National Diagnostic Working Group (NDWG) for inertial confinement fusion (ICF)/high-energy density (HED) science: The whole exceeds the sum of its parts.

The National Diagnostic Working Group (NDWG) has led the effort to fully exploit the major inertial confinement fusion/high-energy density facilities in the US with the best available diagnostics. These diagnostics provide key data used to falsify early theories for ignition and suggest new theories, recently leading to an experiment that exceeds the Lawson condition required for ignition. The factors contributing to the success of the NDWG, collaboration and scope evolution, and the methods of accomplishment of the NDWG are discussed in this Review. Examples of collaborations in neutron and gamma spectroscopy, x-ray and neutron imaging, x-ray spectroscopy, and deep-ultraviolet Thomson scattering are given. An abbreviated history of the multi-decade collaborations and the present semiformal management framework is given together with the latest National Diagnostic Plan.

Differential regulation of MYC expression by PKHD1/Pkhd1 in human and mouse kidneys: phenotypic implications for recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD; MIM#263200) is a severe, hereditary, hepato-renal fibrocystic disorder that leads to early childhood morbidity and mortality. Typical forms of ARPKD are caused by pathogenic variants in the PKHD1 gene, which encodes the fibrocystin/polyductin (FPC) protein. MYC overexpression has been proposed as a driver of renal cystogenesis, but little is known about MYC expression in recessive PKD. In the current study, we provide the first evidence that MYC is overexpressed in kidneys from ARPKD patients and confirm that MYC is upregulated in cystic kidneys from cpk mutant mice. In contrast, renal MYC expression levels were not altered in several Pkhd1 mutant mice that lack a significant cystic kidney phenotype. We leveraged previous observations that the carboxy-terminus of mouse FPC (FPC-CTD) is proteolytically cleaved through Notch-like processing, translocates to the nucleus, and binds to double stranded DNA, to examine whether the FPC-CTD plays a role in regulating MYC/Myc transcription. Using immunofluorescence, reporter gene assays, and ChIP, we demonstrate that both human and mouse FPC-CTD can localize to the nucleus, bind to the MYC/Myc P1 promoter, and activate MYC/Myc expression. Interestingly, we observed species-specific differences in FPC-CTD intracellular trafficking. Furthermore, our informatic analyses revealed limited sequence identity of FPC-CTD across vertebrate phyla and database queries identified temporal differences in PKHD1/Pkhd1 and CYS1/Cys1 expression patterns in mouse and human kidneys. Given that cystin, the Cys1 gene product, is a negative regulator of Myc transcription, these temporal differences in gene expression could contribute to the relative renoprotection from cystogenesis in Pkhd1-deficient mice. Taken together, our findings provide new mechanistic insights into differential mFPC-CTD and hFPC-CTD regulation of MYC expression in renal epithelial cells, which may illuminate the basis for the phenotypic disparities between human patients with PKHD1 pathogenic variants and Pkhd1-mutant mice.

Cystin is required for maintaining fibrocystin (FPC) levels and safeguarding proteome integrity in mouse renal epithelial cells: A mechanistic connection between the kidney defects in cpk mice and human ARPKD.

Autosomal recessive polycystic kidney disease (ARPKD) is caused primarily by mutations in PKHD1, encoding fibrocystin (FPC), but Pkhd1 mutant mice failed to reproduce the human phenotype. In contrast, the renal lesion in congenital polycystic kidney (cpk) mice, with a mutation in Cys1 and cystin protein loss, closely phenocopies ARPKD. Although the nonhomologous mutation diminished the translational relevance of the cpk model, recent identification of patients with CYS1 mutations and ARPKD prompted the investigations described herein. We examined cystin and FPC expression in mouse models (cpk, rescued-cpk (r-cpk), Pkhd1 mutants) and mouse cortical collecting duct (CCD) cell lines (wild type (wt), cpk). We found that cystin deficiency caused FPC loss in both cpk kidneys and CCD cells. FPC levels increased in r-cpk kidneys and siRNA of Cys1 in wt cells reduced FPC. However, FPC deficiency in Pkhd1 mutants did not affect cystin levels. Cystin deficiency and associated FPC loss impacted the architecture of the primary cilium, but not ciliogenesis. No reduction in Pkhd1 mRNA levels in cpk kidneys and CCD cells suggested posttranslational FPC loss. Studies of cellular protein degradation systems suggested selective autophagy as a mechanism. In support of the previously described function of FPC in E3 ubiquitin ligase complexes, we demonstrated reduced polyubiquitination and elevated levels of functional epithelial sodium channel in cpk cells. Therefore, our studies expand the function of cystin in mice to include inhibition of Myc expression via interaction with necdin and maintenance of FPC as functional component of the NEDD4 E3 ligase complexes. Loss of FPC from E3 ligases may alter the cellular proteome, contributing to cystogenesis through multiple, yet to be defined, mechanisms.

