Universal primers for rift valley fever virus whole-genome sequencing.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease causing acute hemorrhagic fever. Accurate identification of mutations and phylogenetic characterization of RVF virus (RVFV) require whole-genome analysis. Universal primers to amplify the entire RVFV genome from clinical samples with low copy numbers are currently unavailable. Thus, we aimed to develop universal primers applicable for all known RVFV strains. Based on the genome sequences available from public databases, we designed eight pairs of universal PCR primers covering the entire RVFV genome. To evaluate primer universality, four RVFV strains (ZH548, Kenya 56 (IB8), BIME-01, and Lunyo), encompassing viral phylogenetic diversity, were chosen. The nucleic acids of the test strains were chemically synthesized or extracted via cell culture. These RNAs were evaluated using the PCR primers, resulting in successful amplification with expected sizes (0.8-1.7 kb). Sequencing confirmed that the products covered the entire genome of the RVFV strains tested. Primer specificity was confirmed via in silico comparison against all non-redundant nucleotide sequences using the BLASTn alignment tool in the NCBI database. To assess the clinical applicability of the primers, mock clinical specimens containing human and RVFV RNAs were prepared. The entire RVFV genome was successfully amplified and sequenced at a viral concentration of 108 copies/mL. Given the universality, specificity, and clinical applicability of the primers, we anticipate that the RVFV universal primer pairs and the developed method will aid in RVFV phylogenomics and mutation detection.

Charge Carrier Dynamics in Non-Fullerene Acceptor-Based Organic Solar Cells: Investigating the Influence of Processing Additives Using Transient Absorption Spectroscopy.

In this study, we present a comprehensive investigation into the charge generation mechanism in bulk-heterojunction organic solar cells employing non-fullerene acceptors (NFAs) both with and without the presence of processing additives. While photovoltaic devices based on Y6 or BTP-eC9 have shown remarkable power conversion efficiencies, the underlying charge generation mechanism in polymer:NFA blends remains poorly understood. To shed light on this, we employ transient absorption (TA) spectroscopy to elucidate the charge transfer pathway within a blend of the donor polymer PM6 and NFAs. Interestingly, the charge carrier lifetimes of neat Y6 and BTP-eC9 are comparable, both reaching up to 20 ns. However, the PM6:BTP-eC9 blend exhibits substantially higher charge carrier generation and a longer carrier lifetime compared to PM6:Y6 blend films, leading to superior performance. By comparing TA data obtained from PM6:Y6 or PM6:BTP-eC9 blend films with and without processing additives, we observe significantly enhanced charge carrier generation and prolonged charge carrier lifetimes in the presence of these additives. These findings underscore the potential of manipulating excited species as a promising avenue for further enhancing the performance of organic solar cells. Moreover, this understanding contributes to the advancement of NFA-based systems and the optimization of charge transfer processes in polymer:NFA blends.

Comparison of RT-qPCR and RT-ddPCR with Rift valley fever virus (RVFV) RNA.

Rift valley fever (RVF) is an important zoonotic disease caused by the Rift valley fever virus (RVFV) which can affect ruminants and humans. In this study, a comparison was done of the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription-droplet digital PCR (RT-ddPCR) assays with synthesized RVFV RNA, cultured viral RNA, and mock clinical RVFV RNA samples. The genomic segments (L, M, and S) of three RVFV strains (BIME01, Kenya56, and ZH548) were synthesized and used as templates for in vitro transcription (IVT). Both the RT-qPCR and RT-ddPCR assays for RVFV did not react with any of the negative reference viral genomes. Thus, both the RT-qPCR and RT-ddPCR assays are specific to RVFV. The comparison of both the RT-qPCR and RT-ddPCR assays with serially diluted templates showed that the LoD of both assays are similar, and a concordant of the results was observed. The LoD of both assays reached the practical measurable minimum concentration. Taken altogether, the sensitivity of the RT-qPCR and RT-ddPCR assays is similar, and the material measured by RT-ddPCR can be used as a reference material for RT-qPCR.

