexpression inhibited ALP activity and calcium deposition in ß-GP-induced VSMCs. Moreover, circHIPK3 could recruit FUS to further stabilize SIRT1 mRNA. CircHIPK3 promoted MFN2 expression to alleviate VSMCs calcification via activating SIRT1/PGC-1α signaling. CONCLUSION: The positive regulation of circHIPK3/FUS/SIRT1/PGC-1α/MFN2 signaling pathway contributed to the alleviate VSMCs calcification, revealing a novel regulatory axis for vascular calcification.

Revolutionising hepatocellular carcinoma surveillance: Harnessing contrast-enhanced ultrasound and serological indicators for postoperative early recurrence prediction.

This study aimed to develop a noninvasive predictive model for identifying early postoperative recurrence of hepatocellular carcinoma (within 2 years after surgery) based on contrast-enhanced ultrasound and serum biomarkers. Additionally, the model's validity was assessedthrough internal and external validation. Clinical data were collected from patients who underwent liver resection at the First Hospital of Quanzhou and Mengchao Hepatobiliary Hospital. The data included general information, contrast-enhanced ultrasound parameters, Liver Imaging Reporting and Data System (LI-RADS) classification, and serum biomarkers. The data from Mengchao Hospital were divided into 2 groups, with a ratio of 6:4, to form the modeling and internal validation sets, respectively. On the other hand, the data from the First Hospital of Quanzhou served as the external validation group. The developed model was named the Hepatocellular Carcinoma Early Recurrence (HCC-ER) prediction model. The predictive efficiency of the HCC-ER model was compared with other established models. The baseline characteristics were found to be well-balanced across the modeling, internal validation, and external validation groups. Among the independent risk factors identified for early recurrence, LI-RADS classification, alpha-fetoprotein, and tumor maximum diameter exhibited hazard ratios of 1.352, 1.337, and 1.135 respectively. Regarding predictive accuracy, the HCC-ER, Tumour-Node-Metastasis, Barcelona Clinic Liver Cancer, and China Liver Cancer models demonstrated prediction errors of 0.196, 0.204, 0.201, and 0.200 in the modeling group; 0.215, 0.215, 0.218, and 0.212 in the internal validation group; 0.210, 0.215, 0.216, and 0.221 in the external validation group. Using the HCC-ER model, risk scores were calculated for all patients, and a cutoff value of 50 was selected. This cutoff effectively distinguished the high-risk recurrence group from the low-risk recurrence group in the modeling, internal validation, and external validation groups. However, the calibration curve of the predictive model slightly overestimated the risk of recurrence. The HCC-ER model developed in this study demonstrated high accuracy in predicting early recurrence within 2 years after hepatectomy. It provides valuable information for developing precise treatment strategies in clinical practice and holds considerable promise for further clinical implementation.

A novel model predicted liver cirrhosis constructed by ultrasound and serological in autoimmune liver hepatitis.

To establish a noninvasive model based on two-dimensional shear wave elasticity (2D-SWE) technology, ultrasound feature and serological indicators to predict cirrhosis in autoimmune hepatitis (AIH) and verified. Patients with AIH confirmed by liver biopsy with liver ultrasound and serological examination were collected from January 2019 to May 2022. Patients were divided into cirrhosis and non-cirrhosis groups. Basic indexes, ultrasound indexes and serological indexes were collected. Multivariable logistic regression used for screening independent risk factors predicting cirrhosis, construct the AIH cirrhosis prediction model, named autoimmune hepatitis cirrhosis (AIHC). Determine best cutoff score according to the Youden index, verified the model's predictive efficacy. One hundred forty-six patients were collected. The following indicators were independent risk factors for predicting cirrhosis: LS (OR: 1.416, P = .015), splenomegaly (OR: 10.446, P = .006), complement C4 (OR: 0.020, P = .009). The best cutoff score was 65, with a sensitivity 88.9% and specificity 75.6%; the area under curve was 0.901, AIHC possessed a higher net reclassification index (NRI) and integrated discrimination improvement compared with other indexes, and AIHC had the best clinical decision curve. The AIHC constructed in this study has better predictive efficacy than other noninvasive indexes, and we visualized the model for easy application, which was worth further promotion in clinical practice.