A 2-4 keV multilayer mirrored channel for the NIF Dante system.

During inertial confinement fusion experiments at the National Ignition Facility (NIF), a capsule filled with deuterium and tritium (DT) gas, surrounded by a DT ice layer and a high-density carbon ablator, is driven to the temperature and densities required to initiate fusion. In the indirect method, 2 MJ of NIF laser light heats the inside of a gold hohlraum to a radiation temperature of 300 eV; thermal x rays from the hohlraum interior couple to the capsule and create a central hotspot at tens of millions degrees Kelvin and a density of 100-200 g/cm3. During the laser interaction with the gold wall, m-band x rays are produced at ∼2.5 keV; these can penetrate into the capsule and preheat the ablator and DT fuel. Preheat can impact instability growth rates in the ablation front and at the fuel-ablator interface. Monitoring the hohlraum x-ray spectrum throughout the implosion is, therefore, critical; for this purpose, a Multilayer Mirror (MLM) with flat response in the 2-4 keV range has been installed in the NIF 37° Dante calorimeter. Precision engineering and x-ray calibration of components mean the channel will report 2-4 keV spectral power with an uncertainty of ±8.7%.

Combined Heart-Lung-Liver Transplantation for Patients With Cystic Fibrosis: The Australian Experience.

BACKGROUND: Combined multivisceral transplantation has emerged as a therapeutic option for a select patient cohort; however, clinical decision-making remains complex and controversial. The aim of this study was to examine patient characteristics, operative complications, and long-term outcomes of all patients who have undergone combined heart-lung-liver transplantation (HLLTx) in Australia. METHODS: In this study, we performed a retrospective analysis of all adult patients who have undergone combined HLLTx in Australia to date. Recipient clinical characteristics, waitlist, and transplant outcomes are described. RESULTS: Eight adult patients have received HLLTx at a single Australian transplant center. Recipients of HLLTx have typically been young (median age, 30.1 years; range, 24-37), underweight (median body mass index, 19.8 kg/m2; range, 16.2-30.4) patients with cystic fibrosis (n = 8, 100%) with severe airflow obstruction (median forced expiratory volume in the first second of expiration, 24% predicted; range, 17%-48%) accompanied by liver cirrhosis confirmed on histopathology (n = 8, 100%). Despite relative preservation of synthetic function and low model for end-stage liver disease scores (median, 8; range, 6-17), all recipients had complications of portal hypertension prior to transplantation, with many patients having suffered life-threatening variceal hemorrhage. In this cohort, HLLTx was associated with overall posttransplant survival of 87.5% at 30 days, 71.4% at 1 year, and 42.9% at 5 years. Listing for combined HLLTx was associated with prolonged waitlist times relative to bilateral sequential single-lung transplantation (median 556 vs 56 days, respectively), however waitlist mortality and/or delisting was comparable between groups. CONCLUSIONS: Taken together, these findings highlight the opportunities and challenges facing combined (heart-) lung and liver transplantation in patients with multiorgan failure.

Interferon Regulatory Factor-5 in Resident Macrophage Promotes Polycystic Kidney Disease.