Seasonal variations of the airborne microbial assemblages of the Seoul subway, South Korea from 16S and ITS gene profiles with chemical analysis.

In this study, we determined the seasonal airborne microbial diversity profiles at SMRT stations by sequencing the 16S rRNA and ITS. Particulate matter samples were collected from air purifiers installed in the platform area of the SMRT subway stations. Three stations that included the most crowded one were selected for the sampling. The sampling was done at each season during 2019. After extracting the total DNA from all seasonal samples, PCR was performed with Illumina overhang adapter primers for the V3-V4 region of the 16S rRNA gene and ITS2 region of the ITS gene. The amplified products were further purified, and sequencing libraries were made. Sequencing was carried with the Illumina Miseq Sequencing system (Illumina, USA) followed by in-depth diversity analyses. The elemental composition of the particulate matter samples collected from the different subway stations were obtained using a WD-XRF spectrometer. The SMRT microbiome showed extensive taxonomic diversity with the most common bacterial genera at the subway stations associated with the skin. Overall, the stations included in this study harbored different phylogenetic communities based on α- and ß-diversity comparisons. Microbial assemblages also varied depending upon the season in which the samples were taken and the station. Major elements present at the subway stations were from aerosols generated between wheels and brake cushions and between the catenaries and the pantographs. This study shows that the microbial composition of the SMRT subway stations comes from a diverse combination of environmental and human sources, the season and the lifestyle of commuters.

Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2.

As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields.

Human Alphacoronavirus Universal Primers for Genome Amplification and Sequencing.

Rapid and accurate sequencing covering the entire genome is essential to identify genetic variations of viral pathogens. However, due to the low viral titers in clinical samples, certain amplification steps are required for viral genome sequencing. At present, there are no universal primers available for alphacoronaviruses and that, since these viruses have diverse strains, new primers specific to the target strain must be continuously developed for sequencing. Thus, in this study, we aimed to develop a universal primer set valid for all human alphacoronaviruses and applicable to samples containing trace amounts of the virus. To this aim, we designed overlapping primer pairs capable of amplifying the entire genome of all known human alphacoronaviruses. The selected primers, named the AC primer set, were composed of 10 primer pairs stretching over the entire genome of alphacoronaviruses, and produced PCR products of the expected size (3-5 kb) from both the HCoV-229E and HCoV-NL63 strains. After genome amplification, an evaluation using various sequencing platforms was carried out. The amplicon library sequencing data were assembled into complete genome sequences in all sequencing strategies examined in this study. The sequencing accuracy varied depending on the sequencing technology, but all sequencing methods showed a sequencing error of less than 0.01%. In the mock clinical specimen, the detection limit was 10-3 PFU/ml (102 copies/ml). The AC primer set and experimental procedure optimized in this study may enable the fast diagnosis of mutant alphacoronaviruses in future epidemics.

Comparison of 16S rRNA Gene Based Microbial Profiling Using Five Next-Generation Sequencers and Various Primers.