Contrast enhanced ultrasound combined with serology predicts hepatocellular carcinoma recurrence: a retrospective observation cohort study.

Objectives: To construct a novel model based on contrast-enhanced ultrasound (CEUS) and serological biomarkers to predict the early recurrence (ER) of primary hepatocellular carcinoma within 2 years after hepatectomy. Methods: A total of 466 patients who underwent CEUS and curative resection between 2016.1.1 and 2019.1.1 were retrospectively recruited from one institution. The training and testing cohorts comprised 326 and 140 patients, respectively. Data on general characteristics, CEUS Liver Imaging Reporting and Data System (LI-RADS) parameters, and serological were collected. Univariate analysis and multivariate Cox proportional hazards regression model were used to evaluate the independent prognostic factors for tumor recurrence, and the Contrast-enhanced Ultrasound Serological (CEUSS) model was constructed. Different models were compared using prediction error and time-dependent area under the receiver operating characteristic curve (AUC). The CEUSS model's performances in ER prediction were assessed. Results: The baseline data of the training and testing cohorts were equal. LI-RADS category, α-fetoprotein level, tumor maximum diameter, total bilirubin level, starting time, iso-time, and enhancement pattern were independent hazards, and their hazards ratios were 1.417, 1.309, 1.133, 1.036, 0.883, 0.985, and 0.70, respectively. The AUCs of CEUSS, BCLC,TNM, and CNLC were 0.706, 0.641, 0.647, and 0.636, respectively, in the training cohort and 0.680, 0.583, 0.607, and 0.597, respectively, in the testing cohort. The prediction errors of CEUSS, BCLC, TNM, and CNLC were 0.202, 0.205, 0.205, and 0.200, respectively, in the training cohort and 0.204, 0.221, 0.219, and 0.211, respectively, in the testing cohort. Conclusions: The CEUSS model can accurately and individually predict ER before surgery and may represent a new tool for individualized treatment.

Parametric Dynamic Distributed Containment Control of Continuous-Time Linear Multi-Agent Systems with Specified Convergence Speed.

This paper focuses on the distributed containment control of continuous-time linear multi-agent systems (MASs) with multiple leaders over fixed topology. A parametric dynamic compensated distributed control protocol is proposed in which both the information from the observer in the virtual layer and actual adjacent agents are employed. The necessary and sufficient conditions of the distributed containment control are derived based on the standard linear quadratic regulator (LQR). On this basis, the dominant poles are configured by using the modified linear quadratic regulator (MLQR) optimal control and Gersgorin's circle criterion, hence the containment control with specified convergence speed of the MAS is achieved. Another main advantage of the proposed design is, in the case of virtual layer failure, by adjusting parameters the dynamic control protocol reduces to static, and the convergence speed can still be specified through the dominant pole assignment method combined with inverse optimal control. Finally, typical numerical examples are presented to demonstrate the effectiveness of theoretical results.

Down-regulating of MFN2 promotes vascular calcification via regulating RAS-RAF-ERK1/2 pathway.

