Constructing An Oxyhalide Interface for 4.8†V-Tolerant High-Nickel Cathodes in All-Solid-State Lithium-Ion Batteries.

All-solid-state lithium batteries (ASSBs) have received increasing attentions as one promising candidate for the next-generation energy storage devices. Among various solid electrolytes, sulfide-based ASSBs combined with layered oxide cathodes have emerged due to the high energy density and safety performance, even at high-voltage conditions. However, the interface compatibility issues remain to be solved at the interface between the oxide cathode and sulfide electrolyte. To circumvent this issue, we propose a simple but effective approach to magic the adverse surface alkali into a uniform oxyhalide coating on LiNi0.8Co0.1Mn0.1O2 (NCM811) via a controllable gas-solid reaction. Due to the enhancement of the close contact at interface, the ASSBs exhibit improved kinetic performance across a broad temperature range, especially at the freezing point. Besides, owing to the high-voltage tolerance of the protective layer, ASSBs demonstrate excellent cyclic stability under high cutoff voltages (500 cycles~94.0 % at 4.5 V, 200 cycles~80.4 % at 4.8 V). This work provides insights into using a high voltage stable oxyhalide coating strategy to enhance the development of high energy density ASSBs.

Efficient and Dense Electron Emission from a SiO2 Tunneling Diode with Low Poisoning Sensitivity.

We report a tunneling diode enabling efficient and dense electron emission from SiO2 with low poisoning sensitivity. Benefiting from the shallow SiO2 channel exposed to vacuum and the low electron affinity of SiO2 (0.9 eV), hot electrons tunneling into the SiO2 channel from the cathode of the diode are efficiently emitted into vacuum with much less restriction in both space and energy than those in previous tunneling electron sources. Monte Carlo simulations on the device performance show an emission efficiency as high as 87.0% and an emission density up to 3.0 × 105 A/cm2. By construction of a tunneling diode based on Si conducting filaments in electroformed SiO2, an emission efficiency up to 83.7% and an emission density up to 4.4 × 105 A/cm2 are experimentally realized. Electron emission from the devices is demonstrated to be independent of vacuum pressure from 10-4 to 10-1 Pa without poisoning.

Neutrophil-to-serum iron ratio as a promising inflammatory biomarker for acute myocardial infarction and Gensini score.

Background: We aim to investigate whether the neutrophil-to-serum iron ratio (N/SI) is a promising biomarker for acute myocardial infarction group (AMI) and Gensini score. Methods: A total of 263 patients were enrolled and divided into four groups. The Gensini score was used to gauge the severity of coronary artery stenosis, and inflammatory biomarkers were calculated. Results: The N/SI was substantially higher in the AMI group than those in other groups, and N/SI was an independent risk factor for AMI. In ROC analyses, N/SI had the highest area under curve (AUC) for AMI among those inflammatory biomarkers. N/SI was also proved to be related with Gensini score. Conclusion: N/SI was discovered to be a new and effective inflammatory biomarker for AMI and Gensini score.

Peoples' health is at risk from heart illnesses. The indicators in patients' blood are often used to evaluate the severity of diseases. The authors collected 263 subjects with heart disease and reviewed their clinical data. Their blood was drawn to measure the neutrophil-to-serum iron ratio, a crucial blood biomarker. In conclusion, the level of neutrophil-to-serum iron ratio in these patients was closely associated with the stage and severity of their disease.

Acupoint Catgut Embedding Improves Lipid Metabolism in Exercise-Induced Fatigue Rats via the PPAR Signaling Pathway.

