Invited Article: A precise instrument to determine the Planck constant, and the future kilogram.

A precise instrument, called a watt balance, compares mechanical power measured in terms of the meter, the second, and the kilogram to electrical power measured in terms of the volt and the ohm. A direct link between mechanical action and the Planck constant is established by the practical realization of the electrical units derived from the Josephson and the quantum Hall effects. We describe in this paper the fourth-generation watt balance at the National Institute of Standards and Technology (NIST), and report our initial determination of the Planck constant obtained from data taken in late 2015 and the beginning of 2016. A comprehensive analysis of the data and the associated uncertainties led to the SI value of the Planck constant, h = 6.626 069 83(22) × 10(-34) J s. The relative standard uncertainty associated with this result is 34 × 10(-9).

Bridging classical and quantum mechanics.

Using a watt balance and a frequency comb, a mass-energy equivalence is derived. The watt balance compares mechanical power measured in terms of the meter, the second, and the kilogram to electrical power measured in terms of the volt and the ohm. A direct link between mechanical action and the Planck constant is established by the practical realization of the electrical units derived from the Josephson and the quantum Hall effects. By using frequency combs to measure velocities and acceleration of gravity, the unit of mass can be realized from a set of three defining constants: the Planck constant h, the speed of light c, and the hyperfine splitting frequency of 133Cs.

Chronocoulometry for quantitative control of mass removal in micro-structures and sensors.

In this work, tungsten wires have been etched in a KOH electrolyte solution. Based on the oxidation state of the electrolytic dissolution reaction's product and time integration of the Faradaic current produced during the reaction, this method is capable of providing a direct measurement of the change in mass of a structure from anodic dissolution. To assess the application of this process for controlled mass removal spanning sub-micrograms to milligrams, two experimental studies and accompanying uncertainty analyses have been undertaken. In the first of these, 5 tungsten wires of length 30 mm were used to remove mass values ranging from 50 to 350 µg. Uncertainty estimates indicate relative combined standard uncertainties of less than 0.3% in the mass changes determined from the measurement of Faradaic current. Comparison of the mass change determined using the electrolytic method, and using a precision ultra-microbalance agreed within this uncertainty. The charge-based method was then applied to modify the dynamic characteristics of a quartz tuning fork oscillator. In these experiments, tungsten fiber attached to one tine of the oscillator was etched in 5 µg increments up to 120 µg of total removed mass. In general, frequency shifts of 2.8 Hz · µg(-1) were observed, indicating sub-microgram resolution for the characterization of probes based on frequency shift and charge-based mass measurement. Taken together, this study provides the basis for a precision method for determining changes in mass based on electrical measurements from an electrochemical system. The utility of this technique is demonstrated through controlled modification of the dynamic properties of a mechanical oscillator.

Electron transport in gold nanowires: stable 1-, 2- and 3-dimensional atomic structures and noninteger conduction states.

Experimental conductivity measurements made during highly stable tensile deformation of Au nanowires show a rich variety of behaviors, including noninteger quantum conductance plateaus, transitions, and slopes. Using tight binding conductance calculations on simulated nanowires previously deformed using density functional theory, we demonstrate that all of these phenomena arise from structural transitions between deeply metastable ordered atomic configurations that self-organize during tensile deformation.

A fiber-optic interferometer with subpicometer resolution for dc and low-frequency displacement measurement.

We have developed a fiber-optic interferometer optimized for best performance in the frequency range from dc to 1 kHz, with displacement linearity of 1% over a range of +/- 25 nm, and noise-limited resolution of 2 pm. The interferometer uses a tunable infrared laser source (nominal 1550 nm wavelength) with high amplitude and wavelength stability, low spontaneous self-emission noise, high sideband suppression, and a coherence control feature that broadens the laser linewidth and dramatically lowers the low-frequency noise in the system. The amplitude stability of the source, combined with the use of specially manufactured "bend-insensitive" fiber and all-spliced fiber construction, results in a robust homodyne interferometer system, which achieves resolution of 40 fm Hz(-1/2) above 20 Hz and approaches the shot-noise-limit of 20 fm Hz(-1/2) at 1 kHz for an optical power of 10 microW, without the need for differential detection. Here we describe the design and construction of the interferometer, as well as modes of operation, and demonstrate its performance.

Spring constant calibration of atomic force microscopy cantilevers with a piezosensor transfer standard.

