Ultrafast and Highly Specific Detection of One-Base Mutated Cell-Free DNA at a Very Low Abundance.

Liquid biopsy as well as genotyping plays important roles in guiding the use of tumor-targeted drugs and monitoring the generation of drug resistance. However, current methods, such as next-generation sequencing (NGS) and pyrosequencing, require long analysis time and complicated steps. To achieve ultrafast and highly specific detection of cell-free DNA (cfDNA) from blood, we improved our recently developed FEN1-aided RPA (FARPA), which combined flap endonuclease 1 (FEN1)-catalyzed invasive reactions with recombinase polymerase amplification (RPA) by inactivating the RPA enzymes before invasive reactions, designing short RPA primers, and changing invasive reaction conditions. Using the L858R and T790M mutations as examples, FARPA was sensitive to detect 5 copies of targeted mutants, specific to sense the mutants with an abundance as low as 0.01% from blood, and ultrafast to get results within 40 min. The method was readily expended to genotyping, and 15 min was enough to report the allele species directly from oral swab samples by coupling quick DNA extraction reagents. Validation was carried out by detecting clinical samples, including 20 cfDNA from patients with non-small cell lung cancer (NSCLC) for liquid biopsy and 43 human genomic DNA (gDNA) purified from blood (33) or lysed from oral swabs (10) for genotyping, giving 100% agreement with NGS and pyrosequencing, respectively. Furthermore, a portable battery-driven device with dual-channel fluorescence detection was successfully constructed to facilitate point-of-care testing (POCT) of liquid biopsy and genotyping, providing doctors with a potential tool to achieve genotyping- or mutant-guided personalized medicine at emergency or source-limited regions.

Brønsted Acid-Catalyzed Intramolecular Tandem Double Cyclization of γ-Hydroxy Acetylenic Ketones with Alkynes into Naphtho[1,2-b]furan-3-ones.

A metal-free and atom-economic route for the synthesis of naphtho[1,2-b]furan-3-ones has been realized via p-TsOH·H2O-catalyzed intramolecular tandem double cyclization of γ-hydroxy acetylenic ketones with alkynes in formic acid. The benzene-linked furanonyl-ynes are the key intermediates obtained by the scission/recombination of C-O double bonds. Further, the structural modifications of the representative product were implemented by reduction, demethylation, substitution, and [5 + 2]-cycloaddition.

Born approximation of trapping forces by acoustical Bessel and vortex fields.

Acoustic radiation forces have been used to trap various objects for fundamental studies and practical applications. Born approximation method, originally introduced to solve quantum scattering problems, is herein extended to analyze trapping forces exerted by two- and three-dimensional acoustic Bessel and vortex fields on spherical and nonspherical objects of arbitrary size. The results are compared with the conventional models like the partial wave expansion and Gorkov force potential. It is shown that for weakly scattering objects (such as common soft biological particles surrounded by fluids), the Born approximation can make predictions for the trapping forces on objects whose characteristic lengths are even up to multiple wavelengths of the sound beams. With the aid of the approximation, the Gorkov force potential is applied to analyze and gain insights into trapping forces on large objects far beyond the original Rayleigh scattering regime. The effects caused by the beam parameters, object shape, and orientation on the trapping behaviors are revealed. This work is useful for the further study of acoustic radiation forces and will guide the experiment of simplified acoustic tweezers on arbitrary-shaped particles.

Flap Endonuclease-Induced Steric Hindrance Change Enables the Construction of Multiplex and Versatile Lateral Flow Strips for DNA Detection.

A lateral flow strip (LFS) is an ideal tool for point-of-care testing (POCT), but traditional LFSs cannot be used for multiplex detection. Herein, a multiplex and versatile LFS based on flap endonuclease 1 (FEN1)-induced steric hindrance change (FISH-LFS) is proposed. In this method, multiplex PCR coupled with cascade invasive reactions was employed to yield single-stranded flaps, which were target-specific but independent of target sequences. Then, the amplicons were applied for FISH-LFS, and the single-stranded flaps would be efficiently captured by the complementary LFS-probes at different test lines. As flaps were cleaved from the specially designed hairpin probes, competition among flaps and hairpin probes would occur in capturing the probes at test lines. We enabled the hairpin probes to flow through the test lines while the flaps to stay at the test lines by making use of the difference in steric hindrance between hairpin probes and flaps. The assay is able to detect as low as two copies of blood pathogens (HBV, HCV, and HIV), to pick up as low as 0.1% mutants from wild-type gDNA, and to genotype 200 copies of SARS-CoV-2 variants α and ß within 75 min at a conventional PCR engine. As the method is free of dye, a portable PCR engine could be used for a cost-effective multiplex detection on site. Results using an ultrafast mobile PCR system for FISH-LFS showed that as fast as 30 min was achieved for detecting three pathogens (HBV, HCV, and HIV) in blood, very suitable for POCT of pathogen screening. The method is convenient in operation, simple in instrumentation, specific in genotyping, and very easy in setting up multiplex POCT assays.