To improve the phenomenon of exercise-induced fatigue that often occurs during horse racing, we previously studied the improvement in exercise tolerance by acupoint catgut embedding preconditioning in an exercise-induced fatigue rat model. We found that acupoint catgut embedding pretreatment effectively improved animal exercise tolerance. Here, by combining transcriptomics and metabolomics, we aimed to explore the underlying mechanisms of this improvement. We used blood biochemical detection combined with ELISA to detect triglyceride (TG), total cholesterol (TC), lactate dehydrogenase (LDH), high-density lipoprotein (HDL), alanine transaminase (ALT), aspartate aminotransferase (AST), and glucose (GLU), arachidonic acid (AA), and free fatty acid (FFA) content and found that acupoint embedding can correct FFA, AA, TG, LDH, and AST in the blood. We used RT-qPCR to measure the expression of genes in tissue from the quadriceps femoris muscle. We found that solute carrier family 27 member 2 (Slc27a2), fatty acid binding protein 1 (Fabp1), apolipoprotein C3 (Apoc3), and lipoprotein lipase (Lpl) genes in the peroxisome proliferator-activated receptor (PPAR) signaling pathway were important. The regulation of lipid metabolism through the PPAR signaling pathway was important for improving the exercise endurance of rats in our exercise-induced fatigue model. Therefore, we conclude that acupoint catgut embedding can not only promote body fat decomposition and reduce lactic acid accumulation but also promote the repair of tissue damage and liver damage caused by exercise fatigue. Acupoint catgut embedding regulates the PPAR signaling pathway by upregulating Lpl expression and downregulating Slc27a2, Fabp1, and Apoc3 expression to further improve body fat metabolism.

In situ study of wet chemical etching of ZnO nanowires with different diameters and polar surfaces by LCTEM.

Understanding how nanomaterials evolve during the etching process is critical in many fields. Herein, the wet chemical etching process of zinc oxide (ZnO) nanowires is studied in situ in radiolytic water via liquid cell transmission electron microscopy (LCTEM). The dissolution rate of thin nanowires is constant with reducing diameter, while thick nanowires (with the original diameter being larger than 95 nm) show complicated etching behaviors. The dissolution rate of thick nanowires is constant at the first stage and then increases. Anisotropic etching occurs at both ends of thick nanowires and distinct tips are formed. Different polarities at the two ends of the nanowire lead to differently shaped tips and different tip formation processes. The arrangement of the sidewall cones determines the macroscopic angle of the final tips. The present results are important for understanding liquid phase etching behavior in different dimensions and with different polar ends.

Temperature-Driven α-ß Phase Transformation and Enhanced Electronic Property of 2H α-In2Se3.

In recent years, thin layered indium selenide (In2Se3) has attracted rapidly increasing attention due to its fascinating properties and promising applications. Here, we report the temperature-driven α-ß phase transformation and the enhanced electronic property of 2H α-In2Se3. We find that 2H α-In2Se3 transforms to ß-In2Se3 when it is heated to a high temperature, and the transformation temperature increases from 550 to 650 K with the thickness decreasing from 67 to 17 nm. Additionally, annealing the sample below the phase transformation temperature can effectively improve the electronic property of a 2H α-In2Se3 field-effect transistor, including increasing the on-state current, decreasing the off-state current, and improving the subthreshold swing. After annealing, not only the contact resistance decreases significantly but also the mobility at 300 K increases more than 2 times to 45.83 cm2 V-1 s-1, which is the highest among the reported values. Our results provide an effective method to improve the electrical property and the stability of the In2Se3 nanodevices.

Near-Infrared Artificial Optical Synapse Based on the P(VDF-TrFE)-Coated InAs Nanowire Field-Effect Transistor.

Optical synapse is the basic component for optical neuromorphic computing and is attracting great attention, mainly due to its great potential in many fields, such as image recognition, artificial intelligence and artificial visual perception systems. However, optical synapse with infrared (IR) response has rarely been reported. InAs nanowires (NWs) have a direct narrow bandgap and a large surface to volume ratio, making them a promising material for IR detection. Here, we demonstrate a near-infrared (NIR) (750 to 1550 nm) optical synapse for the first time based on a poly(vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE))-coated InAs NW field-effect transistor (FET). The responsivity of the P(VDF-TrFE)-coated InAs NW FET reaches 839.3 A/W under 750 nm laser illumination, demonstrating the advantage of P(VDF-TrFE) coverage. The P(VDF-TrFE)-coated InAs NW device exhibits optical synaptic behaviors in response to NIR light pulses, including excitatory postsynaptic current (EPSC), paired-pulse facilitation (PPF) and a transformation from short-term plasticity (STP) to long-term plasticity (LTP). The working mechanism is attributed to the polarization effect in the ferroelectric P(VDF-TrFE) layer, which dominates the trapping and de-trapping characteristics of photogenerated holes. These findings have significant implications for the development of artificial neural networks.