Microbial community analysis based on the 16S rRNA-gene is used to investigate both beneficial and harmful microorganisms in various fields and environments. Recently, the next-generation sequencing (NGS) technology has enabled rapid and accurate microbial community analysis. Despite these advantages of NGS based metagenomics study, sample transport, storage conditions, amplification, library preparation kits, sequencing, and bioinformatics procedures can bias microbial community analysis results. In this study, eight mock communities were pooled from genomic DNA of Lactobacillus acidophilus KCTC 3164T, Limosilactobacillus fermentum KCTC 3112T, Lactobacillus gasseri KCTC 3163T, Lacticaseibacillus paracasei subsp. paracasei KCTC 3510T, Limosilactobacillus reuteri KCTC 3594T, Lactococcus lactis subsp. lactis KCTC 3769T, Bifidobacterium animalis subsp. lactis KCTC 5854T, and Bifidobacterium breve KCTC 3220T. The genomic DNAs were quantified by droplet digital PCR (ddPCR) and were mixed as mock communities. The mock communities were amplified with various 16S rRNA gene universal primer pairs and sequenced by MiSeq, IonTorrent, MGIseq-2000, Sequel II, and MinION NGS platforms. In a comparison of primer-dependent bias, the microbial profiles of V1-V2 and V3 regions were similar to the original ratio of the mock communities, while the microbial profiles of the V1-V3 region were relatively biased. In a comparison of platform-dependent bias, the sequence read from short-read platforms (MiSeq, IonTorrent, and MGIseq-2000) showed lower bias than that of long-read platforms (Sequel II and MinION). Meanwhile, the sequences read from Sequel II and MinION platforms were relatively biased in some mock communities. In the data of all NGS platforms and regions, L. acidophilus was greatly underrepresented while Lactococcus lactis subsp. lactis was generally overrepresented. In all samples of this study, the bias index (BI) was calculated and PCA was performed for comparison. The samples with biased relative abundance showed high BI values and were separated in the PCA results. In particular, analysis of regions rich in AT and GC poses problems for genome assembly, which can lead to sequencing bias. According to this comparative analysis, the development of reference material (RM) material has been proposed to calibrate the bias in microbiome analysis.

Comparison of Digital PCR and Quantitative PCR with Various SARS-CoV-2 Primer-Probe Sets.

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

Kitasatospora acidiphila sp. nov., isolated from pine grove soil, exhibiting antimicrobial potential.

A polyphasic study was carried out to establish the taxonomic position of an acidophilic isolate designated MMS16-CNU292T (=JCM 32302T) from pine grove soil, and provisionally assigned to the genus Kitasatospora. On the basis of 16S rRNA gene sequence similarity, the strain formed a novel evolutionary lineage within Kitasatospora and showed highest similarities to Kitasatospora azatica KCTC 9699T (98.75 %), Kitasatospora kifunensis IFO 15206T (98.74 %), Kitasatospora purpeofusca NRRL B-1817T (98.61 %) and Kitasatospora nipponensis HKI 0315T (98.42 %), respectively. Strain MMS16-CNU292T possessed MK-9(H6) and MK-9(H8) as the major menaquinones, and a major amount of meso-diaminopimelic acid in the cell-wall peptidoglycan. The whole-cell hydrolysates were rich in galactose, glucose and mannose, and the polar lipids mainly consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and phosphatidylinositol mannosides. The major fatty acids were anteiso-C15 : 1-A, anteiso-C15 : 0, and iso-C15 : 0, and the DNA G+C content was 71.5 mol%. The strain exhibited antibacterial activity against a number of bacterial strains, and the activity was generally greater when grown in acidic conditions. The phylogenetic, chemotaxonomic and phenotypic properties enabled distinction of MMS16-CNU292T from related species, and thus the isolate should be recognized as a new species of the genus Kitasatospora, for which the name Kitasatospora acidiphila sp. nov. (type strain=MMS16-CNU292T=KCTC 49011T=JCM 32302T) is proposed.

Sensitivity-Based Fault Detection and Isolation Algorithm for Road Vehicle Chassis Sensors.

Vehicle control systems such as ESC (electronic stability control), MDPS (motor-driven power steering), and ECS (electronically controlled suspension) improve vehicle stability, driver comfort, and safety. Vehicle control systems such as ACC (adaptive cruise control), LKA (lane-keeping assistance), and AEB (autonomous emergency braking) have also been actively studied in recent years as functions that assist drivers to a higher level. These DASs (driver assistance systems) are implemented using vehicle sensors that observe vehicle status and send signals to the ECU (electronic control unit). Therefore, the failure of each system sensor affects the function of the system, which not only causes discomfort to the driver but also increases the risk of accidents. In this paper, we propose a new method to detect and isolate faults in a vehicle control system. The proposed method calculates the constraints and residuals of 12 systems by applying the model-based fault diagnosis method to the sensor of the chassis system. To solve the inaccuracy in detecting and isolating sensor failure, we applied residual sensitivity to a threshold that determines whether faults occur. Moreover, we applied a sensitivity analysis to the parameters semi-correlation table to derive a fault isolation table. To validate the FDI (fault detection and isolation) algorithm developed in this study, fault signals were injected and verified in the HILS (hardware-in-the-loop simulation) environment using an RCP (rapid control prototyping) device.