BACKGROUND: Autosomal dominant polycystic kidney disease is caused by genetic mutations in PKD1 or PKD2. Macrophages and their associated inflammatory cytokines promote cyst progression; however, transcription factors within macrophages that control cytokine production and cystic disease are unknown. METHODS: In these studies, we used conditional Pkd1 mice to test the hypothesis that macrophage-localized interferon regulatory factor-5 (IRF5), a transcription factor associated with production of cyst-promoting cytokines (TNFα, IL-6), is required for accelerated cyst progression in a unilateral nephrectomy (1K) model. Analyses of quantitative real-time PCR (qRT-PCR) and flow-cytometry data 3 weeks post nephrectomy, a time point before the onset of severe cystogenesis, indicate an accumulation of inflammatory infiltrating and resident macrophages in 1K Pkd1 mice compared with controls. qRT-PCR data from FACS cells at this time demonstrate that macrophages from 1K Pkd1 mice have increased expression of Irf5 compared with controls. To determine the importance of macrophage-localized Irf5 in cyst progression, we injected scrambled or IRF5 antisense oligonucleotide (ASO) in 1K Pkd1 mice and analyzed the effect on macrophage numbers, cytokine production, and renal cystogenesis 6 weeks post nephrectomy. RESULTS: Analyses of qRT-PCR and IRF5 ASO treatment significantly reduced macrophage numbers, Irf5 expression in resident-but not infiltrating-macrophages, and the severity of cystic disease. In addition, IRF5 ASO treatment in 1K Pkd1 mice reduced Il6 expression in resident macrophages, which was correlated with reduced STAT3 phosphorylation and downstream p-STAT3 target gene expression. CONCLUSIONS: These data suggest that Irf5 promotes inflammatory cytokine production in resident macrophages resulting in accelerated cystogenesis.

Loss of primary cilia increases polycystin-2 and TRPV4 and the appearance of a nonselective cation channel in the mouse cortical collecting duct.

Flow-related bending of cilia results in Ca2+ influx through a polycystin-1 (Pkd1) and polycystin-2 (Pkd2) complex, both of which are members of the transient receptor potential (TRP) family (TRPP1 and TRPP2, respectively). Deletion of this complex as well as cilia result in polycystic kidney disease. The Ca2+ influx pathway has been previously characterized in immortalized collecting duct cells without cilia and found to be a 23-pS channel that was a multimere of TRPP2 and TRPV4. The purpose of the present study was to determine if this TRPP2 and TRPV4 multimere exists in vivo. Apical channel activity was measured using the patch-clamp technique from isolated split-open cortical collecting ducts from adult conditional knockout mice with (Ift88flox/flox) or without (Ift88-/-) cilia. Single tubules were isolated for measurements of mRNA for Pkd1, Pkd2, Trpv4, and epithelial Na+ channel subunits. The predominant channel activity from Ift88flox/flox mice was from epithelial Na+ channel [5-pS Na+-selective channels with long mean open times (475.7 ± 83.26 ms) and open probability > 0.2]. With the loss of cilia, the predominant conductance was a 23-pS nonselective cation channel (reversal potential near 0) with a short mean open time (72 ± 17 ms), open probability < 0.08, and a characteristic flickery opening. Loss of cilia increased mRNA levels for Pkd2 and Trpv4 from single isolated cortical collecting ducts. In conclusion, 23-pS channels exist in vivo, and activity of this channel is elevated with loss of cilia, consistent with previous finding of an elevated-unregulated Ca2+-permeable pathway at the apical membrane of collecting duct cells that lack cilia.

The Crystal Backlighter Imager: A spherically bent crystal imager for radiography on the National Ignition Facility.