The Role of Macrophage Iron Overload and Ferroptosis in Atherosclerosis.

Ferroptosis is a new type of cell death caused by iron-dependent lipid peroxidation. In recent years, it has been found that ferroptosis can promote the progression of atherosclerosis (AS). Macrophages have been proven to play multiple roles in the occurrence and development of AS. Iron is a necessary mineral that participates in different functions of macrophages under physiological conditions. But iron overload and ferroptosis in macrophages may promote the progression of AS. Herein, we summarize the role of iron overload and ferroptosis in macrophages in AS from the perspective of iron metabolism, and iron overload and ferroptosis are significant contributors to AS development.

Enhancing the electrical performance of InAs nanowire field-effect transistors by improving the surface and interface properties by coating with thermally oxidized Y2O3.

Due to their excellent electrical characteristics, InAs nanowires (NWs) have great potential as conducting channels in integrated circuits. However, the surface effect and loose native oxide coverage can deteriorate the performance of InAs NW transistors. Y2O3, a high-k dielectric with low Gibbs free energy, has been proposed to modify the InAs NW surface. Here, we systematically investigate the effect of Y2O3 coating on the performance of InAs NW field-effect transistors (FETs). We first explore the influence of the thermal oxidation process of Y2O3 on the performance of back-gated FETs. We then observe that the coverage of Y2O3/HfO2 bilayers on the NW decreases the hysteresis (the smallest value reaches 0.1 V), subthreshold swing (SS, down to 169 mV dec-1) and on-state resistance Ron, and increases the field-effect mobility µFE (up to 4876.1 cm2 V-1 s-1) and the on-off ratio, mainly owing to the passivation effect on the NW surface. Finally, paired top-gated NW FETs with a Y2O3/HfO2 bilayer and a single layer of HfO2 dielectric are fabricated and compared. The Y2O3/HfO2 bilayer provides better gate control (SSmin = 113 mV dec-1) under a smaller gate oxide capacitance, with an interface trap density as low as 1.93 × 1012 eV-1 cm-2. The use of the Y2O3/HfO2 stack provides an effective strategy to enhance the performance of III-V-based transistors for future applications.

Comparative study on physicochemical characteristics, α-glucosidase inhibitory effect, and hypoglycemic activity of pectins from normal and Huanglongbing-infected navel orange peels.

This study aimed at comparing the physicochemical characteristics, α-glucosidase inhibitory effect, and hypoglycemic activity of pectins (N-NOP and H-NOP) from peels of normal and Huanglongbing (HLB)-infected Navel oranges. Results indicated the pectins were high methoxy pectins mainly composed of homogalacturonan and rhamnogalacturonan-I. The pectins exhibited similar functional groups, surface morphology, and particle size, and had no triple-helical conformation in solution. They exerted fat and glucose absorption capacities and were mixed-type noncompetitive α-glucosidase inhibitors with IC50 values of 1.182 and 2.524 mg/ml, respectively. Both N-NOP and H-NOP showed hypoglycemic activity in alloxan-induced diabetic mice. Administration of them could promote the synthesis of hepatic glycogen and/or serum insulin to lower blood glucose levels and enhance antioxidant status to alleviate oxidative stress injury in diabetic mice. Moreover, N-NOP had higher yield, molecular weight, ζ-potential, oil holding capacity, α-glucosidase inhibitory effect and in vivo hypoglycemic activity, whereas H-NOP possessed higher uronic acid, degree of esterification, thermal stability, water holding capacity, swelling capacity, and fat absorption capacity. It could be concluded that some similarities and differences existed between N-NOP and H-NOP in physicochemical characteristics, functional properties, α-glucosidase inhibitory effects, and hypoglycemic activity. This study provides references for the basic research and application of pectins from peels of normal and HLB-infected Navel oranges. PRACTICAL APPLICATIONS: Pectin has been widely used in the food and pharmaceutical industries for several decades due to its health benefit, gelling, thickening, and emulsification performances. Diabetes mellitus is a worldwide concern in recent years. Pectins (N-NOP and H-NOP) from peels of normal and Huanglongbing (HLB)-infected Navel oranges possessed in vitro and in vivo hypoglycemic activities, indicating they were potential anti-antidiabetic substitutes of chemical drugs. Moreover, comparative understanding on the physicochemical characteristic, α-glucosidase inhibitory effect and hypoglycemic activity of pectins from peels of normal and Huanglongbing-infected Navel oranges was conducive to the recycling and utilization of Navel orange peels. Recently, the biological activity of pectin from peels of normal Navel oranges has been rarely reported, and the information on pectin from peels of Huanglongbing-infected Navel orange is rare. This study provides references for the basic research and application of pectins from peels of normal and HLB-infected Navel oranges.