Multiplexed and Rapid AST for Escherichia coli Infection by Simultaneously Pyrosequencing Multiple Barcodes Each Specific to an Antibiotic Exposed to a Sample.

Antimicrobial susceptibility testing (AST) is an effective way to guide antibiotic selection. However, conventional culture-based phenotypic AST is time-consuming. The key point to shorten the test is to quantify the small change in the bacterial number after the antibiotic exposure. To achieve rapid AST, we proposed a combination of multiplexed PCR with barcoded pyrosequencing to significantly shorten the time for antibiotic exposure. First, bacteria exposed to each antibiotic were labeled with a unique barcode. Then, the pool of the barcoded products was amplified by PCR with a universal primer pair. Finally, barcodes in the amplicons were individually and quantitatively decoded by pyrosequencing. As pyrosequencing is able to discriminate as low as 5% variation in target concentrations, as short as 7.5 min was enough for cultivation to detect the susceptibility of Escherichia coli to an antibiotic. The barcodes enable more than six kinds of drugs or six kinds of concentrations of a drug to be tested at a time. The susceptibility of 6 antibiotics to 43 E. coli-positive samples from 482 clinical urine samples showed a consistency of 99.3% for drug-resistant samples and of 95.7% for drug-sensitive samples in comparison with the conventional method. In addition, the minimum inhibitory concentration (MIC) of 29 E. coli samples was successfully measured. The proposed AST is dye free (pyrosequencing), multiplexed (six antibiotics), fast (a half-working day for reporting the results), and able to detect the MIC, thus having a great potential for clinical use in quick antibiotic selection.

Multiplex detection of blood-borne pathogens on a self-driven microfluidic chip using loop-mediated isothermal amplification.

Detection of blood-borne pathogens such as hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV) is essential to ensure the safety of blood transfusion. However, traditional PCR-based pathogen nucleic acid detection methods require relatively high experimental facilities and are difficult to apply in areas with limited resources. In this study, a self-driven microfluidic chip was designed to carry out multiplex detection of HBV, HCV and HIV by using loop-mediated isothermal amplification (LAMP). Benefitting from the air permeability of the polydimethylsiloxane material, the chip could accomplish sample loading within 12 min driven by the pressure difference between the reaction chambers and vacuum chambers in the chip without using pumps or any injection devices. Multiplex detection is achieved by presetting LAMP primers specific to different targets in different reaction chambers. Calcein was used as an indicator to indicate the positive amplification reaction, and the result can be recorded by a smartphone camera. After 50 min of isothermal amplification at 63 °C, 2 copies/µL of HBV, HCV and HIV target nucleic acids could be detected. The results of HBV detection of 20 clinical plasma samples by using the chip are consistent with that of the qPCR-based kit, indicating that the LAMP-based self-driven chip has the clinical application potential for blood-borne pathogen detection, especially in resource-limited areas.

Enhancing low-frequency water-column acoustic reflections in marine multichannel seismic data for seismic oceanography.

Reflections of low-frequency, broadband airgun signals from ocean water columns have long been collected in modern marine seismic surveys. However, they were barely examined because of their weak amplitude (following from low acoustic impedance contrast in water columns) and the lack of application interests. Nevertheless, in the past decades, a new cross-discipline "seismic oceanography" has developed the interest to use these water-column reflection signals to image the ocean structures. Motivated by seismic oceanography applications, we examined the acoustic multipath structure of marine seismic survey data with a focus on water-column reflections, and developed a two-step matched filtering approach to enhance water-column reflection and suppress the unwanted bubble waves. The approach was applied to process data collected from the Gulf of Mexico and led to an improvement in imaging mesoscale ocean structures when compared with the traditional matched filtering approach. For the specific data we examined, the results reveal a 11.3-dB improvement of signal-to-noise ratio by removing the noise and a 8-dB improvement of signal-to-reverberation ratio by suppressing bubble waves, while not affecting the information of ocean structures embedded in the signals. This study gains insights into features of water-column acoustic reflections and provides better tools for acoustic imaging of mesoscale ocean structures.

Phase shift approach for engineering desired radiation force: Acoustic pulling force example.

