Studying the Impact of Wall Shear Stress on the Development and Performance of Electrochemically Active Biofilms.

A laminar flow reactor was designed that provides constant and reproducible growth conditions for the bioelectrochemical observation of electroactive bacteria (EAB). Experiments were performed using four reactors in parallel to enable the comparison of EAB growth behavior and bioelectrochemical performance under different hydrodynamic conditions while simultaneously keeping biological conditions identical. With regard to the moderate flow conditions found in wastewater treatment applications, the wall shear stress was adjusted to a range between 0.4 mPa to 2.9 mPa. Chronoamperometric data indicate that early stage current densities are improved by a moderate increase of the wall shear stress. In the same way, current onset times were increasing slightly towards higher values of the applied wall shear stress. Long-term observations of EAB performance showed a decrease in current density and a leveling of the trend observed for the early stages of biofilm growth.

Optimal Geometric Parameters for 3D Electrodes in Bioelectrochemical Systems: A Systematic Approach.

In this study, the performance of electroactive bacteria (EAB), cultivated inside tubular electrode ducts, is systematically investigated to derive predictions on the behavior of EAB under conditions limited by electrochemical losses. A modeling approach is applied to assess the influence of the electrochemical losses on the electrochemical performance and scaling characteristics of complex 3D structures, such as sponges and foams. A modular flow reactor is designed that provides laminar and reproducible flow conditions as a platform for the systematic electrochemical and bioelectrochemical characterization of 3D electrodes in bioelectrochemical systems (BES). The bioelectrochemical experiments are carried out in a set of reactors incorporating cylindrical electrodes exhibiting ducts of 1 cm length and different diameters ranging from 0.1 cm up to 1 cm. Single duct calculations are extrapolated to three dimensions through geometrical considerations; trends in 3D bioanode performance are demonstrated using the resulting simplified 3D structure. The combined experimental and modeling approach constitutes a framework for future studies on systematic electrode design.

The Limits of Three-Dimensionality: Systematic Assessment of Effective Anode Macrostructure Dimensions for Mixed-Culture Electroactive Biofilms.

This study analyzes the biofilm growth and long-term current production of mixed-culture, electrochemically active biofilms (EABs) on macrostructured electrodes under low-shear-force conditions. The channel dimensions were altered systematically in the range 400 µm to 2 mm, and the channel heights were varied between 1 and 4 mm to simulate macrostructures of different scales. Electrodes with finer-structured surfaces produced higher current densities in the short term owing to their large surface area but were outperformed in the long term because the accumulation of biomass led to limitations of mass transfer into the structures. The best long-term performance was observed for electrodes with channel dimensions of 1×4 mm, which showed no significant decrease in performance in the long term. Channels with a diameter of 400 µm were overgrown by the biofilm, which led to a transition from 3 D to 2 D behavior, indicating that structures of this scale might not be suitable for long-term operation under low-shear-stress conditions.

Use of torsional resonators to monitor electroactive biofilms.

Whereas the study of interfaces and thin films with the quartz crystal microbalance (QCM) is well established, biofilms have proven to be a difficult subject for the QCM. The main problem is that the shear wave emanating from the resonator surface does not usually reach to the top of the sample. This problem can be solved with torsional resonators. These have a resonance frequency in the range of tens of kHz, which is much below the frequency of the thickness-shear QCMs. The depth of penetration of the shear wave is correspondingly larger. Data acquisition and data analysis can proceed in analogy to the conventional thickness-shear QCM. Torsional resonators may also be operated as electrochemical QCMs (EQCMs), meaning that a DC electrical potential may be applied to the active electrode and that shifts of frequency and bandwidth may be acquired in parallel to the electrical current. Here we report on the formation of mixed-culture biofilms dominated by the microorganism Geobacter anodireducens. The viscoelastic analysis evidences an increase in rigidity as the films grows. Potential sweeps on electroactive biofilms reveal a softening under negative potentials, that is, under conditions, where the layer's metabolism was slowed down by insufficient oxidative activity of the substrate. For comparison, biofilms were monitored in parallel with a conventional thickness-shear QCM.

A wellness study of 108 individuals using personal, dense, dynamic data clouds.

