The Link between Autosomal Dominant Polycystic Kidney Disease and Chromosomal Instability: Exploring the Relationship.

In autosomal dominant polycystic kidney disease (ADPKD) with germline mutations in a PKD1 or PKD2 gene, innumerable cysts develop from tubules, and renal function deteriorates. Second-hit somatic mutations and renal tubular epithelial (RTE) cell death are crucial features of cyst initiation and disease progression. Here, we use established RTE lines and primary ADPKD cells with disease-associated PKD1 mutations to investigate genomic instability and DNA damage responses. We found that ADPKD cells suffer severe chromosome breakage, aneuploidy, heightened susceptibility to DNA damage, and delayed checkpoint activation. Immunohistochemical analyses of human kidneys corroborated observations in cultured cells. DNA damage sensors (ATM/ATR) were activated but did not localize at nuclear sites of damaged DNA and did not properly activate downstream transducers (CHK1/CHK2). ADPKD cells also had the ability to transform, as they achieved high saturation density and formed colonies in soft agar. Our studies indicate that defective DNA damage repair pathways and the somatic mutagenesis they cause contribute fundamentally to the pathogenesis of ADPKD. Acquired mutations may alternatively confer proliferative advantages to the clonally expanded cell populations or lead to apoptosis. Further understanding of the molecular details of aberrant DNA damage responses in ADPKD is ongoing and holds promise for targeted therapies.

Inhibitor of intramembrane protease RseP blocks the σE response causing lethal accumulation of unfolded outer membrane proteins.

The outer membrane (OM) of Gram-negative bacteria forms a robust permeability barrier that blocks entry of toxins and antibiotics. Most OM proteins (OMPs) assume a ß-barrel fold, and some form aqueous channels for nutrient uptake and efflux of intracellular toxins. The Bam machine catalyzes rapid folding and assembly of OMPs. Fidelity of OMP biogenesis is monitored by the σE stress response. When OMP folding defects arise, the proteases DegS and RseP act sequentially to liberate σE into the cytosol, enabling it to activate transcription of the stress regulon. Here, we identify batimastat as a selective inhibitor of RseP that causes a lethal decrease in σE activity in Escherichia coli, and we further identify RseP mutants that are insensitive to inhibition and confer resistance. Remarkably, batimastat treatment allows the capture of elusive intermediates in the OMP biogenesis pathway and offers opportunities to better understand the underlying basis for σE essentiality.

Engineering Ultra-Low Work Function of Graphene.

Low work function materials are critical for energy conversion and electron emission applications. Here, we demonstrate for the first time that an ultralow work function graphene is achieved by combining electrostatic gating with a Cs/O surface coating. A simple device is built from large-area monolayer graphene grown by chemical vapor deposition, transferred onto 20 nm HfO2 on Si, enabling high electric fields capacitive charge accumulation in the graphene. We first observed over 0.7 eV work function change due to electrostatic gating as measured by scanning Kelvin probe force microscopy and confirmed by conductivity measurements. The deposition of Cs/O further reduced the work function, as measured by photoemission in an ultrahigh vacuum environment, which reaches nearly 1 eV, the lowest reported to date for a conductive, nondiamond material.

Expression of Nek1 during kidney development and cyst formation in multiple nephron segments in the Nek1-deficient kat2J mouse model of polycystic kidney disease.

BACKGROUND: Neks, mammalian orthologs of the fungal protein kinase never-in-mitosis A, have been implicated in the pathogenesis of polycystic kidney disease. Among them, Nek1 is the primary protein inactivated in kat2J mouse models of PKD. RESULT: We report the expression pattern of Nek1 and characterize the renal cysts that develop in kat2J mice. Nek1 is detectable in all murine tissues but its expression in wild type and kat2J heterozygous kidneys decrease as the kidneys mature, especially in tubular epithelial cells. In the embryonic kidney, Nek1 expression is most prominent in cells that will become podocytes and proximal tubules. Kidney development in kat2J homozygous mice is aberrant early, before the appearance of gross cysts: developing cortical zones are thin, populated by immature glomeruli, and characterized by excessive apoptosis of several cell types. Cysts in kat2J homozygous mice form postnatally in Bowman's space as well as different tubular subtypes. Late in life, kat2J heterozygous mice form renal cysts and the cells lining these cysts lack staining for Nek1. The primary cilia of cells lining cysts in kat2J homozygous mice are morphologically diverse: in some cells they are unusually long and in others there are multiple cilia of varying lengths. CONCLUSION: Our studies indicate that Nek1 deficiency leads to disordered kidney maturation, and cysts throughout the nephron.