BACKGROUND: Vascular calcification (VC), as a prevalent feature of atherosclerosis (AS), is a life-threatening pathological change. Mitofusin 2 (MFN2) has been reported to be down-regulated and participate in the pathogenesis of AS. Here, we explored the feasible impacts of MFN2 on VC in AS. METHODS: Atherosclerotic lesion was evaluated by Oil Red O staining. The VC was detected by Alizarin Red S staining, ALP staining, and calcium content in vascular smooth muscle cells (VSMCs) or atherosclerotic mice. The chondrocyte differentiation of VSMCs was measured by Alcian blue staining. Western blotting and qRT-PCR were used to determine the protein and mRNA expression of associated molecules. Intermolecular interaction was measured by ChIP and dual luciferase assays. RESULTS: The expression of MFN2 and E2F1 was reduced in the aorta tissues of AS patients and mice. Silencing of MFN2 drove calcification in VSMCs and aortas of atherosclerotic mice as confirmed by up-regulating RUNX2, OPG levels, and down-regulating SM22α, α-SMA levels. The chondrocyte differentiation of VSMCs was accelerated by MFN2 knockdown through inducing the expression of Aggrecan, Collagen II, and SOX9. In addition, E2F1 promoted the transcription and expression of MFN2 in VSMCs. Overexpression of MFN2 or E2F1 suppressed ox-LDL-induced VSMC calcification. Finally, MFN2 depletion enhanced VSMC calcification via activating RAS-RAF-ERK1/2 pathway. CONCLUSION: Our results suggest that silencing of MFN2 drives VC via activating RAS-RAF-ERK1/2 pathway in the progression of AS, thus MFN2 may be a therapeutic target for AS.

Antioxidant activity, storage stability and in vitro release of epigallocatechin-3-gallate (EGCG) encapsulated in hordein nanoparticles.

Hordein nanoparticles have been successfully fabricated to encapsulate EGCG. The present study aimed to investigate the effect of hordein nanoparticles encapsulation on antioxidant activity, storage stability and in vitro release of EGCG. The antioxidant activities of EGCG before and after encapsulation by hordein nanoparticles had no significant difference. The 28-day storage experiments indicated that EGCG-hordein nanoparticles had a good storage stability in terms of average diameter and zeta potential. Meanwhile, the stability of EGCG was significantly improved by hordein nanoparticles encapsulation. The remaining amount of encapsulated EGCG was 1.31-fold and 1.52-fold higher than that of free EGCG at 4 °C and 25 °C, respectively. The release study in the simulated gastric or intestinal fluids suggested that hordein nanoparticles could slow down the release of EGCG in the simulated gastric or intestinal fluids. These results showed that encapsulation of EGCG using hordein nanoparticles could be a promising approach to improve its stability.

Novel model combining contrast-enhanced ultrasound with serology predicts hepatocellular carcinoma recurrence after hepatectomy.

BACKGROUND: Surgery is the primary curative option in patients with hepatocellular carcinoma (HCC). However, recurrence within 2 years is observed in 30%-50% of patients, being a major cause of mortality. AIM: To construct and verify a non-invasive prediction model combining contrast-enhanced ultrasound (CEUS) with serology biomarkers to predict the early recurrence of HCC. METHODS: Records of 744 consecutive patients undergoing first-line curative surgery for HCC in one institution from 2016-2018 were reviewed, and 292 local patients were selected for analysis. General characteristics including gender and age, CEUS liver imaging reporting and data system (LIRADS) parameters including wash-in time, wash-in type, wash-out time, and wash-out type, and serology biomarkers including alanine aminotransferase, aspartate aminotransferase, platelets, and alpha-fetoprotein (AFP) were collected. Univariate analysis and multivariate Cox proportional hazards regression model were used to evaluate the independent prognostic factors for tumor recurrence. Then a nomogram called CEUS model was constructed. The CEUS model was then used to predict recurrence at 6 mo, 12 mo, and 24 mo, the cut-off value was calculate by X-tile, and each C-index was calculated. Then Kaplan-Meier curve was compared by log-rank test. The calibration curves of each time were depicted. RESULTS: A nomogram predicting early recurrence (ER), named CEUS model, was formulated based on the results of the multivariate Cox regression analysis. This nomogram incorporated tumor diameter, preoperative AFP level, and LIRADS, and the hazard ratio was 1.123 (95% confidence interval [CI]: 1.041-1.211), 1.547 (95%CI: 1.245-1.922), and 1.428 (95%CI: 1.059-1.925), respectively. The cut-off value at 6 mo, 12 mo, and 24 mo was 100, 80, and 50, and the C-index was 0.748 (95%CI: 0.683-0.813), 0.762 (95%CI: 0.704-0.820), and 0.762 (95%CI: 0.706-0.819), respectively. The model showed satisfactory results, and the calibration at 6 mo was desirable; however, the calibration at 12 and 24 mo should be improved. CONCLUSION: The CEUS model enables the well-calibrated individualized prediction of ER before surgery and may represent a novel tool for biomarker research and individual counseling.