Phase shifts from scattering are used to analyze and engineer acoustic radiation forces. With the aid of phase shifts, analytical results for acoustic radiation forces can be simplified into compact and physically meaningful expressions, which can be used to develop a simplified procedure for the engineering of the radiation force. The desired radiation force can be fulfilled by a specific set of phase shifts up to a certain order, and then the required phase shifts can be fulfilled by engineering object and beam parameters. As an example, the phase shift approach is used to engineer the inner-to-outer radius ratio and the outer radius of a spherical shell to show how to use the phase shift method for the design of acoustic radiation forces. The example here is a force that is desired to pull particles against the propagation of a Bessel beam. A small paraxial parameter to pull a spherical shell is satisfied by in-phase scattering of monopole, dipole, quadrupole, octupole, and beyond. The example presented here is relatively simple yet reveals the advantages of the phase shift approach. The phase shift method can provide a simplified route for the design of acoustic tweezers using either traveling beams or standing waves.

Orbital Angular Momentum Reversal and Asymmetry in Acoustic Vortex Beam Reflection.

We present acoustic modeling, measurements, and interpretation of the angular momentum carried in an ultrasonic vortex beam that is obliquely reflected off a flat water-air interface. The experimental measurements observe the theoretically predicted reversals of phase rotation, topological charge, and orbital angular momentum in a reflected vortex beam in direct analogy to optical phenomena. The spatial and temporal evolution of the linear and angular momentum during the reflection are determined by calculating the velocity field from two-dimensional scanned pressure fields. A conversion of the angular momentum indicates a radiation torque along the oblique reflecting surface. We understand this radiation torque originates from the break of rotational symmetry with respect to the incident plane for normal components of the energy flux and linear momentum density at the reflecting surface. Our study provides mechanical evidence on the effect of a flat surface on the reflection of vortex beams and gains insight into the underlying physics, impacting non-contact manipulation of objects and communication.

Carbon tetrachloride (CCl4) accelerated development of non-alcoholic fatty liver disease (NAFLD)/steatohepatitis (NASH) in MS-NASH mice fed western diet supplemented with fructose (WDF).

BACKGROUND: Multiple murine models of nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) have been established by using obesogenic diets and/or chemical induction. MS-NASH mouse (formally FATZO) is a spontaneously developed dysmetabolic strain that can progress from hepatosteatosis to moderate fibrosis when fed a western diet supplemented with 5% fructose (WDF). This study aimed to use carbon tetrachloride (CCl4) to accelerate and aggravate progression of NAFLD/NASH in MS-NASH mouse. METHODS: Male MS-NASH mice at 8 weeks of age were fed WDF for the entire study. Starting at 16 weeks of age, CCl4 was intraperitoneally administered twice weekly at a dose of 0.2 mL/kg for 3 weeks or 0.08 mL/kg for 8 weeks. Obeticholic acid (OCA, 30 mg/kg, QD) was administered in both MS-NASH and C57Bl/6 mice fed WDF and treated with CCl4 (0.08 mL/kg). RESULTS: WDF enhanced obesity and hepatosteatosis, as well as induced moderate fibrosis in MS-NASH mice similar to previous reports. Administration of CCl4 accelerated liver fibrosis with increased bridging and liver hydroxyproline contents, but had no significant impact on liver steatosis and lipid contents. High dose CCl4 caused high mortality and dramatic elevation of ALT and ASL, while low dose CCl4 resulted in a moderate elevation of ALT and AST with low mortality. Compared to C57BI/6 mice with WDF and CCl4 (0.08 mL/kg), MS-NASH mice had more prominent hepatosteatosis and fibrosis. OCA treatment significantly lowered liver triglycerides, steatosis and fibrosis in both MS-NASH and C57Bl/6 mice fed WDF with CCl4 treatment. CONCLUSIONS: CCl4 reduced induction time and exacerbated liver fibrosis in MS-NASH mice on WDF, proving a superior NASH model with more prominent liver pathology, which has been used favorably in pharmaceutical industry for testing novel NASH therapeutics.

The FATZO mouse, a next generation model of type 2 diabetes, develops NAFLD and NASH when fed a Western diet supplemented with fructose.