Structure of kaolinite and influence of stacking faults: reconciling theory and experiment using inelastic neutron scattering analysis.

The structure of kaolinite at the atomic level, including the effect of stacking faults, is investigated using inelastic neutron scattering (INS) spectroscopy and density functional theory (DFT) calculations. The vibrational dynamics of the standard crystal structure of kaolinite, calculated using DFT (VASP) with normal mode analysis, gives good agreement with the experimental INS data except for distinct discrepancies, especially for the low frequency modes (200-400 cm(-1)). By generating several types of stacking faults (shifts in the a,b plane for one kaolinite layer relative to the adjacent layer), it is seen that these low frequency modes are affected, specifically through the emergence of longer hydrogen bonds (O-H⋯O) in one of the models corresponding to a stacking fault of -0.3151a - 0.3151b. The small residual disagreement between observed and calculated INS is assigned to quantum effects (which are not taken into account in the DFT calculations), in the form of translational tunneling of the proton in the hydrogen bonds, which lead to a softening of the low frequency modes. DFT-based molecular dynamics simulations show that anharmonicity does not play an important role in the structural dynamics of kaolinite.

A furnace and environmental cell for the in situ investigation of molten salt electrolysis using high-energy X-ray diffraction.

This paper describes the design, construction and implementation of a relatively large controlled-atmosphere cell and furnace arrangement. The purpose of this equipment is to facilitate the in situ characterization of materials used in molten salt electrowinning cells, using high-energy X-ray scattering techniques such as synchrotron-based energy-dispersive X-ray diffraction. The applicability of this equipment is demonstrated by quantitative measurements of the phase composition of a model inert anode material, which were taken during an in situ study of an operational Fray-Farthing-Chen Cambridge electrowinning cell, featuring molten CaCl(2) as the electrolyte. The feasibility of adapting the cell design to investigate materials in other high-temperature environments is also discussed.

Identification of Two Non-Peptidergic Small Molecule Inhibitors of CBX2 Binding to K27 Trimethylated Oligonucleosomes.

The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively.

A multi modal approach to microstructure evolution and mechanical response of additive friction stir deposited AZ31B Mg alloy.

Current work explored solid-state additive manufacturing of AZ31B-Mg alloy using additive friction stir deposition. Samples with relative densities ≥ 99.4% were additively produced. Spatial and temporal evolution of temperature during additive friction stir deposition was predicted using multi-layer computational process model. Microstructural evolution in the additively fabricated samples was examined using electron back scatter diffraction and high-resolution transmission electron microscopy. Mechanical properties of the additive samples were evaluated by non-destructive effective bulk modulus elastography and destructive uni-axial tensile testing. Additively produced samples experienced evolution of predominantly basal texture on the top surface and a marginal increase in the grain size compared to feed stock. Transmission electron microscopy shed light on fine scale precipitation of Mg[Formula: see text]Al[Formula: see text] within feed stock and additive samples. The fraction of Mg[Formula: see text]Al[Formula: see text] reduced in the additively produced samples compared to feed stock. The bulk dynamic modulus of the additive samples was slightly lower than the feed stock. There was a [Formula: see text] 30 MPa reduction in 0.2% proof stress and a 10-30 MPa reduction in ultimate tensile strength for the additively produced samples compared to feed stock. The elongation of the additive samples was 4-10% lower than feed stock. Such a property response for additive friction stir deposited AZ31B-Mg alloy was realized through distinct thermokinetics driven multi-scale microstructure evolution.

Integrative structural and functional analysis of human malic enzyme 3: A potential therapeutic target for pancreatic cancer.

Malic enzymes (ME1, ME2, and ME3) are involved in cellular energy regulation, redox homeostasis, and biosynthetic processes, through the production of pyruvate and reducing agent NAD(P)H. Recent studies have implicated the third and least well-characterized isoform, mitochondrial NADP+-dependent malic enzyme 3 (ME3), as a therapeutic target for pancreatic cancers. Here, we utilized an integrated structure approach to determine the structures of ME3 in various ligand-binding states at near-atomic resolutions. ME3 is captured in the open form existing as a stable tetramer and its dynamic Domain C is critical for activity. Catalytic assay results reveal that ME3 is a non-allosteric enzyme and does not require modulators for activity while structural analysis suggests that the inner stability of ME3 Domain A relative to ME2 disables allostery in ME3. With structural information available for all three malic enzymes, the foundation has been laid to understand the structural and biochemical differences of these enzymes and could aid in the development of specific malic enzyme small molecule drugs.

