Target Specificity of Cas9 Nuclease via DNA Rearrangement Regulated by the REC2 Domain.

Understanding the underlying principles for the target-specific nuclease activity of CRISPR/Cas9 is a prerequisite to minimize its off-target DNA cleavage for genome engineering applications. Here, we show that the noncatalytic REC2 domain of Cas9 nuclease plays a crucial role in off-target discrimination. Using single-molecule fluorescence methods, we investigate conformational dynamics of the non-target strand (NTS) of DNA interacting with Cas9 and find that REC2 regulates the NTS rearrangement for cleavage reaction with the help of positively charged residues on its surface. This mechanistic model for the target specificity of Cas9 provides molecular insights for the rational approach to Cas9 engineering for highly specific genome editing.

Direct and precise length measurement of single, stretched DNA fragments by dynamic molecular combing and STED nanoscopy.

A combination of DNA stretching method and super-resolution nanoscopy allows an accurate and precise measurement of the length of DNA fragments ranging widely in size from 117 to 23,130 bp. BstEII- and HindIII-treated λDNA fragments were stained with an intercalating dye and then linearly stretched on a coverslip by dynamic molecular combing. The image of individual DNA fragments was obtained by stimulated emission depletion nanoscopy. For DNA fragments longer than ∼1000 bp, the measured lengths of DNA fragments were consistently within ∼0.5 to 1.0 % of the reference values, raising the possibility of this method in a wide range of applications including facile detection for copy number variations and trinucleotide repeat disorder.

Cerebral-perfusion-based single-photon emission computed tomography (SPECT) staging using NeuroGam® in patients with moyamoya disease.

BACKGROUND AND PURPOSE: Cerebral angiography (CA) is the gold standard for moyamoya disease (MMD) staging and diagnosis, but CA findings are not well correlated with clinical symptoms. The purpose of this study was to establish novel cerebral-perfusion-based staging for MMD that is well correlated with clinical symptoms. MATERIALS AND METHODS: From 2010 to 2015, regional cerebrovascular reserve (rCVR) was examined by single-photon emission computed tomography (SPECT) using NeuroGam® (Segamicorp, Houston, TX, USA) in 30 patients (17 women, 13 men; 60 hemispheres; mean 42.0 years old [range 5-60 years old]) with MMD, which was diagnosed by CA and magnetic resonance angiography (MRA). Brain CT or brain magnetic resonance imaging (MRI) was used to evaluate neurological conditions such as transient ischemic attack (TIA), cerebral hemorrhage, and cerebral infarction. A novel staging system for MMD was developed by combining findings from CA, MRI, and SPECT with NeuroGam®. RESULTS: Our novel staging system was strongly associated with clinical symptoms. Twenty-two hemispheres out of 60 were categorized as stage I, 24 hemispheres were categorized as stage II, and 14 hemispheres were categorized as stage III. Hemispheres with higher scores exhibited a higher incidence of clinical symptoms. These findings indicate that cerebral-perfusion-based staging is predictive of MMD clinical symptoms. CONCLUSION: Perfusion-based SPECT staging correlates well with clinical symptoms and may be a reliable alternative to the Suzuki staging by CA.

Analysis of affected and non-affected sides of stroke hemiparalysis patients and correlations between rehabilitation therapy assessments using the bioelectrical impedance analysis method.

[Purpose] The purpose of this study was to demonstrate the use of bioelectrical impedance analysis as an appropriate rehabilitation therapy evaluation tool for stroke hemiplegic patients. [Subjects and Methods] A group of 20 stroke patients diagnosed with stroke hemiplegia who underwent stroke rehabilitation from October to November 2015 participated in this study. Using bioelectrical impedance analysis, stroke hemiparalysis patients were examined, and the affected and non-affected sides were compared. This correlation between impedance measurement values and rehabilitation therapy as an assessment tool was determined. [Results] According to the whole-body bioimpedance measurements, prediction markers, reactances, and phase angles, there were significant differences between the non-affected and affected sides, and bioimpedance had a positive correlation with hand grip power, manual dexterity of hand function, and ability to perform activities of daily living. [Conclusion] There were significant differences between the impedance values of the affected and non-affected sides of hemiplegic stroke patients. These results suggest that bioelectrical impedance analysis can be used as an assessment during the rehabilitation of stroke patients.

