Generation and characterization of mouse knockout for glyoxalase 1.

Glyoxalase 1 (Glo1) is the first enzyme involved in glutathione-dependent detoxification of methylglyoxal, eventually generating d-lactate by the second enzyme glyoxalase 2 (Glo2). An accumulation of intracellular glyoxal and methylglyoxal leads to protein malfunction and mutation via formation of the advanced glycation end products (AGEs). Studies on mouse behavior suggest that methylglyoxal has anxiolytic properties. In this report, we generated and characterized a mouse knockout for Glo1. The knockout mice were viable without a pronounced phenotypic defect. Increased level of AGEs in Glo1 knockout mice was detected by immunoblotting with anti-MGH1 in liver homogenate, but not in brain. Alterations in behavior were observed in open field, light-dark transition, and tail suspension test. Open field data indicate increased exploration for novel environment and entry/stay in center zone in Glo1 knockout mice. In addition, increased light-dark transition and immobility was observed in the knockout mice. These data indicate that Glo1 knockout reduces anxiety-like behavior, but increases depression-like behavior.

Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR Nanocomplex for Targeting Bacterial Pathogens and Antibiotic Resistance.

The overuse of antibiotics plays a major role in the emergence and spread of multidrug-resistant bacteria. A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth. Herein, we introduce a nonviral treatment strategy delivering genome editing material for targeting antibacterial resistance. We apply the CRISPR-Cas9 system, which has been recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo. We utilize polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance. We show that nanosized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage. We also demonstrate that the Cr-Nanocomplex designed to target mecA-the major gene involved in methicillin resistance-can be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allow the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations. The present study shows for the first time that a covalently modified CRISPR system allows nonviral, therapeutic genome editing, and can be potentially applied as a target specific antimicrobial.

Toward a brain functional connectivity mapping modality by simultaneous imaging of coherent brainwaves.

Matching the proton-magnetic-resonance frequency to the frequency of a periodic neural oscillation (e.g., alpha or gamma band waves) by magnetic resonance imaging techniques, enables direct visualization of brain functional connectivity. Functional connectivity has been studied by analyzing the correlation between coherent neural oscillations in different areas of the brain. In electro- or magneto-encephalography, coherent source reconstruction in a source-space is very tricky due to power leaking from the correlation among the sources. For this reason, most studies have been limited to sensor-space analyses, which give doubtful results because of volume current mixing. The direct visualization of coherent brain oscillations can circumvent this problem. The feasibility of this idea was demonstrated by conducting phantom experiments with a SQUID-based, micro-Tesla NMR/MRI system. We introduce an experimental trick, an effective step-up of the measurement B-field in a pulse sequence, to decouple the magnetic resonance signal from the strong magneto-encephalographic signal at the same frequency.

Human DJ-1 and its homologs are novel glyoxalases.

Human DJ-1 is a genetic cause of early-onset Parkinson's disease (PD), although its biochemical function is unknown. We report here that human DJ-1 and its homologs of the mouse and Caenorhabditis elegans are novel types of glyoxalase, converting glyoxal or methylglyoxal to glycolic or lactic acid, respectively, in the absence of glutathione. Purified DJ-1 proteins exhibit typical Michaelis-Menten kinetics, which were abolished completely in the mutants of essential catalytic residues, consisting of cysteine and glutamic acid. The presence of DJ-1 protected mouse embryonic fibroblast and dopaminergically derived SH-SY5Y cells from treatments of glyoxals. Likewise, C. elegans lacking cDJR-1.1, a DJ-1 homolog expressed primarily in the intestine, protected worms from glyoxal-induced death. Sub-lethal doses of glyoxals caused significant degeneration of the dopaminergic neurons in C. elegans lacking cDJR-1.2, another DJ-1 homolog expressed primarily in the head region, including neurons. Our findings that DJ-1 serves as scavengers for reactive carbonyl species may provide a new insight into the causation of PD.

Deposition and transport of Pseudomonas aeruginosa in porous media: lab-scale experiments and model analysis.