Structure of human spermine oxidase in complex with a highly selective allosteric inhibitor.

Human spermine oxidase (hSMOX) plays a central role in polyamine catabolism. Due to its association with several pathological processes, including inflammation and cancer, hSMOX has garnered interest as a possible therapeutic target. Therefore, determination of the structure of hSMOX is an important step to enable drug discovery and validate hSMOX as a drug target. Using insights from hydrogen/deuterium exchange mass spectrometry (HDX-MS), we engineered a hSMOX construct to obtain the first crystal structure of hSMOX bound to the known polyamine oxidase inhibitor MDL72527 at 2.4 Å resolution. While the overall fold of hSMOX is similar to its homolog, murine N1-acetylpolyamine oxidase (mPAOX), the two structures contain significant differences, notably in their substrate-binding domains and active site pockets. Subsequently, we employed a sensitive biochemical assay to conduct a high-throughput screen that identified a potent and selective hSMOX inhibitor, JNJ-1289. The co-crystal structure of hSMOX with JNJ-1289 was determined at 2.1 Å resolution, revealing that JNJ-1289 binds to an allosteric site, providing JNJ-1289 with a high degree of selectivity towards hSMOX. These results provide crucial insights into understanding the substrate specificity and enzymatic mechanism of hSMOX, and for the design of highly selective inhibitors.

Mutation of NIMA-related kinase 1 (NEK1) leads to chromosome instability.

BACKGROUND: NEK1, the first mammalian ortholog of the fungal protein kinase never-in-mitosis A (NIMA), is involved early in the DNA damage sensing/repair pathway. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks after low dose ionizing radiation (IR). NEK1-deficient cells also fail to activate the checkpoint kinases CHK1 and CHK2, and fail to arrest properly at G1/S or G2/M-phase checkpoints after DNA damage. RESULTS: We show here that NEK1-deficient cells suffer major errors in mitotic chromosome segregation and cytokinesis, and become aneuploid. These NEK1-deficient cells transform, acquire the ability to grow in anchorage-independent conditions, and form tumors when injected into syngeneic mice. Genomic instability is also manifest in NEK1 +/- mice, which late in life develop lymphomas with a much higher incidence than wild type littermates. CONCLUSION: NEK1 is required for the maintenance of genome stability by acting at multiple junctures, including control of chromosome stability.

Photon-enhanced thermionic emission for solar concentrator systems.

Solar-energy conversion usually takes one of two forms: the 'quantum' approach, which uses the large per-photon energy of solar radiation to excite electrons, as in photovoltaic cells, or the 'thermal' approach, which uses concentrated sunlight as a thermal-energy source to indirectly produce electricity using a heat engine. Here we present a new concept for solar electricity generation, photon-enhanced thermionic emission, which combines quantum and thermal mechanisms into a single physical process. The device is based on thermionic emission of photoexcited electrons from a semiconductor cathode at high temperature. Temperature-dependent photoemission-yield measurements from GaN show strong evidence for photon-enhanced thermionic emission, and calculated efficiencies for idealized devices can exceed the theoretical limits of single-junction photovoltaic cells. The proposed solar converter would operate at temperatures exceeding 200 degrees C, enabling its waste heat to be used to power a secondary thermal engine, boosting theoretical combined conversion efficiencies above 50%.

Phosphorylation by Nek1 regulates opening and closing of voltage dependent anion channel 1.

VDAC1 is a key component of the mitochondrial permeability transition pore. To initiate apoptosis and certain other forms of cell death, mitochondria become permeable such that cytochrome c and other pre-apoptotic molecules resident inside the mitochondria enter the cytosol and activate apoptotic cascades. We have shown recently that VDAC1 interacts directly with never-in-mitosis A related kinase 1 (Nek1), and that Nek1 phosphorylates VDAC1 on Ser193 to prevent excessive cell death after injury. How this phosphorylation regulates the activity of VDAC1, however, has not yet been reported. Here, we use atomic force microscopy (AFM) and cytochrome c conductance studies to examine the configuration of VDAC1 before and after phosphorylation by Nek1. Wild-type VDAC1 assumes an open configuration, but closes and prevents cytochrome c efflux when phosphorylated by Nek1. A VDAC1-Ser193Ala mutant, which cannot be phosphorylated by Nek1 under identical conditions, remains open and constitutively allows cytochrome c efflux. Conversely, a VDAC1-Ser193Glu mutant, which mimics constitutive phosphorylation by Nek1, remains closed by AFM and prevents cytochrome c leakage in the same liposome assays. Our data provide a mechanism to explain how Nek1 regulates cell death by affecting the opening and closing of VDAC1.