Synthesis and optical characteristics of yttrium-doped zinc oxide nanorod arrays grown by hydrothermal method.

Yttrium-doped ZnO (YZO) nanorods were synthesized by hydrothermal growth on a quartz substrate with various post-annealing temperatures. To investigate the effects of post-annealing on the optical properties and parameters of the nanorods, X-ray diffractometry (XRD), photoluminescence (PL) measurement, and ultraviolet (UV)-visible spectroscopy were used. From the XRD investigation, the full width at half maximum (FWHM) and the dislocation density of the nanorods was found to increase with an increase in the post-annealing temperature. In the PL spectra, the intensity of the near band edge (NBE) emission peak in the UV region also increases with an increase in the temperature of post-annealing. The deep level emission (DLE) peak in the visible region changes with various post-annealing temperatures, and its intensity increases remarkably with post-annealing at 800 degrees C. In this paper, changes in the optical parameters of the nanorods caused by variation in the behavior of Y during post-annealing was investigated, with properties such as absorption coefficients, refractive indices, and dispersion parameters being obtained from transmittance and reflectance analysis.

Influence of Sn doping on structural and optical properties of zinc oxide nanorods prepared via hydrothermal process.

Hydrothermally grown ZnO nanorods were doped with various concentrations of Sn, ranging from 0 to 2.5 at%. Scanning electron microscopy (SEM), X-ray diffractometer (XRD), ultraviolet (UV)-visible spectroscopy, and Photoluminescence (PL) measurements were used to determine the effect of Sn doping on the structural and optical properties. In the SEM images, the nanorods have hexagonal wurtzite structure and the diameter of the nanorods increases with an increase in the Sn content. The optical parameters of the Sn-doped ZnO (SZO) nanorods such as the absorption coefficients, optical bandgaps, Urbach energies, refractive indices, dispersion parameters, dielectric constants, and optical conductivities were determined from the transmittance and reflectance results. In the PL spectra, the intensity of the NBE peak in the UV region decreases and is blue-shifted with an increase in the Sn content, while the DLE peaks of the nanorods in the visible region shift toward the low-energy region with the introduction of Sn.

Enhanced detection of Listeria monocytogenes using tetraethylenepentamine-functionalized magnetic nanoparticles and LAMP-CRISPR/Cas12a-based biosensor.

BACKGROUND: Listeria monocytogenes is a pathogenic bacterium that can lead to severe illnesses, especially among vulnerable populations. Therefore, the development of rapid and sensitive detection methods is vital to prevent and manage foodborne diseases. In this study, we used tetraethylenepentamine (TEPA)-functionalized magnetic nanoparticles (MNPs) and a loop-mediated isothermal amplification (LAMP)-based CRISPR/Cas12a-based biosensor to concentrate and detect, respectively, L. monocytogenes. LAMP enables DNA amplification at a constant temperature, providing a highly suitable approach for point-of-care testing (POCT). The ability of CRISPR/Cas12a to cleave ssDNA reporter, coupled with TEPA-functionalized MNPs effective attachment to negatively charged bacteria, forms a promising biosensor. RESULTS: The LAMP assay was meticulously developed by selecting specific primers and designing crRNA sequences targeting a specific region within the hly gene of L. monocytogenes. We selected primer and refined the amplification conditions by systematically exploring a temperature range from 59 °C to 69 °C, ensuring the attainment of optimal performance. This process was complemented by systematic optimization of LAMP-CRISPR/Cas12a system parameters. In particular, we successfully established the optimal ssDNA reporter concentrations (0-1.2 µM) and Cas12a-mediated trans-cleavage times (0-20 min), crucial components that underpin the effectiveness of the LAMP-CRISPR/Cas12a-based biosensor. For optimizing parameters in capturing L. monocytogenes using TEPA-functionalized MNPs, capture efficiency was significantly enhanced through adjustments in TEPA-functionalized MNPs concentration, incubation times, and magnetic separation duration. Large-volume (20 mL) magnetic separation exhibited a 10-fold sensitivity improvement over conventional methods. Utilizing TEPA-functionalized MNPs, the LAMP-CRISPR/Cas12a-based biosensor achieved detection limits of 100 CFU mL-1 in pure cultures and 100 CFU g-1 in enoki mushrooms. SIGNIFICANCE: The integration of this novel technique with the LAMP-CRISPR/Cas12a-based biosensor enhances the accuracy and sensitivity of L. monocytogenes detection in foods, and it can be a promising biosensor for POCT. The 10-fold increase in sensitivity compared to conventional methods makes this approach a groundbreaking advancement in pathogenic bacteria detection for food safety and public health.