Mesoscopic sliding ferroelectricity enabled photovoltaic random access memory for material-level artificial vision system.

Intelligent materials with adaptive response to external stimulation lay foundation to integrate functional systems at the material level. Here, with experimental observation and numerical simulation, we report a delicate nano-electro-mechanical-opto-system naturally embedded in individual multiwall tungsten disulfide nanotubes, which generates a distinct form of in-plane van der Waals sliding ferroelectricity from the unique combination of superlubricity and piezoelectricity. The sliding ferroelectricity enables programmable photovoltaic effect using the multiwall tungsten disulfide nanotube as photovoltaic random-access memory. A complete "four-in-one" artificial vision system that synchronously achieves full functions of detecting, processing, memorizing, and powering is integrated into the nanotube devices. Both labeled supervised learning and unlabeled reinforcement learning algorithms are executable in the artificial vision system to achieve self-driven image recognition. This work provides a distinct strategy to create ferroelectricity in van der Waals materials, and demonstrates how intelligent materials can push electronic system integration at the material level.

The studies on wet chemical etching via in situ liquid cell TEM.

Wet chemical etching is a widely used process to fabricate fascinating nanomaterials, such as nanoparticles with precisely controlled size and shape. Understanding the etching mechanism and kinetic evolution process is crucial for controlling wet chemical etching. The development of in situ liquid cell transmission electron microscopy (LCTEM) enables the study on wet chemical etching with high temporal and spatial resolutions. However, there still lack a detailed literature review on the wet chemical etching studies by in situ LCTEM. In this review, we summarize the studies on wet etching nanoparticles, one-dimensional nanomaterials and nanoribbons by in situ LCTEM, including etching rate, anisotropic etching, morphology evolution process, and etching mechanism. The challenges and opportunities of in situ LCTEM are also discussed.

Detection of intermediates from the polymerization reaction catalyzed by a D302A mutant of class III polyhydroxyalkanoate (PHA) synthase.

Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of (R)-3-hydroxybutyryl-CoA (HB-CoA) into high molecular weight PHB, biodegradable polymers. The class III PHB synthase from Allochromatium vinosum is composed of a 1:1 mixture of two approximately 40 kDa proteins: PhaC and PhaE. Previous studies using site-directed mutagenesis and a saturated trimer of hydroxybutyryl-CoA have suggested the importance of C149 (in covalent catalysis), H331 (in activation of C149), and D302 (in hydroxyl group activation for ester bond formation) in the polymerization process. All three residues are located on PhaC. We now report that incubation of D302A-PhaCPhaE with [14C]-HB-CoA results in detection, for the first time, of oligomeric HBs covalently bound to PhaC. The reaction products have been analyzed by SDS-PAGE, Westerns with PhaCPhaE antibodies, and autoradiography. Different migratory properties of D302A-PhaC on SDS-PAGE have been observed at [14C]-HB-CoA to enzyme (S/E) ratios between 5 and 100. Trypsin digestion and HPLC analysis of the D302A-PhaCPhaE (from a reaction with a S/E ratio of 5) allowed isolation of multiple radiolabeled peptides. N-Terminal sequencing, MALDI-TOF, and ESI mass spectrometric analysis of these peptides revealed that all of the peptides were identical but were modified by (HB)n ranging in size from n = 3 to n = 10. The in vitro results support the role of D302 in elongation rather than in activating the active site cysteine for acylation. This proposal has been further supported by our in vivo studies on a Wautersia eutropha strain in which the class I synthase gene has been replaced with the D302A-PhaCPhaE gene and the organism examined under PHB production conditions by transmission electron microscopy. Very small granules (<0.05 microm) were observed in contrast to the 0.2-0.5 microm granules observed with the wt strain. Use of the D302A synthase has allowed successful interrogation of the initiation and elongation steps catalyzed by the class III synthase.