Market Impact of Foot-and-Mouth Disease Control Strategies: A UK Case Study.

Foot-and-mouth disease (FMD) poses a serious threat to the agricultural sector due to its highly contagious nature. Outbreaks of FMD can lead to substantial disruptions to livestock markets due to loss of production and access to international markets. In a previously FMD-free country, the use of vaccination to augment control of an FMD outbreak is increasingly being recognized as an alternative control strategy to direct slaughtering [stamping-out (SO)]. The choice of control strategy has implications on production, trade, and hence prices of the sector. Specific choice of eradication strategies depends on their costs and benefits. Economic impact assessments are often based on benefit-cost framework, which provide detailed information on the changes in profit for a farm or budget implications for a government (1). However, this framework cannot capture price effects caused by changes in production due to culling of animals; access to international markets; and consumers' reaction. These three impacts combine to affect equilibrium within commodity markets (2). This paper provides assessment of sectoral level impacts of the eradication choices of FMD outbreaks, which are typically not available from benefit-cost framework, in the context of the UK. The FAPRI-UK model, a partial equilibrium model of the agricultural sector, is utilized to investigate market outcomes of different control strategies (namely SO and vaccinate-to-die) in the case of FMD outbreaks. The outputs from the simulations of the EXODIS epidemiological model (number of animals culled/vaccinated and duration of outbreak) are used as inputs within the economic model to capture the overall price impact of the animal destruction, export ban, and consumers' response.

Dual control of cardiac Na+ Ca2+ exchange by PIP(2): electrophysiological analysis of direct and indirect mechanisms.

Cardiac Na(+)-Ca(2+) exchange (NCX1) inactivates in excised membrane patches when cytoplasmic Ca(2+) is removed or cytoplasmic Na(+) is increased. Exogenous phosphatidylinositol-4,5-bis-phosphate (PIP(2)) can ablate both inactivation mechanisms, while it has no effect on inward exchange current in the absence of cytoplasmic Na(+). To probe PIP(2) effects in intact cells, we manipulated PIP(2) metabolism by several means. First, we used cell lines with M1 (muscarinic) receptors that couple to phospholipase C's (PLCs). As expected, outward NCX1 current (i.e. Ca(2+) influx) can be strongly inhibited when M1 agonists induce PIP(2) depletion. However, inward currents (i.e. Ca(2+) extrusion) without cytoplasmic Na(+) can be increased markedly in parallel with an increase of cell capacitance (i.e. membrane area). Similar effects are incurred by cytoplasmic perfusion of GTPgammaS or the actin cytoskeleton disruptor latrunculin, even in the presence of non-hydrolysable ATP (AMP-PNP). Thus, G-protein signalling may increase NCX1 currents by destabilizing membrane cytoskeleton-PIP(2) interactions. Second, to increase PIP(2) we directly perfused PIP(2) into cells. Outward NCX1 currents increase as expected. But over minutes currents decline substantially, and cell capacitance usually decreases in parallel. Third, using BHK cells with stable NCX1 expression, we increased PIP(2) by transient expression of a phosphatidylinositol-4-phosphate-5-kinase (hPIP5KIbeta) and a PI4-kinase (PI4KIIalpha). NCX1 current densities were decreased by > 80 and 40%, respectively. Fourth, we generated transgenic mice with 10-fold cardiac-specific overexpression of PI4KIIalpha. This wortmannin-insensitive PI4KIIalpha was chosen because basal cardiac phosphoinositides are nearly insensitive to wortmannin, and surface membrane PI4-kinase activity, defined functionally in excised patches, is not blocked by wortmannin. Both phosphatidylinositol-4-phosphate (PIP) and PIP(2) were increased significantly, while NCX1 current densities were decreased by 78% with no loss of NCX1 expression. Most mice developed cardiac hypertrophy, and immunohistochemical analysis suggests that NCX1 is redistributed away from the outer sarcolemma. Cholera toxin uptake was increased 3-fold, suggesting that clathrin-independent endocytosis is enhanced. We conclude that direct effects of PIP(2) to activate NCX1 can be strongly modulated by opposing mechanisms in intact cells that probably involve membrane cytoskeleton remodelling and membrane trafficking.