The Crystal Backlighter Imager (CBI) is a quasi-monochromatic, near-normal incidence, spherically bent crystal imager developed for the National Ignition Facility (NIF), which will allow inertial confinement fusion capsule implosions to be radiographed close to stagnation. This is not possible using the standard pinhole-based area-backlighter configuration, as the self-emission from the capsule hotspot overwhelms the backlighter signal in the final stages of the implosion. The CBI mitigates the broadband self-emission from the capsule hot spot by using the extremely narrow bandwidth inherent to near-normal-incidence Bragg diffraction. Implementing a backlighter system based on near-normal reflection in the NIF chamber presents unique challenges, requiring the CBI to adopt novel engineering and operational strategies. The CBI currently operates with an 11.6 keV backlighter, making it the highest energy radiography diagnostic based on spherically bent crystals to date. For a given velocity, Doppler shift is proportional to the emitted photon energy. At 11.6 keV, the ablation velocity of the backlighter plasma results in a Doppler shift that is significant compared to the bandwidth of the instrument and the width of the atomic line, requiring that the shift be measured to high accuracy and the optics aligned accordingly to compensate. Experiments will be presented that used the CBI itself to measure the backlighter Doppler shift to an accuracy of better than 1 eV. These experiments also measured the spatial resolution of CBI radiographs at 7.0 µm, close to theoretical predictions. Finally, results will be presented from an experiment in which the CBI radiographed a capsule implosion driven by a 1 MJ NIF laser pulse, demonstrating a significant (>100) improvement in the backlighter to self-emission ratio compared to the pinhole-based area-backlighter configuration.

Mesencephalic astrocyte-derived neurotropic factor is an important factor in chondrocyte ER homeostasis.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) resident protein that can be secreted due to an imperfect KDEL motif. MANF plays a cytoprotective role in several soft tissues and is upregulated in conditions resulting from intracellular retention of mutant protein, including two skeletal diseases, metaphyseal chondrodysplasia, Schmid type (MCDS) and multiple epiphyseal dysplasia (MED). The role of MANF in skeletal tissue homeostasis is currently unknown. Interestingly, cartilage-specific deletion of Manf in a mouse model of MED resulted in increased disease severity, suggesting its upregulation may be chondroprotective. Treatment of MED chondrocytes with exogenous MANF led to a decrease in the cellular levels of BiP (GRP78), confirming MANF's potential to modulate ER stress responses. However, it did not alleviate the intracellular retention of mutant matrilin-3, suggesting that it is the intracellular MANF that is of importance in the pathobiology of skeletal dysplasias. The Col2Cre-driven deletion of Manf from mouse cartilage resulted in a chondrodysplasia-like phenotype. Interestingly, ablation of MANF in cartilage did not have extracellular consequences but led to an upregulation of several ER-resident chaperones including BiP. This apparent induction of ER stress in turn led to dysregulated chondrocyte apoptosis and decreased proliferation, resulting in reduced long bone growth. We have previously shown that ER stress is an underlying disease mechanism for several skeletal dysplasias. The cartilage-specific deletion of Manf described in this study phenocopies our previously published chondrodysplasia models, further confirming that ER stress itself is sufficient to disrupt skeletal growth and thus represents a potential therapeutic target.

Truncating PKHD1 and PKD2 mutations alter energy metabolism.

Deficiency in polycystin 1 triggers specific changes in energy metabolism. To determine whether defects in other human cystoproteins have similar effects, we studied extracellular acidification and glucose metabolism in human embryonic kidney (HEK-293) cell lines with polycystic kidney and hepatic disease 1 ( PKHD1) and polycystic kidney disease (PKD) 2 ( PKD2) truncating defects along multiple sites of truncating mutations found in patients with autosomal recessive and dominant PKDs. While neither the PKHD1 or PKD2 gene mutations nor their position enhanced cell proliferation rate in our cell line models, truncating mutations in these genes progressively increased overall extracellular acidification over time ( P < 0.001 for PKHD1 and PKD2 mutations). PKHD1 mutations increased nonglycolytic acidification rate (1.19 vs. 1.03, P = 0.002), consistent with an increase in tricarboxylic acid cycle activity or breakdown of intracellular glycogen. In addition, they increased basal and ATP-linked oxygen consumption rates [7.59 vs. 5.42 ( P = 0.015) and 4.55 vs. 2.98 ( P = 0.004)]. The PKHD1 and PKD2 mutations also altered mitochondrial morphology, resembling the effects of polycystin 1 deficiency. Together, these data suggest that defects in major PKD genes trigger changes in mitochondrial energy metabolism. After validation in in vivo models, these initial observations would indicate potential benefits of targeting energy metabolism in the treatment of PKDs.

Sub-nanosecond single line-of-sight (SLOS) x-ray imagers (invited).

