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%.

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.

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.

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.

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.

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.

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.

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.

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.

Oxidation Temperature Effects on ZnO Thin Films Prepared from Zn Thin Films on Quartz Substrates.

We investigated the structural and optical properties of the ZnO thin films formed by oxidation of Zn thin films. Zn thin films were deposited by thermal evaporation and were then annealed from 300 to 800 degrees C to prepare ZnO thin films. We found that ZnO thin films were formed by thermal oxidation of Zn thin films at oxidation temperatures over 400 degrees C. The grain size of ZnO thin films increased with the oxidation temperature and the highest ZnO (002) intensity was obtained at 600 degrees C. In the PL spectra, the intensity of the near-band-edge peak increased with the oxidation temperatures until 400 degrees C. However, these values gradually decreased with a further increase in the oxidation temperatures over 400 degrees C. The transmittance of the ZnO thin films was more than 90% for the visible wavelength region, and the optical band gap was red-shifted with increase in the oxidation temperature.

Effect of Oxidation Temperature on Characteristics of Thermally Oxidized ZnO Thin Films on Mica Substrates.

Muscovite mica is one of the promising alternatives to polymer substrates because of its good thermal resistivity, flexibility, and transparency. In this study, metallic Zn films with a thickness of 300 nm were deposited on mica substrates through thermal evaporation; the thin films were then oxidized by annealing at temperatures ranging from 350 to 550 degrees C. The structural and optical properties of thermally oxidized ZnO thin films were investigated. Diffraction peaks for ZnO (100) and (002) planes were observed only for the ZnO thin films oxidized at temperatures above 450 degrees C. These films consisted of relatively rough film-like structures, and the average transmittance of the films was greater than 70% in the visible region. The highest near-band-edge emission was observed for the ZnO thin films oxidized at 500 degrees C. Upon increasing the oxidation temperatures to 500 degrees C, the optical band gap was blue-shifted.

Toward single-molecule optical mapping of the epigenome.

The past decade has seen an explosive growth in the utilization of single-molecule techniques for the study of complex systems. The ability to resolve phenomena otherwise masked by ensemble averaging has made these approaches especially attractive for the study of biological systems, where stochastic events lead to inherent inhomogeneity at the population level. The complex composition of the genome has made it an ideal system to study at the single-molecule level, and methods aimed at resolving genetic information from long, individual, genomic DNA molecules have been in use for the last 30 years. These methods, and particularly optical-based mapping of DNA, have been instrumental in highlighting genomic variation and contributed significantly to the assembly of many genomes including the human genome. Nanotechnology and nanoscopy have been a strong driving force for advancing genomic mapping approaches, allowing both better manipulation of DNA on the nanoscale and enhanced optical resolving power for analysis of genomic information. During the past few years, these developments have been adopted also for epigenetic studies. The common principle for these studies is the use of advanced optical microscopy for the detection of fluorescently labeled epigenetic marks on long, extended DNA molecules. Here we will discuss recent single-molecule studies for the mapping of chromatin composition and epigenetic DNA modifications, such as DNA methylation.

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.

Optical parameters of boron-doped ZnO nanorods grown by low-temperature hydrothermal reaction.

Sol-gel spin-coating was used to deposit ZnO seed layers onto quartz substrates, and ZnO nanorods doped with various concentrations of B (i.e., BZO nanorods) ranging from 0 to 2.0 at% were hydrothermally grown on the ZnO seed layers. The effects of B doping on the absorption coefficient, optical band gap, Urbach energy, refractive index, extinction coefficient, single-oscillator energy, dispersion energy, average oscillator strength, average oscillator wavelength, dielectric constant, and optical conductivity of the hydrothermally grown BZO nanorods were investigated. The optical band gaps were 3.255, 3.243, 3.254, 3.258, and 3.228 eV for the nanorods grwon at 0, 0.5, 1.0, 1.5 and 2.0 at% B, respectively. B doping increased the Urbach energy from 40.7 to 65.1 meV for the nanorods grown at 0 and 2.0 at% B, respectively, and significantly affected the dispersion energy, the single-oscillator energy, the average oscillator wavelength, the average oscillator strength, the refractive index, and the optical conductivity of the hydrothermally grown BZO nanorods.

A convenient and efficient purification method for chemically labeled oligonucleotides.