Combining density functional theory (DFT) and pair distribution function (PDF) analysis to solve the structure of metastable materials: the case of metakaolin.

Understanding the atomic structure of complex metastable (including glassy) materials is of great importance in research and industry, however, such materials resist solution by most standard techniques. Here, a novel technique combining thermodynamics and local structure is presented to solve the structure of the metastable aluminosilicate material metakaolin (calcined kaolinite) without the use of chemical constraints. The structure is elucidated by iterating between least-squares real-space refinement using neutron pair distribution function data, and geometry optimisation using density functional modelling. The resulting structural representation is both energetically feasible and in excellent agreement with experimental data. This accurate structural representation of metakaolin provides new insight into the local environment of the aluminium atoms, with evidence of the existence of tri-coordinated aluminium. By the availability of this detailed chemically feasible atomic description, without the need to artificially impose constraints during the refinement process, there exists the opportunity to tailor chemical and mechanical processes involving metakaolin and other complex metastable materials at the atomic level to obtain optimal performance at the macro-scale.

Density functional modeling of the local structure of kaolinite subjected to thermal dehydroxylation.

Understanding the atomic-level changes that occur as kaolinite is converted (thermally dehydroxylated) to metakaolin is critical to the optimization of this large-scale industrial process. Metakaolin is X-ray amorphous; therefore, conventional crystallographic techniques do not reveal the changes in local structure during its formation. Local structure-based experimental techniques are useful in understanding the atomic structure but do not provide the thermodynamic information which is necessary to ensure plausibility of refined structures. Here, kaolinite dehydroxylation is modeled using density functional theory, and a stepwise methodology, where several water molecules are removed from the structure, geometry optimization is carried out, and then the process is repeated. Hence, the structure remains in an energetically and thermodynamically feasible state while transitioning from kaolinite to metakaolin. The structures generated during the dehydroxylation process are validated by comparison with X-ray and neutron pair distribution function data. Thus, this study illustrates one possible route by which dehydroxylation of kaolinite can take place, revealing a chemically, energetically, and experimentally plausible structure of metakaolin. This methodology of density functional modeling of the stepwise changes in a material is not limited in application to kaolinite or other aluminosilicates and provides an accurate representation of the local structural changes occurring in materials used in industrially important processes.

The Children's Hospital Boston non-routine event reporting program.

Several authors have described methods to track perfusion and cardiac surgical morbidity and mortality as well as perfusion accidents. There is currently not a standard definition of a perfusion accident nor is there a standard reporting threshold for events which do not directly cause known morbidity. We propose the term non-routine events (NREs) instead of accidents, and provide a working definition and reporting threshold for such. This paper describes the program which we developed to track perfusion NREs within the Cardiovascular Program at Children's Hospital, Boston. NREs are categorized by type (technique, equipment, or patient-related) and bypass period (pre-cardiopulmonary bypass, bypass, or post-cardiopulmonary). NRE outcomes are also classified by the level of discussion or change in perfusion practice after multidisciplinary review. We have documented during a 44 month interval that 42% (29/69) of reported NREs occur during the bypass period and are equipment related and thus, efforts to improve practice should focus there. We have also seen a generally decreasing incidence of NREs requiring either a change in perfusion practice or a new protocol during this time period. We believe that our regular multidisciplinary meetings to discuss NREs have increased awareness among the entire team about potential problems in the program and that intuitively, it has improved patient safety.

What is the structure of kaolinite? Reconciling theory and experiment.