OView-AI Supporter for Classifying Pneumonia, Pneumothorax, Tuberculosis, Lung Cancer Chest X-ray Images Using Multi-Stage Superpixels Classification.

The deep learning approach has recently attracted much attention for its outstanding performance to assist in clinical diagnostic tasks, notably in computer-aided solutions. Computer-aided solutions are being developed using chest radiography to identify lung diseases. A chest X-ray image is one of the most often utilized diagnostic imaging modalities in computer-aided solutions since it produces non-invasive standard-of-care data. However, the accurate identification of a specific illness in chest X-ray images still poses a challenge due to their high inter-class similarities and low intra-class variant abnormalities, especially given the complex nature of radiographs and the complex anatomy of the chest. In this paper, we proposed a deep-learning-based solution to classify four lung diseases (pneumonia, pneumothorax, tuberculosis, and lung cancer) and healthy lungs using chest X-ray images. In order to achieve a high performance, the EfficientNet B7 model with the pre-trained weights of ImageNet trained by Noisy Student was used as a backbone model, followed by our proposed fine-tuned layers and hyperparameters. Our study achieved an average test accuracy of 97.42%, sensitivity of 95.93%, and specificity of 99.05%. Additionally, our findings were utilized as diagnostic supporting software in OView-AI system (computer-aided application). We conducted 910 clinical trials and achieved an AUC confidence interval (95% CI) of the diagnostic results in the OView-AI system of 97.01%, sensitivity of 95.68%, and specificity of 99.34%.

Four-color alternating-laser excitation single-molecule fluorescence spectroscopy for next-generation biodetection assays.

BACKGROUND: Single-molecule detection (SMD) technologies are well suited for clinical diagnostic applications by offering the prospect of minimizing precious patient sample requirements while maximizing clinical information content. Not yet available, however, is a universal SMD-based platform technology that permits multiplexed detection of both nucleic acid and protein targets and that is suitable for automation and integration into the clinical laboratory work flow. METHODS: We have used a sensitive, specific, quantitative, and cost-effective homogeneous SMD method that has high single-well multiplexing potential and uses alternating-laser excitation (ALEX) fluorescence-aided molecule sorting extended to 4 colors (4c-ALEX). Recognition molecules are tagged with different-color fluorescence dyes, and coincident confocal detection of ≥2 colors constitutes a positive target-detection event. The virtual exclusion of the majority of sources of background noise eliminates washing steps. Sorting molecules with multidimensional probe stoichiometries (S) and single-molecule fluorescence resonance energy transfer efficiencies (E) allows differentiation of numerous targets simultaneously. RESULTS: We show detection, differentiation, and quantification-in a single well-of (a) 25 different fluorescently labeled DNAs; (b) 8 bacterial genetic markers, including 3 antibiotic drug-resistance determinants found in 11 septicemia-causing Staphylococcus and Enterococcus strains; and (c) 6 tumor markers present in blood. CONCLUSIONS: The results demonstrate assay utility for clinical molecular diagnostic applications by means of multiplexed detection of nucleic acids and proteins and suggest potential uses for early diagnosis of cancer and infectious and other diseases, as well as for personalized medicine. Future integration of additional technology components to minimize preanalytical sample manipulation while maximizing throughput should allow development of a user-friendly ("sample in, answer out") point-of-care platform for next-generation medical diagnostic tests that offer considerable savings in costs and patient sample.