Personal data for 108 individuals were collected during a 9-month period, including whole genome sequences; clinical tests, metabolomes, proteomes, and microbiomes at three time points; and daily activity tracking. Using all of these data, we generated a correlation network that revealed communities of related analytes associated with physiology and disease. Connectivity within analyte communities enabled the identification of known and candidate biomarkers (e.g., gamma-glutamyltyrosine was densely interconnected with clinical analytes for cardiometabolic disease). We calculated polygenic scores from genome-wide association studies (GWAS) for 127 traits and diseases, and used these to discover molecular correlates of polygenic risk (e.g., genetic risk for inflammatory bowel disease was negatively correlated with plasma cystine). Finally, behavioral coaching informed by personal data helped participants to improve clinical biomarkers. Our results show that measurement of personal data clouds over time can improve our understanding of health and disease, including early transitions to disease states.

RNA Sequencing Identifies Novel Translational Biomarkers of Kidney Fibrosis.

CKD is the gradual, asymptomatic loss of kidney function, but current tests only identify CKD when significant loss has already happened. Several potential biomarkers of CKD have been reported, but none have been approved for preclinical or clinical use. Using RNA sequencing in a mouse model of folic acid-induced nephropathy, we identified ten genes that track kidney fibrosis development, the common pathologic finding in patients with CKD. The gene expression of all ten candidates was confirmed to be significantly higher (approximately ten- to 150-fold) in three well established, mechanistically distinct mouse models of kidney fibrosis than in models of nonfibrotic AKI. Protein expression of these genes was also high in the folic acid model and in patients with biopsy-proven kidney fibrosis. mRNA expression of the ten genes increased with increasing severity of kidney fibrosis, decreased in response to therapeutic intervention, and increased only modestly (approximately two- to five-fold) with liver fibrosis in mice and humans, demonstrating specificity for kidney fibrosis. Using targeted selected reaction monitoring mass spectrometry, we detected three of the ten candidates in human urine: cadherin 11 (CDH11), macrophage mannose receptor C1 (MRC1), and phospholipid transfer protein (PLTP). Furthermore, urinary levels of each of these three proteins distinguished patients with CKD (n=53) from healthy individuals (n=53; P<0.05). In summary, we report the identification of urinary CDH11, MRC1, and PLTP as novel noninvasive biomarkers of CKD.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

Cyclic voltammetric study of cobalt poly-4-t-butylpyridine ligand complexes on glassy carbon electrodes: electrolyte dependence and mechanistic considerations.

Polypyridyl complexes of Co(II/III) have been gaining prominence as potential replacements for I(-)/I(3)(-) as mediators in dye sensitized solar cells. In that regard, homoleptic pseudo-octahedral complexes of 4,4'-di-t-butyl-2,2'-bipyridine, [Co(DTB)(3)](2+/3+), and 4,4',4″-tri-t-butyl-2,2':6,2″-terpyridine, [Co(TTT)(2)](2+/3+), have been of particular interest. These complexes show extreme electrode surface and electrolyte dependent electrochemical behavior. Below, we report on the cyclic voltammetric behavior of these two complexes at glassy carbon electrodes in two different electrolytes. The electrochemical data suggests that the Co(II/III) electron transfer is significantly nonadiabatic, especially for [Co(DTB)(3)](2+/3+) in LiClO(4) electrolyte.

Perturbing peptide cation-radical electronic states by thioxoamide groups: formation, dissociations, and energetics of thioxopeptide cation-radicals.