Class III polyhydroxybutyrate synthase: involvement in chain termination and reinitiation.

Polyhydroxybutyrate (PHB) synthase catalyzes the polymerization of (R)-3-hydroxybutyryl-CoA (CoA = coenzyme A) into high molecular weight PHB. Recombinant wild-type (wt) class III synthase from Allochromatium vinosum (PhaCPhaE(Av)), antibodies to this synthase and to PHB, and [(14)C]hydroxybutyryl-CoA (HB-CoA) have been used to detect oligomeric hydroxybutyrate (HB) units covalently bound to the synthase using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Although a distribution of products is typically observed, short (HB)(n)-bound synthases (designated species I) are most prevalent at low substrate to enzyme (S/E) ratios. Species I is similar to (HB)(n)-PhaC(Av) (n = 3-10 at minimum) recently identified using D302A-PhaCPhaE(Av) (Tian, J., Sinskey, A. J., and Stubbe, J. (2005) Biochemistry 44, 1495-1503). Species I is shown to be an intermediate in the elongation process of PHB synthesis in vitro. The reaction catalyzed by the wt synthase in vitro was further studied under two sets of conditions: at high (70000) and low (<200) S/E ratios. At high S/E ratios, kinetic analysis of the reaction of HB-CoA with the wt synthase monitored using antibodies to PhaCPhaE(Av) and Western blotting revealed the disappearance of PhaC(Av) at early time points and its reappearance as the molecular weight of the PHB approached 1.8 MDa. At low S/E ratios, species I was observed to increase with time after complete consumption of all of the HB-CoA. The results from studies under both sets of conditions suggest that an inherent property of the synthase is chain termination and reinitiation.

Kinetic studies of polyhydroxybutyrate granule formation in Wautersia eutropha H16 by transmission electron microscopy.

Wautersia eutropha, formerly known as Ralstonia eutropha, a gram-negative bacterium, accumulates polyhydroxybutyrate (PHB) as insoluble granules inside the cell when nutrients other than carbon are limited. In this paper, we report findings from kinetic studies of granule formation and degradation in W. eutropha H16 obtained using transmission electron microscopy (TEM). In nitrogen-limited growth medium, the phenotype of the cells at the early stages of granule formation was revealed for the first time. At the center of the cells, dark-stained "mediation elements" with small granules attached were observed. These mediation elements are proposed to serve as nucleation sites for granule initiation. TEM images also revealed that when W. eutropha cells were introduced into nitrogen-limited medium from nutrient-rich medium, the cell size increased two- to threefold, and the cells underwent additional volume changes during growth. Unbiased stereology was used to analyze the two-dimensional TEM images, from which the average volume of a W. eutropha H16 cell and the total surface area of granules per cell in nutrient-rich and PHB production media were obtained. These parameters were essential in the calculation of the concentration of proteins involved in PHB formation and utilization and their changes with time. The extent of protein coverage of the granule surface area is presented in the accompanying paper.

Nontemplate-dependent polymerization processes: polyhydroxyalkanoate synthases as a paradigm.

This review focuses on nontemplate-dependent polymerases that use water-soluble substrates and convert them into water-insoluble polymers that form granules or inclusions within the cell. The initial part of the review summarizes briefly the current knowledge of polymer formation catalyzed by starch and glycogen synthases, polyphosphate kinase (a polymerase), cyanophycin synthetases, and rubber synthases. Specifically, our current understanding of their mechanisms of initiation, elongation (including granule formation), termination, remodeling, and polymer reutilization will be presented. General underlying principles that govern these types of polymerization reactions will be enumerated as a paradigm for all nontemplate-dependent polymerizations. The bulk of the review then focuses on polyhydroxyalkanoate (PHA) synthases that generate polyoxoesters. These enzymes are of interest as they generate biodegradable polymers. Our current knowledge of PHA production and utilization in vitro and in vivo as well as the contribution of many proteins to these processes will be reviewed.

Transcriptional analysis of Ralstonia eutropha genes related to poly-(R)-3-hydroxybutyrate homeostasis during batch fermentation.