Identification of myricetin and scutellarein as novel chemical inhibitors of the SARS coronavirus helicase, nsP13.

Severe acute respiratory syndrome (SARS) is an infectious disease with a strong potential for transmission upon close personal contact and is caused by the SARS-coronavirus (CoV). However, there are no natural or synthetic compounds currently available that can inhibit SARS-CoV. We examined the inhibitory effects of 64 purified natural compounds against the activity of SARS helicase, nsP13, and the hepatitis C virus (HCV) helicase, NS3h, by conducting fluorescence resonance energy transfer (FRET)-based double-strand (ds) DNA unwinding assay or by using a colorimetry-based ATP hydrolysis assay. While none of the compounds, examined in our study inhibited the DNA unwinding activity or ATPase activity of human HCV helicase protein, we found that myricetin and scutellarein potently inhibit the SARS-CoV helicase protein in vitro by affecting the ATPase activity, but not the unwinding activity, nsP13. In addition, we observed that myricetin and scutellarein did not exhibit cytotoxicity against normal breast epithelial MCF10A cells. Our study demonstrates for the first time that selected naturally-occurring flavonoids, including myricetin and scultellarein might serve as SARS-CoV chemical inhibitors.

Three-dimensional EM structure of an intact activator-dependent transcription initiation complex.

We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a premelted transcription bubble. A 20-A electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP alpha subunit C-terminal domain (alphaCTD), interactions of alphaCTD with sigma(70) region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP beta', beta, and sigma(70) subunits.

Nonradioactive, ultrasensitive site-specific protein-protein photocrosslinking: interactions of alpha-helix 2 of TATA-binding protein with general transcription factor TFIIA and transcriptional repressor NC2.

We have developed an approach that enables nonradioactive, ultrasensitive (attamole sensitivity) site-specific protein-protein photocrosslinking, and we have applied the approach to the analysis of interactions of alpha-helix 2 (H2) of human TATA-element binding protein (TBP) with general transcription factor TFIIA and transcriptional repressor NC2. We have found that TBP H2 can be crosslinked to TFIIA in the TFIIA-TBP-DNA complex and in higher order transcription-initiation complexes, and we have mapped the crosslink to the 'connector' region of the TFIIA alpha/beta subunit (TFIIAalpha/beta). We further have found that TBP H2 can be crosslinked to NC2 in the NC2-TBP-DNA complex, and we have mapped the crosslink to the C-terminal 'tail' of the NC2 alpha-subunit (NC2alpha). Interactions of TBP H2 with the TFIIAalpha/beta connector and the NC2alpha C-terminal tail were not observed in crystal structures of TFIIA-TBP-DNA and NC2-TBP-DNA complexes, since relevant segments of TFIIA and NC2 were not present in truncated TFIIA and NC2 derivatives used for crystallization. We propose that interactions of TBP H2 with the TFIIAalpha/beta connector and the NC2alpha C-terminal tail provide an explanation for genetic results suggesting importance of TBP H2 in TBP-TFIIA interactions and TBP-NC2 interactions, and provide an explanation-steric exclusion-for competition between TFIIA and NC2.

Lighting up individual DNA binding proteins with quantum dots.