We describe a method to calibrate the spring constants of cantilevers for atomic force microscopy (AFM). The method makes use of a "piezosensor" composed of a piezoresistive cantilever and accompanying electronics. The piezosensor was calibrated before use with an absolute force standard, the NIST electrostatic force balance (EFB). In this way, the piezosensor acts as a force transfer standard traceable to the International System of Units. Seven single-crystal silicon cantilevers with rectangular geometries and nominal spring constants from 0.2 to 40 Nm were measured with the piezosensor method. The values obtained for the spring constant were compared to measurements by four other techniques: the thermal noise method, the Sader method, force loading by a calibrated nanoindentation load cell, and direct calibration by force loading with the EFB. Results from different methods for the same cantilever were generally in agreement, but differed by up to 300% from nominal values. When used properly, the piezosensor approach provides spring-constant values that are accurate to +/-10% or better. Methods such as this will improve the ability to extract quantitative information from AFM methods.

Ischemic epigenetics and the transplanted kidney.

The primary purpose of this investigation was to study oxidative demethylation of DNA following ischemia/reperfusion injury (I/RI) that putatively influences posttransplant gene expression in transplanted kidneys. Our hypothesis was that as a result of I/RI, oxidative damage, which is inherent in solid organ transplantation, may lead to aberrant demethylation of cytosine-guanine (CpG) sites within gene promoter regions of DNA. The methylated CpG sites normally contribute to the binding of proteins that render DNA inaccessible to transcription factors. Therefore, conversion of methylated cytosines to nonmethylated cytosines by oxidative damage in postischemic organs might facilitate enhanced gene expression in donor organs by exposing the demethylated CpG site in a gene promoter to DNA-binding proteins that enhance gene transcription. In this study, we investigated the demethylation of a specific CpG within the IFNgamma response element resident in the promoter region of the C3 gene in the rat kidney. In response to 24 hours of cold ischemia and a subsequent 2 hours of reperfusion in an isolated ex-vivo circuit, we observed a significant change in the ratio of methylated to unmethylated cytosines at this site. Epigenetic modifications to donor DNA have not been previously investigated, but our own data suggests that they have the potential to modify gene expression posttransplantation. Since epigenetic modification may become stable and heritable upon mitosis, such changes to the donor organ DNA may persist with enormous implications for transplant outcomes.

Comparative efficacy of renal preservation solutions to limit functional impairment after warm ischemic injury.

In kidney transplantation, cold storage is the dominant modality used to prolong organ viability ex vivo, but is inevitably followed by a period of warm ischemia. Preservation fluids limit tissue damage during the ischemic period, but there is little information on the influence of preservation fluids on the physiologic consequences of warm ischemia alone, or on the comparative ability of such preservation fluids to limit warm ischemic injury. In this study, warm ischemia was induced in rat kidneys by crossclamping the left renal pedicle for 45 min with contralateral nephrectomy. The ischemic kidneys were flushed with Euro-Collins (EC), hyper osmolar citrate (HOC), University of Wisconsin (UW), or phosphate buffered sucrose (PBS)140 solution. Over a period of 2 h after reperfusion, urine and blood samples were collected and physiological parameters related to the function of the postischemic kidneys were assessed. The data show that postischemic renal function can be influenced by the choice of preservation fluid. Essentially, the continued use of EC as a renal preservation solution finds little support in these data, and, while HOC and UW solutions were better able to limit the decline in renal function after warm ischemia than EC, the solution most able to limit functional impairment after warm ischemia was PBS140. This analysis compares the efficacies of the commonly used preservation solutions and could form the basis for future solid-organ transplant studies that may ultimately allow us to propose best-practice guidelines and an optimum platform for improved preservation solutions.

Properties of the cysteine-less Pho84 phosphate transporter of Saccharomyces cerevisiae.

The derepressible Pho84 high-affinity phosphate permease of Saccharomyces cerevisiae, encoded by the PHO84 gene belongs to a family of phosphate:proton symporters (PHS). The protein contains 12 native cysteine residues of which five are predicted to be located in putative transmembrane regions III, VI, VIII, IX, and X, and the remaining seven in the hydrophilic domains of the protein. Here we report on the construction of a Pho84 transporter devoid of cysteine residues (C-less) in which all 12 native residues were replaced with serines using PCR mutagenesis and the functional consequences of this. Our results clearly demonstrate that the C-less Pho84 variant is able to support growth of yeast cells to the same extent as the wild-type Pho84 and is stably expressed under derepressible conditions and is fully active in proton-coupled phosphate transport across the yeast plasma membrane.

In situ localization of C3 synthesis in experimental acute renal allograft rejection.