We developed an efficient, cost-effective, and rapid purification method for chemically-labeled oligonucleotides that requires less time than conventional procedures such as ethanol precipitation or size-exclusion chromatography. Based on the hydrophilic and hydrophobic properties of DNA and amine-reactive fluorophores, we show that n-butanol saturated with distilled water may be used to remove unreacted fluorophores by sequestering them in the organic phase, while labeled DNA remains in the aqueous phase. This phase extraction method is simple, fast, and allows for processing multiple samples simultaneously, a necessity for high-throughput labeling strategies.

Preparation of non-aggregated fluorescent nanodiamonds (FNDs) by non-covalent coating with a block copolymer and proteins for enhancement of intracellular uptake.

Fluorescent nanodiamonds (FNDs) are very promising fluorophores for use in biosystems due to their high biocompatibility and photostability. To overcome their tendency to aggregate in physiological solutions, which severely limits the biological applications of FNDs, we developed a new non-covalent coating method using a block copolymer, PEG-b-P(DMAEMA-co-BMA), or proteins such as BSA and HSA. By simple mixing of the block copolymer with FNDs, the cationic DMAEMA and hydrophobic BMA moieties can strongly interact with the anionic and hydrophobic moieties on the FND surface, while the PEG block can form a shell to prevent the direct contact between FNDs. The polymer-coated FNDs, along with BSA- and HSA-coated FNDs, showed non-aggregation characteristics and maintained their size at the physiological salt concentration. The well-dispersed, polymer- or protein-coated FNDs in physiological solutions showed enhanced intracellular uptake, which was confirmed by CLSM. In addition, the biocompatibility of the coated FNDs was expressly supported by a cytotoxicity assay. Our simple non-covalent coating with the block copolymer, which can be easily modified by various chemical methods, projects a very promising outlook for future biomedical applications, especially in comparison with covalent coating or protein-based coating.

Recent changes in risk factors of chronic subdural hematoma.

OBJECTIVE: Chronic subdural hematoma (CSDH) is a typical disease that is encountered frequently in neurosurgical practice. The medications which could cause coagulopathies were known as one of the risk factors of CSDH, such as anticoagulants (ACs) and antiplatelet agents (APs). Recently, the number of patients who are treated with ACs/APs is increasing, especially in the elderly population. With widespread use of these drugs, there is a need to study the changes in risk factors of CSDH patients. METHODS: We retrospectively reviewed 290 CSDH patients who underwent surgery at our institute between 1996 and 2010. We classified them into three groups according to the time of presentation (Group A : the remote period group, 1996-2000, Group B : the past period group, 2001-2005, and Group C : the recent period group, 2006-2010). Also, we performed the comparative analysis of independent risk factors between three groups. RESULTS: Among the 290 patients, Group A included 71 patients (24.5%), Group B included 98 patients (33.8%) and Group C included 121 patients (41.7%). Three patients (4.2%) in Group A had a history of receiving ACs/APs, 8 patients (8.2%) in Group B, and 19 patients (15.7%) in Group C. Other factors such as head trauma, alcoholism, epilepsy, previous neurosurgery and underlying disease having bleeding tendency were also evaluated. In ACs/APs related cause of CSDH in Group C, significantly less proportion of the patients are associated with trauma or alcohol compared to the non-medication group. CONCLUSION: In this study, the authors concluded that ACs/APs have more importance as a risk factor of CSDH in the recent period compared to the past. Therefore, doctors should prescribe these medications carefully balancing the potential risk and benefit.

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.

Pervaporative separation of bioethanol using a polydimethylsiloxane/polyetherimide composite hollow-fiber membrane.

Pervaporation is one of the most promising separation processes for the purification of ethanol. In this study, a composite hollow-fiber membrane with a thin polydimethylsiloxane (PDMS) active layer on a polyetherimide (PEI) macroporous support was used for pervaporative separation of ethanol produced by Saccharomyces cerevisiae from glucose fermentation broth. The pervaporation performance for ethanol/water binary mixtures was strongly dependent on the feed concentration and operating temperature for ethanol concentrations of 1-10%. The composite hollow-fiber membrane was stable over the long-term (about 160 days) with an ethanol permeation flux of 60-62 g/m(2)h and a separation factor of 7-9. In comparison with published results for PDMS composite membranes, the PDMS/PEI hollow-fiber composite membrane had relatively good pervaporation performance with a total flux of 231-252 g/m(2)h.