In this study, the deposition and transport of Pseudomonas aeruginosa on sandy porous materials have been investigated under static and dynamic flow conditions. For the static experiments, both equilibrium and kinetic batch tests were performed at a 1:3 and 3:1 soil:solution ratio. The batch data were analysed to quantify the deposition parameters under static conditions. Column tests were performed for dynamic flow experiments with KCl solution and bacteria suspended in (1) deionized water, (2) mineral salt medium (MSM) and (3) surfactant + MSM. The equilibrium distribution coefficient (K(d)) was larger at a 1:3 (2.43 mL g(-1)) than that at a 3:1 (0.28 mL g(-1)) soil:solution ratio. Kinetic batch experiments showed that the reversible deposition rate coefficient (k(att)) and the release rate coefficient (k(det)) at a soil:solution ratio of 3:1 were larger than those at a 1:3 ratio. Column experiments showed that an increase in ionic strength resulted in a decrease in peak concentration of bacteria, mass recovery and tailing of the bacterial breakthrough curve (BTC) and that the presence of surfactant enhanced the movement of bacteria through quartz sand, giving increased mass recovery and tailing. Deposition parameters under dynamic condition were determined by fitting BTCs to four different transport models, (1) kinetic reversible, (2) two-site, (3) kinetic irreversible and (4) kinetic reversible and irreversible models. Among these models, Model 4 was more suitable than the others since it includes the irreversible sorption term directly related to the mass loss of bacteria observed in the column experiment. Applicability of the parameters obtained from the batch experiments to simulate the column breakthrough data is evaluated.

Chemical Analysis of an Isotopically Labeled Molecule Using Two-Dimensional NMR Spectroscopy at 34 µT.

Low-field nuclear magnetic resonance (NMR) spectroscopy, conducted at or below a few millitesla, provides only limited spectral information due to its inability to resolve chemical shifts. Thus, chemical analysis based on this technique remains challenging. One potential solution to overcome this limitation is the use of isotopically labeled molecules. However, such compounds, particularly their use in two-dimensional (2D) NMR techniques, have rarely been studied. This study presents the results of both experimental and simulated correlation spectroscopy (COSY) on 1-13C-ethanol at 34.38 µT. The strong heteronuclear coupling in this molecule breaks the magnetic equivalence, causing all J-couplings, including homonuclear coupling, to split the 1H spectrum. The obtained COSY spectrum clearly shows the spectral details. Furthermore, we observed that homonuclear coupling between 1H spins generated cross-peaks only when the associated 1H spins were coupled to identical 13C spin states. Our findings demonstrate that a low-field 2D spectrum, even with a moderate spectral line width, can reveal the J-coupling networks of isotopically labeled molecules.

Detection of the 40 Hz auditory steady-state response with optically pumped magnetometers.

Magnetoencephalography (MEG) is a functional neuroimaging technique that noninvasively detects the brain magnetic field from neuronal activations. Conventional MEG measures brain signals using superconducting quantum interference devices (SQUIDs). SQUID-MEG requires a cryogenic environment involving a bulky non-magnetic Dewar flask and the consumption of liquid helium, which restricts the variability of the sensor array and the gap between the cortical sources and sensors. Recently, miniature optically pumped magnetometers (OPMs) have been developed and commercialized. OPMs do not require cryogenic cooling and can be placed within millimeters from the scalp. In the present study, we arranged six OPM sensors on the temporal area to detect auditory-related brain responses in a two-layer magnetically shielded room. We presented the auditory stimuli of 1 kHz pure-tone bursts with 200 ms duration and obtained the M50 and M100 components of auditory-evoked fields. We delivered the periodic stimuli with a 40 Hz repetition rate and observed the gamma-band power changes and inter-trial phase coherence of auditory steady-state responses at 40 Hz. We found that the OPM sensors have a performance comparable to that of conventional SQUID-MEG sensors, and our results suggest the feasibility of using OPM sensors for functional neuroimaging and brain-computer interface applications.

Non-invasive magnetocardiography for the early diagnosis of coronary artery disease in patients presenting with acute chest pain.

BACKGROUND: Accurate identification of patients with acute coronary syndrome (ACS) is often difficult especially when an electrocardiogram (ECG) does not show typical elevation of ST segment. The aim of the present study was therefore to evaluate the efficacy of magnetocardiography (MCG) for diagnosis of ACS in patients with acute chest pain presenting without ST segment elevation. METHODS AND RESULTS: In the present retrospective study 364 patients with the suspected ACS without ST segment elevation were selected. Significant coronary artery disease (CAD) was defined as a stenosis > or =50% in at least one of 16 segments of the 3 major coronary arteries and their branches. The MCG recordings were obtained at resting state using a 64-channel MCG system in a magnetically shielded room. The patients were classified on the basis of the probability distribution. The presence of significant CAD was identified with a sensitivity of 84.0% and a specificity of 85.0%, compared to 44.7% and 89.8% on ECG. In the subgroup of patients without specific findings on ECG or biomarker test, MCG had a sensitivity of 73.5% and a specificity of 82.3%. CONCLUSIONS: MCG was acceptably sensitive and specific in identifying patients with ACS even in the absence of specific findings on ECG and positive biomarker tests. Thus, MCG seems beneficial for the early triage of patients with acute chest pain.