Recent evidence has implicated complement in renal transplant injury and identified the kidney as a source of complement components. We therefore investigated the local gene expression of complement component C3, pivotal to complement activation pathways and a mediator of inflammatory injury, in a rat renal transplant model. By reverse transcriptase-polymerase chain reaction, the expression of C3 mRNA increased in two phases. The first phase coincided with post-ischemic injury over 2 days post-transplantation and was localized by in situ hybridization to vessels and glomerular mesangial cells in allogeneic and syngeneic (control) kidney transplants. In allografts only, a second phase was found in tubular epithelial cells, glomerular parietal cells, vessel walls and some infiltrating cells, which peaked on day 4 together with rapid influx of leukocytes, tubule cell damage, the induction of interleukin-2 and interferon-gamma mRNA, and the up-regulation of tumor necrosis factor-alpha and interleukin-1beta mRNA in the graft. In vitro studies showed that interleukin-2 and interferon-gamma up-regulate C3 production in renal tubule cells. We conclude that post-ischemic injury led to transient up-regulation of glomerular expression of C3 mRNA. Subsequent cellular rejection was associated with tubulointerstitial/glomerular parietal cell expression of C3 mRNA. This differential expression of local C3, immediately post-transplant or associated with acute rejection, may have implications for putative therapeutic complement inhibition in clinical transplantation.

Predominant role for C5b-9 in renal ischemia/reperfusion injury.

Previous work has indicated that complement is a mediator of ischemia/reperfusion (I/R) injury. To investigate the components of complement responsible for this effect, we examined a model of renal I/R injury in C3-, C4-, C5-, and C6-deficient mice. We occluded the renal arteries and veins (40-58 minutes) and, after reperfusion (0-72 hours), assessed renal structural and functional injury. C3-, C5-, and C6-deficient mice were protected from renal I/R injury, whereas C4-deficient mice were not protected. C6-deficient mice treated with antibody to block C5a generation showed no additional protection from I/R injury. Reconstitution with C6 alone restored the I/R injury in C6-deficient mice. Tubular epithelial cells were the main structures damaged by complement-mediated attack, and, in contrast, the renal vessels were spared. Neutrophil infiltration and myeloperoxidase activity were reduced in C-deficient mouse kidney, but by a similar extent in C3-deficient and C6-deficient mice. We conclude that the membrane attack complex of complement (in which C5 and C6 participate) may account for the effect of complement on mouse renal I/R injury. Neither C5a-mediated neutrophil infiltration nor the classic pathway, in which C4 participates, appears to contribute to I/R injury in this model. By contrast with other organs, such as the heart, the primary effect of complement in the ischemic area is on the parenchymal cell rather than the vascular endothelial cell. The membrane attack complex of complement is a potential target for prevention of I/R injury in this model.

Targeting the complement system.

Interest has blossomed in the development of complement inhibitors, in parallel with a growth in our understanding of the biology of the complement cascade. The first generation of designed inhibitors was based on naturally occurring complement receptors and regulatory molecules. These agents provided useful tools for exploring the role of complement in experimental models of disease, but may have limited therapeutic application in humans because of their short half-lives, limited bioavailability and possible antigenicity. More recently, humanized antibodies and synthetic molecules that block the activation of complement have been developed, which look as though they may overcome some of these difficulties. The possibility for precision inhibition of a limited part of the complement cascade, or for inhibition confined to a single organ, may offer effective therapeutic results, while avoiding the disadvantages of nonselective complement blockade. This review examines the recent evidence that complement inhibition will reduce tissue damage resulting from organ transplantation, ischaemia-reperfusion injury, cancer, glomerulonephritis and the use of extracorporeal circuits.

Strategies for targeting complement inhibitors in ischaemia/reperfusion injury.

A transplanted organ suffers inherently from an ischaemic insult and subsequent reperfusion injury. The severity of such early events is thought to influence the success of the transplant procedure, not only in the immediate post-transplant period, but also to predispose the graft to both acute and chronic rejection. In this paper, we review the influence of the complement system upon ischaemia,reperfusion injury. The recognition of the involvement of complement has led to novel strategies to try to modulate ischaemia/reperfusion injury, some of which we have summarized. Finally, we note our own strategy to target complement inhibition in ischaemic tissues.

Influence of complement on the allospecific antibody response to a primary vascularized organ graft.