Thioxodipeptides Gly-thio-Lys (GtK), Ala-thio-Lys (AtK), and Ala-thio-Arg (AtR) in which the amide group has been modified to a thioxoamide were made into dications by electrospray ionization and converted to cation-radicals, (GtK + 2H)(+•), (AtK + 2H)(+•), and (AtR + 2H)(+•), by electron transfer dissociation (ETD) tandem mass spectrometry using fluoranthene anion-radical as an electron donor. The common and dominant dissociation of these cation-radicals was the loss of a hydrogen atom. The dissociation products were characterized by collision-induced dissociation (CID) multistage tandem mass spectrometry up to CID-MS(5). The ground electronic states of several (GtK + 2H)(+•), (AtK + 2H)(+•), and (AtR + 2H)(+•) conformers were explored by extensive ab initio and density functional theory calculations of the potential energy surface. In silico electron transfer to the precursor dications, (GtK + 2H)(2+), (AtK + 2H)(2+), and (AtR + 2H)(2+), formed zwitterionic intermediates containing thioenol anion-radical and ammonium cation groups that were local energy minima on the potential energy surface of the ground electronic state. The zwitterions underwent facile isomerization by N-terminal ammonium proton migration to the thioenol anion-radical group forming aminothioketyl intermediates. Combined potential energy mapping and RRKM calculations of dissociation rate constants identified N-C(α) bond cleavages as the most favorable dissociation pathways, in a stark contrast to the experimental results. This discrepancy is interpreted as being due to the population upon electron transfer of low-lying excited electronic states that promote loss of hydrogen atoms. For (GtK + 2H)(+•), these excited states were characterized by time-dependent density functional theory as A-C states that had large components of Rydberg-like 3s molecular orbitals at the N-terminal and lysine ammonium groups that are conducive to hydrogen atom loss.

Assigning structures to gas-phase peptide cations and cation-radicals. An infrared multiphoton dissociation, ion mobility, electron transfer, and computational study of a histidine peptide ion.

Infrared multiphoton dissociation (IRMPD) spectroscopy, using a free-electron laser, and ion mobility measurements, using both drift-cell and traveling-wave instruments, were used to investigate the structure of gas-phase peptide (AAHAL + 2H)(2+) ions produced by electrospray ionization. The experimental data from the IRMPD spectra and collisional cross section (Ω) measurements were consistent with the respective infrared spectra and Ω calculated for the lowest-energy peptide ion conformer obtained by extensive molecular dynamics searches and combined density functional theory and ab initio geometry optimizations and energy calculations. Traveling-wave ion mobility measurements were employed to obtain the Ω of charge-reduced peptide cation-radicals, (AAHAL + 2H)(+●), and the c(3), c(4), z(3), and z(4) fragments from electron-transfer dissociation (ETD) of (AAHAL + 2H)(2+). The experimental Ω for the ETD charge-reduced and fragment ions were consistent with the values calculated for fully optimized ion structures and indicated that the ions retained specific hydrogen bonding motifs from the precursor ion. In particular, the Ω for the doubly protonated ions and charge-reduced cation-radicals were nearly identical, indicating negligible unfolding and small secondary structure changes upon electron transfer. The experimental Ω for the (AAHAL + 2H)(+●) cation-radicals were compatible with both zwitterionic and histidine radical structures formed by electron attachment to different sites in the precursor ion, but did not allow their distinction. The best agreement with the experimental Ω was found for ion structures fully optimized with M06-2X/6-31+G(d,p) and using both projection approximation and trajectory methods to calculate the theoretical Ω values.

Tunable charge tags for electron-based methods of peptide sequencing: design and applications.

Charge tags using basic auxiliary functional groups 6-aminoquinolinylcarboxamido, 4-aminopyrimidyl-1-methylcarboxamido, 2-aminobenzoimidazolyl-1-methylcarboxamido, and the fixed-charge 4-(dimethylamino)pyridyl-1-carboxamido moiety are evaluated as to their properties in electron transfer dissociation mass spectra of arginine C-terminated peptides. The neutral tags have proton affinities that are competitive with those of amino acid residues in peptides. Charge reduction by electron transfer from fluoranthene anion-radicals results in peptide backbone dissociations that improve sequence coverage by providing extensive series of N-terminal c-type fragments without impeding the formation of C-terminal z fragments. Comparison of ETD mass spectra of free and tagged peptides allows one to resolve ambiguities in fragment ion assignment through mass shifts of c ions. Simple chemical procedures are reported for N-terminal tagging of Arg-containing tryptic peptides.

The early life of a peptide cation-radical. Ground and excited-state trajectories of electron-based peptide dissociations during the first 330 femtoseconds.