A new generation of fast-gated x-ray framing cameras have been developed that are capable of capturing multiple frames along a single line-of-sight with 30 ps temporal resolution. The instruments are constructed by integrating pulse-dilation electron imaging with burst mode hybrid-complimentary metal-oxide-semiconductor sensors. Two such instruments have been developed, characterized, and fielded at the National Ignition Facility and the OMEGA laser. These instruments are particularly suited for advanced x-ray imaging applications in Inertial Confinement Fusion and High energy density experiments. Here, we discuss the system architecture and the techniques required for tuning the instruments to achieve optimal performance. Characterization results are also presented along with planned future improvements to the design.

The single-line-of-sight, time-resolved x-ray imager diagnostic on OMEGA.

The single-line-of-sight, time-resolved x-ray imager (SLOS-TRXI) on OMEGA is one of a new generation of fast-gated x-ray cameras comprising an electron pulse-dilation imager and a nanosecond-gated, burst-mode, hybrid complementary metal-oxide semiconductor sensor. SLOS-TRXI images the core of imploded cryogenic deuterium-tritium shells in inertial confinement fusion experiments in the ∼4- to 9-keV photon energy range with a pinhole imager onto a photocathode. The diagnostic is mounted on a fixed port almost perpendicular to a 16-channel, framing-camera-based, time-resolved Kirkpatrick-Baez microscope, providing a second time-gated line of sight for hot-spot imaging on OMEGA. SLOS-TRXI achieves ∼40-ps temporal resolution and better than 10-µm spatial resolution. Shots with neutron yields of up to 1 × 1014 were taken without observed neutron-induced background signal. The implosion images from SLOS-TRXI show the evolution of the stagnating core.

The dilation aided single-line-of-sight x-ray camera for the National Ignition Facility: Characterization and fielding.

Crystal x-ray imaging is frequently used in inertial confinement fusion and laser-plasma interaction applications as it has advantages compared to pinhole imaging, such as higher signal throughput, better achievable spatial resolution, and chromatic selection. However, currently used x-ray detectors are only able to obtain a single time resolved image per crystal. The dilation aided single-line-of-sight x-ray camera described here was designed for the National Ignition Facility (NIF) and combines two recent diagnostic developments, the pulse dilation principle used in the dilation x-ray imager and a ns-scale multi-frame camera that uses a hold and readout circuit for each pixel. This enables multiple images to be taken from a single-line-of-sight with high spatial and temporal resolution. At the moment, the instrument can record two single-line-of-sight images with spatial and temporal resolution of 35 µm and down to 35 ps, respectively, with a planned upgrade doubling the number of images to four. Here we present the dilation aided single-line-of-sight camera for the NIF, including the x-ray characterization measurements obtained at the COMET laser, as well as the results from the initial timing shot on the NIF.

Characterization and calibration of a multilayer coated Wolter optic for an imager on the Z-machine at Sandia National Laboratories.

The need for a time-resolved monochromatic x-ray imaging diagnostic at photon energies >15 keV has motivated the development of a Wolter optic to study x-ray sources on the Z-machine at Sandia National Laboratories. The work is performed in both the LLNL's x-ray calibration facility and SNL's micro-focus x-ray lab. Characterizations and calibrations include alignment, measurement of throughput within the field of view (FOV), the point-spread function within the FOV both in and out of focus, and bandpass in the FOV. These results are compared with ray tracing models, showing reasonable agreement.

Design and raytrace simulations of a multilayer-coated Wolter x-ray optic for the Z machine at Sandia National Laboratories.

Recent breakthroughs in the fabrication of small-radii Wolter optics for astrophysics allow high energy density facilities to consider such optics as novel x-ray diagnostics at photon energies of 15-50 keV. Recently, the Lawrence Livermore National Laboratory, Sandia National Laboratories (SNL), the Smithsonian Astrophysical Observatory, and the NASA Marshall Space Flight Center jointly developed and fabricated the first custom Wolter microscope for implementation in SNL's Z machine with optimized sensitivity at 17.5 keV. To achieve spatial resolution of order 100-200 microns over a field of view of 5 × 5 × 5 mm3 with high throughput and narrow energy bandpass, the geometry of the optic and its multilayer required careful design and optimization. While the geometry mainly influences resolution and the field of view of the diagnostic, the mirror coating determines the spectral response and throughput. Here we outline the details of the design and fabrication process for the first multilayer-coated Wolter I optic for SNL's Z machine (Z Wolter), including its W/Si multilayer, and present results of raytrace simulations completed to predict and verify the performance of the optic.