Dynamic nuclear polarisation of liquids at one microtesla using circularly polarised RF with application to millimetre resolution MRI.

Magnetic resonance imaging in ultra-low fields is often limited by mediocre signal-to-noise ratio hindering a higher resolution. Overhauser dynamic nuclear polarisation (O-DNP) using nitroxide radicals has been an efficient solution for enhancing the thermal nuclear polarisation. However, the concurrence of positive and negative polarisation enhancements arises in ultra-low fields resulting in a significantly reduced net enhancement, making O-DNP far less attractive. Here, we address this issue by applying circularly polarised RF. O-DNP with circularly polarised RF renders a considerably improved enhancement factor of around 150,000 at 1.2 µT. A birdcage coil was adopted into an ultra-low field MRI system to generate the circularly polarised RF field homogeneously over a large volume. We acquired an MR image of a nitroxide radical solution with an average in-plane resolution of 1 mm. De-noising through compressive sensing further improved the image quality.

[Clinical outcomes of lamivudine therapy and HLA alleles in chronic hepatitis B patients].

BACKGROUND/AIMS: The human leukocyte antigen (HLA) system is an integral component of immune response. Highly polymorphic HLA genes may play a pivotal role in the response of antiviral therapy. We investigated the effects of HLA gene polymorphism on the clinical outcome of chronic hepatitis B patients who received lamivudine treatment. METHODS: Depending on their clinical response to lamivudine therapy, a total of sixty one patients were divided into following groups; non-responders, viral breakthroughers, relapsers, and seroconverters. HLA-A, -B, -Cw, -DRB and HLA-DRB1 alleles typing was performed on each group through the polymerase chain reaction and the sequence-specific oligonucleotide hybridization method. The distribution patterns of HLA-A, HLA-B, HLA-Cw, HLA-DRB, and HLA-DRB1 were then analysed. RESULTS: When non-responders were compared to the other groups, high frequencies in HLA-Cw1, HLA-DRB14 and HLA-DRB4 (p=0.015, 0.033 and 0.004 respectively) were evident. When seroconverters were compared to viral breakthroughers, high frequencies in HLA-A2 and HLA-DRB4 (p=0.048, 0.025 respectively) were evident. CONCLUSIONS: Our data suggests that HLA-A2, HLA-Cw1, HLA-DRB14 genes are related to the clinical outcomes of lamivudine treatment in chronic hepatitis B patients. These genes may be used in the prediction of the clinical outcome of lamivudine therapy in chronic hepatitis B patients.

High-Energy, Short-Duration Bursts of Coherent Terahertz Radiation from an Embedded Plasma Dipole.

Emission of radiation from electrons undergoing plasma oscillations (POs) at the plasma frequency has attracted interest because of the existence of intriguing and non-trivial coupling mechanism between the electrostatic PO and the emitted electromagnetic wave. While broadband emission from plasma waves in inhomogeneous plasma is well known, the underlying physics of narrowband emission at the plasma frequency observed in experiments and in solar radio-bursts is obscure. Here we show that a spatially-localized plasma dipole oscillation (PDO) can be generated when electrons are trapped in a moving train of potential wells produced by the ponderomotive force of two slightly detuned laser pulses that collide in plasma and give rise to a burst of quasi-monochromatic radiation. The energy radiated in the terahertz spectral region can reach an unprecedented several millijoules, which makes it suitable for applications requiring short pulses of high-intensity, narrowband terahertz radiation.

Analysis of Fixed-Dose Combination Products Approved by the US Food and Drug Administration, 2010-2015: Implications for Designing a Regulatory Shortcut to New Drug Application.