The ability to determine the precise loci and occupancy of DNA-binding proteins is instrumental to our understanding of cellular processes like gene expression and regulation. We propose a single-molecule approach for the direct visualization of proteins bound to their template DNA. Fluorescent quantum dots (QD) are used to label proteins bound to DNA, allowing multicolor, nanometer-resolution localization. Protein-DNA complexes are linearly extended and imaged to determine the precise location of the protein binding sites. The method is demonstrated by detecting individual QD-labeled T7-RNA polymerases on the T7 bacteriophage genome. This work demonstrates the potential of this approach to precisely read protein binding position or, alternatively, "write" such information on extended DNA with QDs via sequence-specific molecular recognition.

Addition of an N-Terminal Poly-Glutamate Fusion Tag Improves Solubility and Production of Recombinant TAT-Cre Recombinase in Escherichia coli.

Cre recombinase is widely used to manipulate DNA sequences for both in vitro and in vivo research. Attachment of a trans-activator of transcription (TAT) sequence to Cre allows TATCre to penetrate the cell membrane, and the addition of a nuclear localization signal (NLS) helps the enzyme to translocate into the nucleus. Since the yield of recombinant TAT-Cre is limited by formation of inclusion bodies, we hypothesized that the positively charged arginine-rich TAT sequence causes the inclusion body formation, whereas its neutralization by the addition of a negatively charged sequence improves solubility of the protein. To prove this, we neutralized the positively charged TAT sequence by proximally attaching a negatively charged poly-glutamate (E12) sequence. We found that the E12 tag improved the solubility and yield of E12-TAT-NLS-Cre (E12-TAT-Cre) compared with those of TAT-NLS-Cre (TATCre) when expressed in E. coli. Furthermore, the growth of cells expressing E12-TAT-Cre was increased compared with that of the cells expressing TAT-Cre. Efficacy of the purified TATCre was confirmed by a recombination test on a floxed plasmid in a cell-free system and 293 FT cells. Taken together, our results suggest that attachment of the E12 sequence to TAT-Cre improves its solubility during expression in E. coli (possibly by neutralizing the ionic-charge effects of the TAT sequence) and consequently increases the yield. This method can be applied to the production of transducible proteins for research and therapeutic purposes.

In vivo assembly and single-molecule characterization of the transcription machinery from Shewanella oneidensis MR-1.

Harnessing the new bioremediation and biotechnology applications offered by the dissimilatory metal-reducing bacteria, Shewanella oneidensis MR-1, requires a clear understanding of its transcription machinery, a pivotal component in maintaining vitality and in responding to various conditions, including starvation and environmental stress. Here, we have reconstituted the S. oneidensis RNA polymerase (RNAP) core in vivo by generating a co-overexpression construct that produces a long polycistronic mRNA encoding all of the core subunits (alpha, beta, beta', and omega) and verified that this reconstituted core is capable of forming fully functional holoenzymes with the S. oneidensis sigma factors sigma(70), sigma(38), sigma(32), and sigma(24). Further, to demonstrate the applications for this reconstituted core, we report the application of single-molecule fluorescence resonance energy transfer (smFRET) assays to monitor the mechanisms of transcription by the S. oneidensis sigma(70)-RNAP holoenyzme. These results show that the reconstituted transcription machinery from S. oneidensis, like its Escherichia coli counterpart, "scrunches" the DNA into its active center during initial transcription, and that as the holoenzyme transitions into elongation, the release of sigma(70) is non-obligatory.

Static and kinetic site-specific protein-DNA photocrosslinking: analysis of bacterial transcription initiation complexes.

Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking - involving rapid-quench-flow mixing and pulsed-laser irradiation - permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes.

General and facile purification of dye-labeled oligonucleotides by pH-controlled extraction.

Previously, we reported a method for facile purification of oligonucleotides labeled with hydrophobic dyes, based on the solubility difference between the hydrophilic DNA and unreacted dye. Here, we present a new purification method applicable to any dye regardless of its hydrophobicity. We exploited the population shift of a fluorescent dye in a low-pH aqueous solution from its anionic form toward its neutral form. When the pH of an aqueous solution containing dye-labeled DNA and unreacted free dye is lowered, and the solution is mixed with a hydrophobic organic solvent (butanol), the neutral free dye is preferentially dissolved in the organic phase, leaving behind the hydrophilic dye-labeled DNA in the aqueous phase. We experimentally verified that our new method results in high yields of dye-labeled oligonucleotides and the efficient removal of free dye.