We report a new approach to investigating the mechanisms of fast peptide cation-radical dissociations based on an analysis of time-resolved reaction progress by Ehrenfest dynamics, as applied to an Ala-Arg cation-radical model system. Calculations of stationary points on the ground electronic state that were carried out with effective CCSD(T)/6-311++G(3df,2p) could not explain the experimental branching ratios for loss of a hydrogen atom, ammonia, and N-C(α) bond dissociation in (AR + 2H)(+•). The Ehrenfest dynamics results indicate that the ground and low-lying excited electronic states of (AR + 2H)(+•) follow different reaction courses in the first 330 femtoseconds after electron attachment. The ground (X) state undergoes competing loss of N-terminal ammonia and isomerization to an aminoketyl radical intermediate that depend on the vibrational energy of the charge-reduced ion. The A and B excited states involve electron capture in the Arg guanidine and carboxyl groups and are non-reactive on the short time scale. The C state is dissociative and progresses to a fast loss of an H atom from the Arg guanidine group. Analogous results were obtained by using the B3LYP and CAM-B3LYP density functionals for the excited state dynamics and including the universal M06-2X functional for ground electronic state calculations. The results of this Ehrenfest dynamics study indicate that reaction pathway branching into the various dissociation channels occurs in the early stages of electron attachment and is primarily determined by the electronic states being accessed. This represents a new paradigm for the discussion of peptide dissociations in electron based methods of mass spectrometry.

Electron-capture and -transfer dissociation of peptides tagged with tunable fixed-charge groups: structures and dissociation energetics.

Pyridiniummethylcarbonyl moieties that were previously designed on the basis of electronic structure analysis are now utilized as fixed-charge tags with tunable electronic properties to be used for N-terminal peptide derivatization and sequencing by electron-transfer dissociation. Dipeptides AK and KA were derivatized at the peptide N-terminus with 4-dimethylaminopyridinium-N-acetyl (DMAP-ac) and pyridinium-N-acetyl (pyrid-ac) tags of increasing intrinsic recombination energies. Upon the capture of a free electron or electron transfer from fluoranthene anions, (DMAP-ac-AK+H)(2+), (DMAP-ac-KA+H)(2+), (pyrid-ac-AK+H)(2+) and (pyrid-ac-KA+H)(2+) ions, as well as underivatized (AK+2H)(2+), completely dissociated. The fixed-charge tags steered the dissociation upon electron transfer to form abundant backbone N-C(α) bond cleavages, whereas the underivatized peptide mainly underwent H-atom and side-chain losses. Precursor ion structures for the tagged peptides were analyzed by an exhaustive conformational search combined with B3LYP/6-31+G(d,p) geometry optimization and single-point energy calculations in order to select the global energy minima. Structures, relative energies, transition states, ion-molecule complexes, and dissociation products were identified for several charge-reduced species from the tagged peptides. The electronic properties of the charge tags and their interactions with the peptide moieties are discussed. Electrospray ionization and electron-transfer dissociation of larger peptides are illustrated with a DMAP-tagged pentapeptide.

Dipole-guided electron capture causes abnormal dissociations of phosphorylated pentapeptides.

Electron transfer and capture mass spectra of a series of doubly charged ions that were phosphorylated pentapeptides of a tryptic type (pS,A,A,A,R) showed conspicuous differences in dissociations of charge-reduced ions. Electron transfer from both gaseous cesium atoms at 100 keV kinetic energies and fluoranthene anion radicals in an ion trap resulted in the loss of a hydrogen atom, ammonia, and backbone cleavages forming complete series of sequence z ions. Elimination of phosphoric acid was negligible. In contrast, capture of low-energy electrons by doubly charged ions in a Penning ion trap induced loss of a hydrogen atom followed by elimination of phosphoric acid as the dominant dissociation channel. Backbone dissociations of charge-reduced ions also occurred but were accompanied by extensive fragmentation of the primary products. z-Ions that were terminated with a deaminated phosphoserine radical competitively eliminated phosphoric acid and H(2)PO(4) radicals. A mechanism is proposed for this novel dissociation on the basis of a computational analysis of reaction pathways and transition states. Electronic structure theory calculations in combination with extensive molecular dynamics mapping of the potential energy surface provided structures for the precursor phosphopeptide dications. Electron attachment produces a multitude of low lying electronic states in charge-reduced ions that determine their reactivity in backbone dissociations and H- atom loss. The predominant loss of H atoms in ECD is explained by a distortion of the Rydberg orbital space by the strong dipolar field of the peptide dication framework. The dipolar field steers the incoming electron to preferentially attach to the positively charged arginine side chain to form guanidinium radicals and trigger their dissociations.