An x-ray optic calibration facility for high energy density diagnostics.

A facility to calibrate x-ray imaging optics was built at Lawrence Livermore National Laboratory to support high energy density (HED) and inertial confinement fusion (ICF) diagnostics such as those at the National Ignition Facility and the Sandia Z-Machine. Calibration of the spectral reflectivity and resolution of these x-ray diagnostics enable absolute determination of the x-ray flux and wavelengths generated in the HED and ICF experiments. Measurement of the optic point spread function is used to determine spatial resolution of the optic. This facility was constructed to measure (1) the x-ray reflectivity to ±5% over a spectral range from 5 to 60 keV; (2) point spread functions with a resolution of 50 µm (currently) and 13 µm (future) in the image plane; and (3) optic distance relative to the x-ray source and detector to within ±100 µm in each dimension. This article describes the capabilities of the calibration facility, concept of operations, and initial data from selected x-ray optics.

A Wolter imager on the Z machine to diagnose warm x-ray sources.

A new Wolter x-ray imager has been developed for the Z machine to study the emission of warm (>15 keV) x-ray sources. A Wolter optic has been adapted from observational astronomy and medical imaging, which uses curved x-ray mirrors to form a 2D image of a source with 5 × 5 × 5 mm3 field-of-view and measured 60-300-µm resolution on-axis. The mirrors consist of a multilayer that create a narrow bandpass around the Mo Kα lines at 17.5 keV. We provide an overview of the instrument design and measured imaging performance. In addition, we present the first data from the instrument of a Mo wire array z-pinch on the Z machine, demonstrating improvements in spatial resolution and a 350-4100× increase in the signal over previous pinhole imaging techniques.

On the system stability and calibration of the image plate/scanner system for plasma diagnosis at the National Ignition Facility.

At the National Ignition Facility (NIF), storage phosphor image plates (IP) are used extensively for recording x-rays, charged particles, and neutrons. For x-ray imaging and spectroscopy, absolute and relative calibrations are important for extracting plasma information from the diagnostics. We use Fuji MS, SR, and TR image plates that have been cut to fit custom diagnostic envelopes. The image plates are scanned on a General Electric FLA 7000 IP flying spot scanner. Calibrations for sensitivity, spatial scale, and temperature dependent fade are applied. During a set of recent calibrations, we noticed large shifts in the absolute calibration of the image plate system. The possible source of these shifts is discussed. We discuss scanner stability and a method for calibration. We discuss the fade and temperature effects of the image plates and how this correction is applied within the NIF environment. We also compare our NIF GE FLA 7000 IP scanner with a new General Electric Amersham Typhoon IP scanner.

Methylome of human skeletal muscle after acute & chronic resistance exercise training, detraining & retraining.

DNA methylation is an important epigenetic modification that can regulate gene expression following environmental encounters without changes to the genetic code. Using Infinium MethylationEPIC BeadChip Arrays (850,000 CpG sites) we analysed for the first time, DNA isolated from untrained human skeletal muscle biopsies (vastus lateralis) at baseline (rest) and immediately following an acute (single) bout of resistance exercise. In the same participants, we also analysed the methylome following a period of muscle growth (hypertrophy) evoked via chronic (repeated bouts-3 sessions/wk) resistance exercise (RE) (training) over 7-weeks, followed by complete exercise cessation for 7-weeks returning muscle back to baseline levels (detraining), and finally followed by a subsequent 7-week period of RE-induced hypertrophy (retraining). These valuable methylome data sets described in the present manuscript and deposited in an open-access repository can now be shared and re-used to enable the identification of epigenetically regulated genes/networks that are modified after acute anabolic stimuli and hypertrophy, and further investigate the phenomenon of epigenetic memory in skeletal muscle.