BACKGROUND: Metabolic disorders such as insulin resistance, obesity, and hyperglycemia are prominent risk factors for the development of non-alcoholic fatty liver disease (NAFLD)/steatohepatitis (NASH). Dietary rodent models employ high fat, high cholesterol, high fructose, methionine/choline deficient diets or combinations of these to induce NAFLD/NASH. The FATZO mice spontaneously develop the above metabolic disorders and type 2 diabetes (T2D) when fed with a normal chow diet. The aim of the present study was to determine if FATZO mice fed a high fat and fructose diet would exacerbate the progression of NAFLD/NASH. METHODS: Male FATZO mice at the age of 8 weeks were fed with high fat Western diet (D12079B) supplemented with 5% fructose in the drinking water (WDF) for the duration of 20 weeks. The body weight, whole body fat content, serum lipid profiles and liver function markers were examined monthly along with the assessment of liver histology for the development of NASH. In addition, the effects of obeticholic acid (OCA, 30 mg/kg, QD) on improvement of NASH progression in the model were evaluated. RESULTS: Compared to normal control diet (CD), FATZO mice fed with WDF were heavier with higher body fat measured by qNMR, hypercholesterolemia and had progressive elevations in AST (~ 6 fold), ALT (~ 6 fold), liver over body weight (~ 2 fold) and liver triglyceride (TG) content (1.4-2.9 fold). Histological examination displayed evidence of NAFLD/NASH, including hepatic steatosis, lobular inflammation, ballooning and fibrosis in FATZO mice fed WDF. Treatment with OCA for 15 weeks in FATZO mice on WDF significantly alleviated hypercholesterolemia and elevation of AST/ALT, reduced liver weight and liver TG contents, attenuated hepatic ballooning, but did not affect body weight and blood TG levels. CONCLUSION: WDF fed FATZO mice represent a new model for the study of progressive NAFLD/NASH with concurrent metabolic dysregulation.

Effect of addition of melatonin on liquid storage of ram semen at 4°C.

Adding a certain amount of antioxidants to semen extender has been shown to improve semen quality. The aim of present study was to elucidate whether the supplementation of melatonin to the Tris-based extender (CTR) could enhance the quality of ram spermatozoa during storage at 4°C. Ram semen samples were collected and diluted with CTR extender containing different concentrations (0, 0.05 (M 0.05), 0.1 (M 0.1), 0.2 (M 0.2) or 0.4 (M 0.4) mM) of melatonin. Sperm routine indicators, mitochondrial activity, total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content were analysed in control and melatonin treatment groups. The higher per cent of motility, plasma membrane integrity, mitochondrial activity and T-AOC activity was observed in M 0.05, M 0.1 and M 0.2 groups compared to control group at 5 days of storage (p < 0.05), while lower percentage of MDA content was observed among these groups (p < 0.05). In addition, there were no significant differences in acrosome integrity among the control and M 0.05, M 0.1 and M 0.2 groups during the experiment. The above results show that the addition of 0.05, 0.1, 0.2 mM of melatonin is beneficial to the preservation of ram semen during liquid storage at 4°C mainly through antioxidative stress.

Trehalose protects testicular tissue of dairy goat upon cryopreservation.

The aim of this study was to investigate whether the addition of trehalose to cryomedia reduces cellular damage and improves gene expression in cryopreserved dairy goat testicular tissues. Testicular tissues were cryopreserved in cryomedia without or with trehalose at a concentration of 5%, 10%, 15%, 20% or 25%. Cryopreserved testicular tissues were analysed for TUNEL-positive cell number, expression of BAX, BCL-2, CREM, BOULE and HSP70-2. Isolated Leydig cells from cryopreserved tissue were cultured, and spent medium was evaluated for testosterone level. The results showed that though the TUNEL-positive cell number increased in cryopreserved testicular tissues, the presence of trehalose reduced apoptotic cell number significantly. Quantitative real-time polymerase chain reaction results showed that although the expression of BAX was upregulated following cryopreservation, the presence of trehalose downregulates it in cryopreserved testicular tissues. Expression of BCL-2, CREM, BOULE and HSP70-2 was downregulated following cryopreservation but the presence of trehalose significantly upregulated their expression in cryopreserved testicular tissues. Leydig cells isolated from testicular tissues cryopreserved with trehalose produced higher testosterone than the one without it (control). These results suggest that trehalose has a protective role in cryopreservation of dairy goat testicular tissue, and the most suitable trehalose concentration for cryopreservation is 15%.

Generalized optical theorem for an arbitrary incident field.

A formalism of optical theorem extended for an arbitrarily shaped wave field is presented. The formalism concerns only time-harmonic scattering in free space. The theorem relates the extinction cross section to the imaginary part of the total scattering amplitude at the forward direction of the individual plane wave components multiplied by the corresponding plane wave amplitude in the angular spectrum of the incident wave. A differential extinction cross section is defined from the theorem. An alternative formalism relating the total cross section to plane wave scattering is also presented. Physical interpretation is provided and applications are discussed.