Poly-(R)-3-hydroxybutyrate (PHB) homeostasis in Ralstonia eutropha takes place at the interface of the cytosol and the hydrophobic PHB granule. PHB synthesis and degradation are therefore intimately linked to the process of granule assembly and breakdown. Unraveling this time-dependent three-dimensional process requires an understanding of the kinetics of synthesis of relevant proteins. Reverse transcriptase quantitative PCR and quantitative Western blotting were carried out on batch cultures of R. eutropha H16 in order to gain insight into how expression of the PHB-related genes phaA, phaB, phaC, phaP, phaR, phaZ1a, phaZ1b, and phaZ1c changed during a cell growth phase, a PHB production phase, and a PHB utilization phase. phaA, phaB, phaC, phaR, and phaZ1a were transcribed throughout cell growth, PHB production, and PHB degradation. PHB-mediated induction of PhaP expression was shown to occur at the transcriptional level, with transcript levels increasing during PHB production and decreasing during PHB utilization. Levels of PhaP correlated strongly with levels of PHB. Levels of phaZ1b transcript and protein increased sharply during production and decreased during degradation, but transcript accumulation did not depend on PHB production as in the case of phaP. No evidence of phaZ1c expression was found under the experimental conditions used in this study.

Analysis of transient polyhydroxybutyrate production in Wautersia eutropha H16 by quantitative Western analysis and transmission electron microscopy.

Polyhydroxybutyrates (PHBs) are polyoxoesters generated from (R)3-hydroxybutyryl coenzyme A by PHB synthase. During the polymerization reaction, the polymers undergo a phase transition and generate granules. Wautersia eutropha can transiently accumulate PHB when it is grown in a nutrient-rich medium (up to 23% of the cell dry weight in dextrose-free tryptic soy broth [TSB]). PHB homeostasis under these growth conditions was examined by quantitative Western analysis to monitor the proteins present, their levels, and changes in their levels over a 48-h growth period. The proteins examined include PhaC (the synthase), PhaP (a phasin), PhaR (a transcription factor), and PhaZ1(a), PhaZ1(b), and PhaZ1(c) (putative intracellular depolymerases), as well as PhaZ2 (a hydroxybutyrate oligomer hydrolase). The results show that PhaC and PhaZ1(a) were present simultaneously. No PhaZ1(b) or PhaZ1(c) was detected at any time throughout growth. PhaZ2 was observed and exhibited an expression pattern different from that of PhaZ1(a). The levels of PhaP changed dramatically and corresponded kinetically to the levels of PHB. Transmission electron microscopy (TEM) provided the dimensions of the average cell and the average granule at 4 h and 24 h of growth (J. Tian, A. J. Sinskey, and J. Stubbe, J. Bacteriol. 187:3814-3824, 2005). This information allowed us to calculate the amount of each protein and number of granules per cell and the granule surface coverage by proteins. The molecular mass of PHB (10(6) Da) was determined by dynamic light scattering at 4 h, the time of maximum PHB accumulation. At this time, the surface area of the granules was maximally covered with PhaP (27 to 54%), and there were one or two PhaP molecules/PHB chain. The ratio of PHB chains to PhaC was approximately 60, which required reinitiation of polymer formation on PhaC. The TEM studies of wild-type and deltaphaR strains in TSB provided further support for an alternative mechanism of granule formation.

Polyhydroxyalkanoate (PHA) hemeostasis: the role of PHA synthase.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers with properties of thermoplastics Production of these polymers in an economically competitive fashion via bioengineering requires an understanding of the biosynthetic pathway and its regulation. This review summarizes our current knowledge of the mechanism of the class I and III PHA synthases: the initiation, elongation and termination processes. It also summarizes our current understanding of the phase transition from soluble substrates (coenzyme A esters of beta-hydroxyalkanoates) to insoluble granules and our understanding of the requirement for a transcription factor, phasin proteins, and depolymerases in PHA homeostasis.

Ralstonia eutropha H16 encodes two and possibly three intracellular Poly[D-(-)-3-hydroxybutyrate] depolymerase genes.

Intracellular poly[D-(-)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share approximately 30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (Delta phaZ1, Delta phaZ2, Delta phaZ3, Delta phaZ1 Delta phaZ2, Delta phaZ1 Delta phaZ3, Delta phaZ2 Delta phaZ3, and Delta phaZ1 Delta phaZ2 Delta phaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated approximately 80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.