Controlling the Temperature and Speed of the Phase Transition of VO2 Microcrystals.

We investigated the control of two important parameters of vanadium dioxide (VO2) microcrystals, the phase transition temperature and speed, by varying microcrystal width. By using the reflectivity change between insulating and metallic phases, phase transition temperature is measured by optical microscopy. As the width of square cylinder-shaped microcrystals decreases from ∼70 to ∼1 µm, the phase transition temperature (67 °C for bulk) varied as much as 26.1 °C (19.7 °C) during heating (cooling). In addition, the propagation speed of phase boundary in the microcrystal, i.e., phase transition speed, is monitored at the onset of phase transition by using the high-speed resistance measurement. The phase transition speed increases from 4.6 × 10(2) to 1.7 × 10(4) µm/s as the width decreases from ∼50 to ∼2 µm. While the statistical description for a heterogeneous nucleation process explains the size dependence on phase transition temperature of VO2, the increase of effective thermal exchange process is responsible for the enhancement of phase transition speed of small VO2 microcrystals. Our findings not only enhance the understanding of VO2 intrinsic properties but also contribute to the development of innovative electronic devices.

Pulsatile tinnitus arising from aberrant internal carotid artery at nasopharynx.

Pulsatile tinnitus arising from an aberrant internal carotid artery at the level of the nasopharynx is rarely found. We present a case of a 74-year-old woman complaining of pulsatile tinnitus in the right ear. The tinnitus was not audible by auscultation. Endoscopic examination revealed pulsating swelling of the nasopharyngeal wall on the right side. Computed tomography angiography of the head and neck region showed medial and superficial transposition of the right internal carotid artery at the level of the nasopharynx. The artery was located right next to the orifice of the Eustachian tube and coursed along the distal portion of the tube. The patient's perception of tinnitus was improved after explaining the cause of the tinnitus and reassuring her about the-condition.

Is preoperative computed tomographic density measurement of soft tissues helpful in the diagnosis of cholesteatoma?

OBJECTIVES: We undertook to verify the usefulness of computed tomography Hounsfield units (HU) in differentiating cholesteatoma from inflammatory tissue. METHODS: In 91 enrolled cases, the lesions were classified according to the gross pathology, and the specific locations of each lesion were documented by 1 surgeon within 1 day after the operation. The densities in HU of cholesteatoma and non-cholesteatoma lesions were retrospectively measured 3 times by the same examiner, and the difference between the two groups was analyzed. The interobserver reliability among the 3 examiners was assessed to verify the confidence level of the HU measurements in preoperative detection of cholesteatoma. RESULTS: The mean HU values of cholesteatoma were 35.7 to 66.6 HU, and those of non-cholesteatoma lesions were 32.9 to 51.3 HU. A general linear model-repeated-measures analysis of variance did not show any significant difference between the cholesteatoma and non-cholesteatoma lesions (p = 0.305). The general linear model-repeated-measures analysis of variance showed a significant difference of the measured HU levels among the 3 examiners (p = 0.021). CONCLUSIONS: This study showed that the HU values on preoperative computed tomography did not suffice for the detection of cholesteatoma lesions. A clinician's physical examination together with an interpretation of computed tomography is still the "gold standard" method.

T-S model based indirect adaptive fuzzy control using online parameter estimation.