Acoustic Purcell Effect for Enhanced Emission.

We observe that our experimentally measured emission power enhancement of a speaker inside a previously proposed metacavity agrees with our numerically calculated enhancement of the density of states (DOS) of the source-cavity system. We interpret the agreement by formulating a relation between the emitted sound power and the acoustic DOS. The formulation is an analog to Fermi's golden rule in quantum emission. The formulation complements the radiation impedance theory in traditional acoustics for describing sound emission. Our study bridges the gap between acoustic DOS and the acoustic Purcell effect for sound emission enhancement.

Low-density point eating algorithm for surface reconstruction from dense scans.

We present a low-density point eating algorithm for surface reconstruction from dense scans. First, the density map for each scan is estimated and the boundary densities are down-weighted. Subsequently, the poorly scanned low-density overlapping points are eaten up based on a user-specified threshold. Finally, the overlapping areas are thinned by using the moving least-squares operator and the homogeneous points are weighted averaged. The new algorithm can extract smooth but detailed point set surfaces that are as close as possible to the ground truth. The good performance of the new algorithm is demonstrated by comparison with several advanced surface reconstruction algorithms.

From acoustic radiation pressure to three-dimensional acoustic radiation forces.

This paper presents three-dimensional acoustic radiation forces exerted by an arbitrary field on an obstacle as a sum of two parts due to momentum transfers by ingoing and outgoing spherical waves, respectively. This view of momentum conservation establishes a connection of the force with asymmetry of far fields and acoustic radiation pressure exerted by a plane wave on a planar reflector. These geometrical interpretations and physical aspects are useful for finding and interpreting special force conditions by examining far-field patterns and scattering functions of sound fields. Multipole expansion of the force as a function of beam shape coefficients and scattering functions is simplified for connection with complex phase shifts of scattering functions. A relation of the scattering function dependence in the small-particle limit is identified.

A general theory of arbitrary Bessel beam scattering and interactions with a sphere.

This paper presents an analytical theory that formulates interactions of an arbitrary-order Bessel beam with an arbitrarily located sphere as a superposition of interactions with a series of Bessel beams of different orders whose axis is through the object's center. The analysis is via a parallel-axis relation that is derived to represent the incident Bessel beam as a superposition of a series of Bessel beams of different orders along a parallel, shifted axis. By the superposition, summing on-axial formulas gives off-axial formulas, including multipole expansion of the incident beam, scattered fields, powers of scattering and absorption, and axial radiation forces. Features of the off-axial scattering and interactions are hence accessed from prior studies on the axially centered illuminations. Dependence on the object location is contained in a weighting function in the superposition that also depends on the beam orders. Transverse and azimuthal forces are interpreted as a result of interactions between the beams of different orders in the superposition.

Exposure-fusion-based dot-grid image acquisition and recognition for sheet metal strain analysis.

Dot-grid images are usually captured for grid strain analysis during sheet metal forming. Due to the strong reflective characteristic of the metallic surfaces, the recorded dot-grid images often have poor quality, low positioning accuracy, and low recognition rate. Therefore, an exposure-fusion-based dot-grid image acquisition and recognition approach is proposed. First, multiple dot-grid images are captured at different exposure levels. Subsequently, the recorded multi-exposure dot-grid images are fused into a new high-quality dot-grid image based on exposure fusion technology. Finally, a dot-grid image recognition procedure is developed to detect the dot-grids in the new dot-grid image. Both synthetic and real dot-grid images were tested to verify the performance of the novel approach. When synthetic dot-grid images were tested, the maximum positioning error was up to 6.044 pixels if they were recognized in the traditional way, whereas the maximum positioning error was reduced to 0.132 pixels if the novel approach was adopted. When real dot-grid images were tested, the lowest recognition rate is only 50.52% if they were recognized in the traditional way. Nevertheless, the recognition rate can reach about 91% if the novel approach was employed. These experimental results show the superiorities of the novel approach.

Enhancing monochromatic multipole emission by a subwavelength enclosure of degenerate Mie resonances.

Sound emission is inefficient at low frequencies as limited by source size. This letter presents enhancing emission of monochromatic monopole and multipole sources by enclosing the source with a subwavelength circular enclosure filled of an anisotropic material of a low radial sound speed. The anisotropy is associated with an infinite tangential density along the azimuth. Numerical simulations show that emission gain is produced at frequencies surrounding degenerate Mie resonant frequencies of the enclosure, and meanwhile the radiation directivity pattern is well preserved. The degeneracy is theoretically analyzed. A realization of the material is suggested by using a space-coiling structure.