BACKGROUND: Fixed-dose combination (FDC) drugs have been an attractive product in pharmaceutical markets because of their unique advantages, but general guidance directing the clinical development of FDC drugs is not yet available in the US. METHOD: All drug approval reports of FDC products approved by the US FDA from January 2010 to December 2015 were intensively analyzed to investigate the regulatory requirements of the US FDA. RESULT: Through analyzing 63 approved FDCs out of 655 New Drug Application (NDA) approvals, it was found that completion of the full phases of clinical trials was not always required for approval by the FDA, which indicates that some phases of clinical studies can be possibly exempted, if justified. CONCLUSION: The results imply that pharmaceutical companies can accelerate FDC development and enter the market earlier if scientific regulatory rationales are established.

Quantifying bacterial attachment and detachment using leaching solutions of various ionic strengths after bacterial pulse.

In this study, we quantified the attachment and detachment of bacteria during transport in order to elucidate the contributions of reversible attachment on bacterial breakthrough curves. The first set of breakthrough experiment was performed for a laboratory sand column using leaching solutions of deionized water and mineral salt medium (MSM) of 200 mM with reference to KCl solution by employing Pseudomonas putida as a model bacterium. In the second set of experiment, the ionic strengths of leaching solutions immediately after bacterial pulse were lowered to tenfold and 100-fold diluted system (2 and 20 mM MSM) to focus on the influence of physicochemical factor. Results have shown that bacterial retention occurred in the sand column due to the physical deposition and physicochemical attachment. The physicochemical attachment was attributed to the high ionic strength (200 mM MSM) of leaching solution and the formation of primary energy minimum. Replacing the 200 mM leaching solution with the lower ionic strengths after pulse resulted in the increased tailing of breakthrough curve due to the detachment from the attached bacteria. The detachment could be well explained by DLVO theory, which showed the formation of energy barrier and disappearance of the secondary minimum as the ionic strength gradually decreased. Analysis of mass recovery revealed that 12-20% of the attachment was due to physical and physicochemical attachment, respectively, where the latter consisted of 25-75% of irreversible and reversible attachment respectively.

Development of a bio-magnetic measurement system and sensor configuration analysis for rats.

Magnetoencephalography (MEG) based on superconducting quantum interference devices enables the measurement of very weak magnetic fields (10-1000 fT) generated from the human or animal brain. In this article, we introduce a small MEG system that we developed specifically for use with rats. Our system has the following characteristics: (1) variable distance between the pick-up coil and outer Dewar bottom (∼5 mm), (2) small pick-up coil (4 mm) for high spatial resolution, (3) good field sensitivity (45∼ 80fT/cm/Hz), (4) the sensor interval satisfies the Nyquist spatial sampling theorem, and (5) small source localization error for the region to be investigated. To reduce source localization error, it is necessary to establish an optimal sensor layout. To this end, we simulated confidence volumes at each point on a grid on the surface of a virtual rat head. In this simulation, we used locally fitted spheres as model rat heads. This enabled us to consider more realistic volume currents. We constrained the model such that the dipoles could have only four possible orientations: the x- and y-axes from the original coordinates, and two tangentially layered dipoles (local x- and y-axes) in the locally fitted spheres. We considered the confidence volumes according to the sensor layout and dipole orientation and positions. We then conducted a preliminary test with a 4-channel MEG system prior to manufacturing the multi-channel system. Using the 4-channel MEG system, we measured rat magnetocardiograms. We obtained well defined P-, QRS-, and T-waves in rats with a maximum value of 15 pT/cm. Finally, we measured auditory evoked fields and steady state auditory evoked fields with maximum values 400 fT/cm and 250 fT/cm, respectively.

Three-dimensional reconstruction of a cardiac outline by magnetocardiography.

A 3-D cardiac visualization is significantly helpful toward clinical applications of magnetocardiography (MCG), but the cardiac reconstruction requires a segmentation process using additional image modalities. This paper proposes a 3-D cardiac outline reconstruction method using only MCG measurement data without further imaging techniques. The cardiac outline was reconstructed by a combination of both spatial filtering and coherence mapping method. The strength of cardiac activities was first estimated by the array-gain constraint minimum-norm spatial filter with recursively updated gram matrix (AGMN-RUG). Then, waveforms were reconstructed at whole source grids, and the maximum source points of an atrium and ventricle were selected as a reference, respectively. Next, the coherence between each maximum source point and whole source points was compared by the coherence mapping method. A reconstructed cardiac outline was validated by comparing with an overlapped volume ratio when the reconstructed volume was identically matched with the original volume. The results obtained by the AGMN-RUG were compared to the results by other spatial filters. The accuracy of numerical simulation and phantom experiment by the AGMN-RUG was superior 10% and 8%, respectively, than the accuracy by the standardized low-resolution electromagnetic tomography. This accuracy demonstrated the efficacy of the proposed 3-D cardiac reconstruction method.