The induction of antibody responses against T cell-dependent antigens has been reported to be influenced by complement. We therefore asked if the primary induction of alloantibodies against transplantation antigens, an important determinant of transplant outcome, is complement sensitive and whether this has functional implications. We transplanted rat kidney allografts into fully major histocompatibility complex-mismatched recipients, in which complement activation was inhibited by daily injection of soluble recombinant human complement receptor type 1 (sCR1). Control allograft recipients were injected with saline. Animals in the control group showed a marked antibody response against donor-specific antigens and an increase in the proportion of activated B and T splenocytes by day 5 after transplantation. Complement-inhibited rats showed a reduced level of antibody binding on target cells sharing the same histocompatibility antigens as the donor strain (p < 0.001), and a reduced level of activated splenic B (p < 0.01) and T (p < 0.01) cells. In a functional assay, the plasma of complement-inhibited rats showed reduced cytotoxic activity against donor-specific cells, and their grafts contained less bound antibody than controls. Analysis beyond 6 days was obscured due to the development of antibodies against sCR1. We conclude that complement activation facilitates the induction of the alloantibody response. Sparing of vascular injury and prolongation of graft survival, previously reported in complement-inhibited rats (Pratt J. R. et al., Am. J. Path. 1996, 149: 2055), could therefore be due to down-regulation of the B cell response as well as reduced complement-dependent cytotoxicity. Inhibition of complement may provide an ancillary approach to the prevention of allospecific antibody formation and the prolongation of allograft survival in primary kidney grafting.

Developmental regulation of the plant mitochondrial matrix located HSP70 chaperone and its role in protein import.

Changes in the level of the mitochondrial chaperone mtHSP70 have been investigated in pea (Pisum sativum) leaf mitochondria by Western blot analysis and quantified by scanning densitometry. As pea leaves develop (from 6 days to 30 days of age) the levels of mtHSP70 decrease. Analysis of the levels of the alpha subunit of the F1ATPase show that the levels of this protein remain constant throughout the same developmental period, whereas the levels of the alternative oxidase increase. In vitro import of the alternative oxidase precursor protein into pea leaf mitochondria from day 6 to day 30 leaves and quantification by scanning densitometry indicates that protein import efficiency decreases with increasing maturity of the plant cell. Results are discussed in terms of how changing levels of the mtHSP70 chaperone, as a result of plant cell development, influence the efficiency of protein import.

Expression of tissue type plasminogen activator and type 1 plasminogen activator inhibitor, and persistent fibrin deposition in chronic renal allograft failure.

Persistent fibrin deposition has been observed in kidneys undergoing chronic rejection, and has been suggested to contribute to the obliteration of the vasculature in these grafts. The mechanisms leading to it are not clear. Fibrinolysis, the process to remove fibrin in tissues, is initiated by tissue type plasminogen activator (tPA) and suppressed by type 1 plasminogen activator inhibitor (PAI-1). To investigate their roles in chronic rejection and fibrin deposition, we serially examined the expression of tPA and PAI-1 in an unmodified chronic rejection model, using a Fisher 344 to Lewis rat renal transplant, at 0, 2, 4, 6, 10, 12, 16 and 20 weeks post-transplantation (N = 4 rats/time point in each group). We also analyzed fibrin deposition and the development of chronic changes in the grafts. Our results show that tPA was up-regulated only in the acute phase of rejection (P < 0.05), whereas PAI-1 was induced and persistently expressed during the progressive phase of chronic rejection, together with persistent fibrin deposition in the grafts. Immunohistochemistry showed PAI-1 was mainly localized to the damaged/proliferative vascular intima. The results suggest that persistent induction of PAI-1 may be responsible for the continuance of fibrin deposition, which is associated with irreversible damage and chronic graft loss.

Effects of complement inhibition with soluble complement receptor-1 on vascular injury and inflammation during renal allograft rejection in the rat.

Complement is both an effector of the humoral immune response and a stimulator of leukocyte activation. To examine the influence of complement on the allograft response, we inhibited complement using recombinant human soluble complement receptor-1 (sCR1; TP10), in an unsensitized model of rat renal allograft rejection. Lewis to DA renal transplant recipients were treated daily with 25 mg/kg sCR1 or saline and sacrificed on days 1 to 5 after transplant. Transplanted organs were examined histologically and immunohistochemically for leukocyte subset markers and for the third component of complement, C3, and membrane attack complex deposition. A second set of recipients was followed from day 5 to day 9 to assess graft survival. sCR1-treated recipients displayed > 90% inhibition of plasma complement activity and a marked reduction in tissue C3 and membrane attack complex deposition. Inactivation of complement reduced the vascular injury such that there was almost complete sparing of vascular damage in day 5 sCR1-treated rats. There was a significant reduction in infiltrating leukocytes by day 5 after transplant, and complement inhibition delayed the time to reach a histologically defined end point of graft survival from 5 days in controls to 9 days in the sCR1-treated group. These results imply that the vascular and cell-mediated injury arises, in part, from complement activation. The partial inhibition of these injuries by sCR1 may have functional implications for strategies to inhibit allograft rejection.