Density functional modeling of the crystalline layered aluminosilicate mineral kaolinite is conducted, first to reconcile discrepancies in the literature regarding the exact geometry of the inner and inner surface hydroxyl groups, and second to investigate the performance of selected exchange-correlation functionals in providing accurate structural information. A detailed evaluation of published experimental and computational structures is given, highlighting disagreements in space groups, hydroxyl bond lengths, and bond angles. A major aim of this paper is to resolve these discrepancies through computations. Computed structures are compared via total energy calculations and validated against experimental structures by comparing computed neutron diffractograms, and a final assessment is performed using vibrational spectra from inelastic neutron scattering. The density functional modeling is carried out at a sufficiently high level of theory to provide accurate structure predictions while keeping computational requirements low enough to enable the use of the structures in large-scale calculations. It is found that the best functional to use for efficient density functional modeling of kaolinite using the DMol3 software package is the BLYP functional. The computed structure for kaolinite at 0 K has C1 symmetry, with the inner hydroxyl group angled slightly above the a,b plane and the inner surface hydroxyls aligned close to perpendicular to that plane.

Raptor-rictor axis in TGFbeta-induced protein synthesis.

Transforming growth factor-beta (TGFbeta) stimulates pathological renal cell hypertrophy for which increased protein synthesis is critical. The mechanism of TGFbeta-induced protein synthesis is not known, but PI 3 kinase-dependent Akt kinase activity is necessary. We investigated the contribution of downstream effectors of Akt in TGFbeta-stimulated protein synthesis. TGFbeta increased inactivating phosphorylation of Akt substrate tuberin in a PI 3 kinase/Akt dependent manner, resulting in activation of mTOR kinase. mTOR activity increased phosphorylation of S6 kinase and the translation repressor 4EBP-1, which were sensitive to inhibition of both PI 3 kinase and Akt. mTOR inhibitor rapamycin and a dominant negative mutant of mTOR suppressed TGFbeta-induced phosphorylation of S6 kinase and 4EBP-1. PI 3 kinase/Akt and mTOR regulated dissociation of 4EBP-1 from eIF4E to make the latter available for binding to eIF4G. mTOR and 4EBP-1 modulated TGFbeta-induced protein synthesis. mTOR is present in two multi protein complexes, mTORC1 and mTORC2. Raptor and rictor are part of mTORC1 and mTORC2, respectively. shRNA-mediated downregulation of raptor inhibited TGFbeta-stimulated mTOR kinase activity, resulting in inhibition of phosphorylation of S6 kinase and 4EBP-1. Raptor shRNA also prevented protein synthesis in response to TGFbeta. Downregulation of rictor inhibited serine 473 phosphorylation of Akt without any effect on phosphorylation of its substrate, tuberin. Furthermore, rictor shRNA increased phosphorylation of S6 kinase and 4EBP-1 in TGFbeta-independent manner, resulting in increased protein synthesis. Thus mTORC1 function is essential for TGFbeta-induced protein synthesis. Our data also provide novel evidence that rictor negatively regulates TORC1 activity to control basal protein synthesis, thus conferring tight control on cellular hypertrophy.

Surface Photovoltage-Induced Ultralow Work Function Material for Thermionic Energy Converters.

Low work function materials are essential for efficient thermionic energy converters (TECs), electronics, and electron emission devices. Much effort has been put into finding thermally stable material combinations that exhibit low work functions. Submonolayer coatings of alkali metals have proven to significantly reduce the work function; however, a work function less than 1 eV has not been reached. We report a record-low work function of 0.70 eV by inducing a surface photovoltage (SPV) in an n-type semiconductor with an alkali metal coating. Ultraviolet photoelectron spectroscopy indicates a work function of 1.06 eV for cesium/oxygen-activated GaAs consistent with density functional theory model predictions. By illuminating with a 532 nm laser we induce an additional shift down to 0.70 eV due to the SPV. Further, we apply the SPV to the collector of an experimental TEC and demonstrate an I-V curve shift consistent with the collector work function reduction. This method opens an avenue toward efficient TECs and next-generation electron emission devices.

Birefringence and optical power confinement in horizontal multi-slot waveguides made of Si and SiO2.

Through simulations and measurements, we show that in multi-slot thin film waveguides, the TM polarized modes can be confined mostly in the low refractive index layers of the waveguide. The structure consisted of alternating layers of a-Si and SiO(2), in the thickness range between 3 and 40 nm, for which the slots were the SiO(2) layers. Simulations were performed using the transfer matrix method and experiments using the m-line technique at 1.55 mum. The dependence of the birefringence and of the power confinement in the slots was studied as a function of the waveguide thickness, the Si and SiO(2) layer thicknesses, and the SiO(2) / Si layer thickness ratio. We find a large birefringence-a refractive index difference between TE and TM modes-as large as 0.8. For TM polarized modes, up to ~ 85% of the total power in the fundamental mode can be confined in the slots.