A parameter estimation scheme with an appropriate adaptive law for updating the parameters is designed and analyzed based on the Lyapunov theory for the general MIMO Takagi-Sugeno (T-S) fuzzy models. The parameters of the Takagi-Sugeno fuzzy models can be estimated by observing the behavior of the system and with the online parameter estimator, any type of fuzzy controllers works adaptively to the parameter perturbation. In order to show the applicability of the proposed estimator, an existing fuzzy state feedback controller is adopted and indirect adaptive fuzzy control design with the proposed estimator is shown. From the numerical simulations and experiments, it is shown that the derived adaptive law works for the estimation model to follows the parameterized plant model and the overall control system has robustness to the parameter perturbation.

Algorithm for detecting human faces based on convex-hull.

In this paper, we proposed a new method to detect faces in color based on the convex-hull. We detect two kinds of regions that are skin and hair likeness region. After preprocessing, we apply the convex-hull to their regions and can find a face from their intersection relationship. The proposed algorithm can accomplish face detection in an image involving rotated and turned faces as well as several faces. To validity the effectiveness of the proposed method, we make experiment with various cases.

Preparation and Structures of the 2.2.2-Cryptand(1+) Salts of the [Sb(2)Se(4)](2)(-), [As(2)S(4)](2)(-), [As(10)S(3)](2)(-), and [As(4)Se(6)](2)(-) Anions.

2.2.2-Cryptand(1+) salts of the [Sb(2)Se(4)](2)(-), [As(2)S(4)](2)(-), [As(10)S(3)](2)(-), and [As(4)Se(6)](2)(-) anions have been synthesized from the reduction of binary chalcogenide compounds by K in NH(3)(l) in the presence of the alkali-metal-encapsulating ligand 2.2.2-cryptand (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane), followed by recrystallization from CH(3)CN. The [Sb(2)Se(4)](2)(-) anion, which has crystallographically imposed symmetry 2, consists of two discrete edge-sharing SbSe(3) pyramids with terminal Se atoms cis to each other. The Sb-Se(t) bond distance is 2.443(1) Å, whereas the Sb-Se(b) distance is 2.615(1) Å (t = terminal; b = bridge). The Se(b)-Sb-Se(t) angles range from 104.78(4) to 105.18(5) degrees, whereas the Se(b)-Sb-Se(b) angles are 88.09(4) and 88.99(4) degrees. The (77)Se NMR data for this anion in solution are consistent with its X-ray structure (delta 337 and 124 ppm, 1:1 intensity, -30 degrees C, CH(3)CN/CD(3)CN). Similar to this [Sb(2)Se(4)](2)(-) anion, the [As(2)S(4)](2)(-) anion consists of two discrete edge-sharing AsS(3) pyramidal units. The As-S(t) bond distances are 2.136(7) and 2.120(7) Å, whereas the As-S(b) distances range from 2.306(7) to 2.325(7) Å. The S(b)-As-S(t) angles range from 106.2(3) to 108.2(3) degrees, and the S(b)-As-S(b) angles are 88.3(2) and 88.9(2) degrees. The [As(10)S(3)](2)(-) anion has an 11-atom As(10)S center composed of six five-membered edge-sharing rings. One of the three waist positions is occupied by a S atom, and the other two waist positions feature As atoms with exocyclic S atoms attached, making each As atom in the structure three-coordinate. The As-As bond distances range from 2.388(3) to 2.474(3) Å. The As-S(t) bond distances are 2.181(5) and 2.175(4) Å, and the As-S(b) bond distance is 2.284(6) Å. The [As(4)Se(6)](2)(-) anion features two AsSe(3) units joined by Se-Se bonds with the two exocyclic Se atoms trans to each other. The average As-Se(t) bond distance is 2.273(2) Å, whereas the As-Se(b) bond distances range from 2.357(3) to 2.462(2) Å. The Se(b)-As-Se(t) angles range from 101.52(8) to 105.95(9) degrees, and the Se(b)-As-Se(b) angles range from 91.82(7) to 102.97(9) degrees. The (77)Se NMR data for this anion in solution are consistent with its X-ray structure (delta 564 and 317 ppm, 3:1 intensity, 25 degrees C, DMF/CD(3)CN).