The histidine effect. Electron transfer and capture cause different dissociations and rearrangements of histidine peptide cation-radicals.

Electron-transfer and -capture dissociations of doubly protonated peptides gave dramatically different product ions for a series of histidine-containing pentapeptides of both non-tryptic (AAHAL, AHAAL, AHADL, AHDAL) and tryptic (AAAHK, AAHAK, AHAAK, HAAAK, AAAHR, AAHAR, AHAAR, HAAAR) type. Electron transfer from gaseous Cs atoms and fluoranthene anions triggered backbone dissociations of all four N-C(alpha) bonds in the peptide ions in addition to loss of H and NH(3). Substantial fractions of charge-reduced cation-radicals did not dissociate on an experimental time scale ranging from 10(-6) to 10(-1) s. Multistage tandem mass spectrometric (MS(n)) experiments indicated that the non-dissociating cation-radicals had undergone rearrangements. These were explained as being due to proton migrations from N-terminal ammonium and COOH groups to the C-2' position of the reduced His ring, resulting in substantial radical stabilization. Ab initio calculations revealed that the charge-reduced cation-radicals can exist as low-energy zwitterionic amide pi* states which were local energy minima. These states underwent facile exothermic proton migrations to form aminoketyl radical intermediates, whereas direct N-C(alpha) bond cleavage in zwitterions was disfavored. RRKM analysis indicated that backbone N-C(alpha) bond cleavages did not occur competitively from a single charge-reduced precursor. Rather, these bond cleavages proceeded from distinct intermediates which originated from different electronic states accessed by electron transfer. In stark contrast to electron transfer, capture of a free electron by the peptide ions mainly induced radical dissociations of the charge-carrying side chains and loss of a hydrogen atom followed by standard backbone dissociations of even-electron ions. The differences in dissociation are explained by different electronic states being accessed upon electron transfer and capture.

Car-Parrinello density matrix search with a first principles fictitious electron mass method for electronic wave function optimization.

In spite of its success in molecular dynamics and the advantage of being a first order propagation technique, the Car-Parrinello method and its variations have not been successful in self-consistent-field (SCF) wave function optimization due to slow convergence. In this article, we introduce a first principles fictitious mass scheme to weigh each individual density element differently and instantaneously. As an alternative to diagonalization in SCF, the Car-Parrinello scheme is implemented as a density matrix search method. Not only does the fictitious mass scheme developed herein allow a very fast SCF convergence, but also the Car-Parrinello density matrix search (CP-DMS) exhibits linear scaling with respect to the system size for alanine helical chain test molecules. The excellent performance of CP-DMS holds even for very challenging compact three-dimensional quantum particles. While the conventional diagonalization based SCF method has difficulties optimizing electronic wave functions for CdSe quantum dots, CP-DMS shows both smooth and faster convergence.

First order simultaneous optimization of molecular geometry and electronic wave function.

We present a very efficient simultaneous optimization method for both molecular geometry and electronic wave function. We introduce a simultaneous least squares scheme to minimize errors in a full local quadratic space of both geometric and wave function vectors. Several self-consistent-field iterations are taken until a uniform search direction is achieved in every geometry optimization step. Not only does this new simultaneous optimization method significantly reduce the number of self-consistent-field iterations to reach convergence, it is able to overcome shallow potential wells to find a better stationary point. This new method exhibits up to approximately 60% savings in computational cost compared to conventional geometry optimization methods.

Analytical theory of the stochastic dynamics of the power stroke in nonprocessive motor proteins.

Statistical distributions of the structural states of individual molecules of nonprocessive motor complexes such as actomyosins are examined theoretically by considering a two-state stochastic model coupled by chemical reactions along the reaction coordinate representing the internal conformational states of the motor. The use of a conformational reaction coordinate allows for the approximation of taking the rate constants as local in their dependence on the reaction coordinate, and yields a simple analytic solution of the stationary states. The approximation is also tested against numerical solutions with a nonlocal form of rate constants. The theory is well-suited for computational treatments based on atomic structures of protein constituents using free energy molecular dynamics simulations. With empirical sets of free energy functions, stationary distributions of forces exerted by a motor head compare well with known experimental data.