Modulation of cardiac PIP2 by cardioactive hormones and other physiologically relevant interventions.

Phosphatidylinositol 4,5-bisphosphate (PIP2) affects profoundly several cardiac ion channels and transporters, and studies of PIP2-sensitive currents in excised patches suggest that PIP2 can be synthesized and broken down within 30 s. To test when, and if, total phosphatidylinositol 4-phosphate (PIP) and PIP(2) levels actually change in intact heart, we used a new, nonradioactive HPLC method to quantify anionic phospholipids. Total PIP and PIP2 levels (10-30 micromol/kg wet weight) do not change, or even increase, with activation of Galpha(q)/phospholipase C (PLC)-dependent pathways by carbachol (50 microM), phenylephrine (50 microM), and endothelin-1 (0.3 microM). Adenosine (0.2 mM) and phorbol 12-myristate 13-acetate (1microM) both cause 30% reduction of PIP2 in ventricles, suggesting that diacylglycerol (DAG)-dependent mechanisms negatively regulate cardiac PIP2. PIP2, but not PIP, increases reversibly by 30% during electrical stimulation (2 Hz for 5 min) in guinea pig left atria; the increase is blocked by nickel (2 mM). Both PIP and PIP2 increase within 3 min in hypertonic solutions, roughly in proportion to osmolarity, and similar effects occur in multiple cell lines. Inhibitors of several volume-sensitive signaling mechanisms do not affect these responses, suggesting that PIP2 metabolism might be sensitive to membrane tension, per se.

Nonradioactive analysis of phosphatidylinositides and other anionic phospholipids by anion-exchange high-performance liquid chromatography with suppressed conductivity detection.

Phosphatidylinositol 4,5-biphosphate (PIP(2)) modulates the function of numerous ion transporters and channels, as well as cell signaling and cytoskeletal proteins. To study PIP(2) levels of cells without radiolabeling, we have developed a new method to quantify anionic phospholipid species. Phospholipids are extracted and deacylated to glycero-head groups, which are then separated by anion-exchange HPLC and detected by suppressed conductivity measurements. The major anionic head groups can be quantified in single runs with practical detection limits of about 100 pmol, and the D3 isoforms of phosphatidylinositol phosphate (PIP) and PIP(2) are detected as shoulder peaks. In HeLa, Hek 293 and COS cells, as well as intact heart, PIP(2) amounts to 0.5 to 1.5% of total anionic phospholipid (10 to 30 micromol/liter cell water or 0.15 to 0.45 nmol/mg protein). In cell cultures, overexpression of Type I PIP5-kinase specifically increases PIP(2), whereas overexpression of Type II PI4-kinase can increase both PIP and PIP(2). Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and the D3 isomers of PIP(2) are detected after treatment of cells with pervanadate; in yeast, overexpression of a phosphatidylinositol 3-kinase (VPS34) specifically increases phosphatidylinositol 3-phosphate (PI3P). Using isolated cardiac membranes, lipid kinase and lipid phosphatase activities can be monitored with the same methods. Upon addition of ATP, PIP increases while PIP(2) remains low; exogenous PIP(2) is rapidly degraded to PIP and phosphatidylinositol (PI). In summary, the HPLC methods described here can be used to probe multiple aspects of phosphatidylinositide (Ptide) metabolism without radiolabeling.