Dynamic nuclear polarization in the hyperfine-field-dominant region.

Dynamic nuclear polarization (DNP) allows measuring enhanced nuclear magnetic resonance (NMR) signals. Though the efficiency of DNP has been known to increase at low fields, the usefulness of DNP has not been throughly investigated yet. Here, using a superconducting quantum interference device-based NMR system, we performed a series of DNP experiments with a nitroxide radical and measured DNP spectra at several magnetic fields down to sub-microtesla. In the DNP spectra, the large overlap of two peaks having opposite signs results in net enhancement factors, which are significantly lower than theoretical expectations and nearly invariant with respect to magnetic fields below the Earth's field. The numerical analysis based on the radical's Hamiltonian provides qualitative explanations of such features. The net enhancement factor reached 325 at maximum experimentally, but our analysis reveals that the local enhancement factor at the center of the rf coil is 575, which is unaffected by detection schemes. We conclude that DNP in the hyperfine-field-dominant region yields sufficiently enhanced NMR signals at magnetic fields above 1 µT.

T 1 relaxation measurement of ex-vivo breast cancer tissues at ultralow magnetic fields.

We investigated T 1 relaxations of ex-vivo cancer tissues at low magnetic fields in order to check the possibility of achieving a T 1 contrast higher than those obtained at high fields. The T 1 relaxations of fifteen pairs (normal and cancerous) of breast tissue samples were measured at three magnetic fields, 37, 62, and 122 µT, using our superconducting quantum interference device-based ultralow field nuclear magnetic resonance setup, optimally developed for ex-vivo tissue studies. A signal reconstruction based on Bayesian statistics for noise reduction was exploited to overcome the low signal-to-noise ratio. The ductal and lobular-type tissues did not exhibit meaningful T 1 contrast values between normal and cancerous tissues at the three different fields. On the other hand, an enhanced T 1 contrast was obtained for the mucinous cancer tissue.

Stereospecific mechanism of DJ-1 glyoxalases inferred from their hemithioacetal-containing crystal structures.

UNLABELLED: DJ-1 family proteins have recently been characterized as novel glyoxalases, although their cofactor-free catalytic mechanisms are not fully understood. Here, we obtained crystals of Arabidopsis thaliana DJ-1d (atDJ-1d) and Homo sapiens DJ-1 (hDJ-1) covalently bound to glyoxylate, an analog of methylglyoxal, forming a hemithioacetal that presumably mimics an intermediate structure in catalysis of methylglyoxal to lactate. The deuteration level of lactate supported the proton transfer mechanism in the enzyme reaction. Differences in the enantiomeric specificity of d/l-lactacte formation observed for the DJ-1 superfamily proteins are explained by the presence of a His residue in the active site with essential Cys and Glu residues. The model for the stereospecificity was further evaluated by a molecular modeling simulation with methylglyoxal hemithioacetal superimposed on the glyoxylate hemithioacetal. The mechanism of DJ-1 glyoxalase provides a basis for understanding the His residue-based stereospecificity. DATABASE: Structural data have been submitted to the Protein Data Bank under accession numbers 4OFW (structure of atDJ-1d), 4OGF (structure of hDJ-1 with glyoxylate) and 4OGG (structure of atDJ-1d with glyoxylate).

Two-dimensional NMR spectroscopy of (13)C methanol at less than 5 µT.

Two-dimensional (2D) spectroscopy is one of the most significant applications of nuclear magnetic resonance (NMR). Here, we demonstrate that the 2D NMR can be performed even at a low magnetic field of less than 5µT, which is ten times less than the Earth's magnetic field. The pulses used in the experiment were composed of circularly polarized fields for coherent as well as wideband excitations. Since the excitation band covers the entire spectral range, the simplest two-pulse sequence delivered the full 2D spectrum. At 5µT, methanol with (13)C enriched up to 99% belongs to a strongly coupled regime, and thus its 2D spectrum exhibits complicated spectral correlations, which can be exploited as a fingerprint in chemical analysis. In addition, we show that, with compressive sensing, the acquisition of the 2D spectrum can be accelerated to take only 45% of the overall duration.