Gene profiling during regression of pressure overload-induced cardiac hypertrophy.

Regression of cardiac hypertrophy and improvement of the functional capacity of failing hearts have reportedly been achieved by mechanical unloading in cardiac work. In this study, cardiac hypertrophy was first induced in rats by transverse aortic constriction and then mechanically unloaded by relieving the constriction after significant cardiac hypertrophy had developed. Hypertrophy was significantly regressed at the cellular and molecular levels at day 1, 3, and 7 after constriction relief. Gene profiling analysis revealed that 52 genes out of 9,911 genes probed on a gene array were specifically upregulated during the early regression period. Among these regression-induced genes, Eyes absent 2 (eya2) was of particular interest because it is a transcriptional cofactor involved in mammalian organogenesis as well as Drosophila eye development. Adenovirus-mediated overexpression of eya2 in rat neonatal cardiomyocytes completely abrogated phenylephrine-induced development of cardiomyocyte hypertrophy as determined by cell size, sarcomere rearrangement and fetal gene re-expression. Our data strongly suggest that transcriptional programs distinct from those mediating cardiac hypertrophy may be operating during the regression of hypertrophy, and eya2 may be a key regulator of one of these programs.

Effects of Precursor Concentration on Structural and Optical Properties of ZnO Thin Films Grown on Muscovite Mica Substrates by Sol-Gel Spin-Coating.

The structural and optical properties of the ZnO thin films grown on mica substrates for different precursor concentrations were investigated. The surface morphologies of all the samples indicated that they consisted of granular structures with spherical nano-sized crystallites. The thickness of the ZnO thin films increased significantly and the optical band gap exhibited a blue shift with an increase in the precursor concentration. It is remarkable that the highest I(NBE)/I(DLE) ratio was observed for the ZnO thin film with 0.8 M precursor concentration, even though cracks formed on the surface of this film.

Novel oral formulation of paclitaxel inhibits neointimal hyperplasia in a rat carotid artery injury model.

BACKGROUND: Paclitaxel has been shown to inhibit vascular smooth muscle cell migration and proliferation contributing to neointimal formation. This study tested whether novel oral formulations of paclitaxel can prevent neointimal formation in a rat carotid artery injury model. METHODS AND RESULTS: Oral formulations of paclitaxel (0, 5, 7.5, or 10 mg/kg) were administered to 40 rats by gavage for 5 days after injury. The peak plasma levels of paclitaxel administered at 5, 7.5, and 10 mg/kg were 61+/-16, 89+/-22, and 108+/-28 nmol/L, respectively. Treatment effects were assessed 11 days after injury. The angiographic minimum luminal diameters of the oral paclitaxel groups treated at 5, 7.5, and 10 mg/kg were 6.28+/-2.09, 6.97+/-1.79, and 7.97+/-1.57 AU, and these were significantly larger than that of the control group (4.67+/-1.45 AU). The oral paclitaxel groups (5, 7.5, 10 mg/kg; 0.05+/-0.05, 0.04+/-0.03, 0.05+/-0.03 mm2) showed significant neointimal formation reductions versus the control group (0.13+/-0.05 mm2). All rats survived to study completion. Only 2 animals in the 10 mg/kg group experienced weight loss ( approximately 10%) and loose stools between 4 and 6 days after injury. All other animals appeared healthy during the study. For comparison purposes, intraperitoneal formulations of paclitaxel (0 or 2 mg/kg) were administered by injection to 15 rats. We confirmed that the intraperitoneal administration of paclitaxel also effectively inhibited neointimal formation. CONCLUSIONS: Oral formulations of paclitaxel provide an effective means of inhibiting proliferative response to vascular injury in the rat. Thus, oral formulations of paclitaxel may prevent human restenosis without significant toxicity.