Development of RPA-Cas12a assay for rapid and sensitive detection of Pneumocystis jirovecii.

Pneumocystis jirovecii is a prevalent opportunistic fungal pathogen that can lead to life-threatening Pneumocystis pneumonia in immunocompromised individuals. Given that timely and accurate diagnosis is essential for initiating prompt treatment and enhancing patient outcomes, it is vital to develop a rapid, simple, and sensitive method for P. jirovecii detection. Herein, we exploited a novel detection method for P. jirovecii by combining recombinase polymerase amplification (RPA) of nucleic acids isothermal amplification and the trans cleavage activity of Cas12a. The factors influencing the efficiency of RPA and Cas12a-mediated trans cleavage reaction, such as RPA primer, crRNA, the ratio of crRNA to Cas12a and ssDNA reporter concentration, were optimized. Our RPA-Cas12a-based fluorescent assay can be completed within  30-40 min, comprising a 25-30 min RPA reaction and a 5-10 min trans cleavage reaction. It can achieve a lower detection threshold of 0.5 copies/µL of target DNA with high specificity. Moreover, our RPA-Cas12a-based fluorescent method was examined using 30 artificial samples and demonstrated high accuracy with a diagnostic accuracy of 93.33%. In conclusion, a novel, rapid, sensitive, and cost-effective RPA-Cas12a-based detection method was developed and demonstrates significant potential for on-site detection of P. jirovecii in resource-limited settings.

P T-symmetric photonic lattices with type-II Dirac cones.

The type-II Dirac cone is a special feature of the band structure, whose Fermi level is represented by a pair of crossing lines. It has been demonstrated that such a structure is useful for investigating topological edge solitons and, more specifically, for mimicking the Klein tunneling. However, it is still not clear what the interplay between type-II Dirac cones and the non-Hermiticity mechanism will result in. Here, this question is addressed; in particular, we report the P T-symmetric photonic lattices with type-II Dirac cones for the first time to our knowledge. We identify a slope-exceptional ring and name it the type-II exceptional ring. We display the restoration of the P T symmetry of the lattice by reducing the separation between the sites in the unit cell. Curiously, the amplitude of the beam during propagation in the non-Hermitian lattice with P T symmetry only decays because of diffraction, whereas in the P T symmetry-broken lattice it will be amplified, even though the beam still diffracts. This work establishes the link between the non-Hermiticity mechanism and the violation of Lorentz invariance in these physical systems.

Deciphering antifungal and antibiofilm mechanisms of isobavachalcone against Cryptococcus neoformans through RNA-seq and functional analyses.

Cryptococcus neoformans has been designated as critical fungal pathogens by the World Health Organization, mainly due to limited treatment options and the prevalence of antifungal resistance. Consequently, the utilization of novel antifungal agents is crucial for the effective treatment of C. neoformans infections. This study exposed that the minimum inhibitory concentration (MIC) of isobavachalcone (IBC) against C. neoformans H99 was 8 µg/mL, and IBC dispersed 48-h mature biofilms by affecting cell viability at 16 µg/mL. The antifungal efficacy of IBC was further validated through microscopic observations using specific dyes and in vitro assays, which confirmed the disruption of cell wall/membrane integrity. RNA-Seq analysis was employed to decipher the effect of IBC on the C. neoformans H99 transcriptomic profiles. Real-time quantitative reverse transcription PCR (RT-qPCR) analysis was performed to validate the transcriptomic data and identify the differentially expressed genes. The results showed that IBC exhibited various mechanisms to impede the growth, biofilm formation, and virulence of C. neoformans H99 by modulating multiple dysregulated pathways related to cell wall/membrane, drug resistance, apoptosis, and mitochondrial homeostasis. The transcriptomic findings were corroborated by the antioxidant analyses, antifungal drug sensitivity, molecular docking, capsule, and melanin assays. In vivo antifungal activity analysis demonstrated that IBC extended the lifespan of C. neoformans-infected Caenorhabditis elegans. Overall, the current study unveiled that IBC targeted multiple pathways simultaneously to inhibit growth significantly, biofilm formation, and virulence, as well as to disperse mature biofilms of C. neoformans H99 and induce cell death.

Establishment of RT-RPA-Cas12a assay for rapid and sensitive detection of human rhinovirus B.

Human rhinovirus B (HRV-B) is a major human viral pathogen that can be responsible for various kinds of infections. Due to the health risks associated with HRV-B, it is therefore crucial to explore a rapid, specific, and sensitive method for surveillance. Herein, we exploited a novel detection method for HRV-B by combining reverse-transcription recombinase polymerase amplification (RT-RPA) of nucleic acids isothermal amplification and the trans-cleavage activity of Cas12a. Our RT-RPA-Cas12a-based fluorescent assay can be completed within 35-45 min and obtain a lower detection threshold to 0.5 copies/µL of target RNA. Meanwhile, crRNA sequences without a specific protospacer adjacent motif can effectively activate the trans-cleavage activity of Cas12a. Moreover, our RT-RPA-Cas12a-based fluorescent method was examined using 30 clinical samples, and exhibited high accuracy with positive and negative predictive agreement of 90% and 100%, respectively. Taken together, a novel promising, rapid and effective RT-RPA-Cas12a-based detection method was explored and shows promising potential for on-site HRV-B infection in resource-limited settings.

Vector valley Hall edge solitons in distorted type-II Dirac photonic lattices.

Topological edge states have recently garnered a lot of attention across various fields of physics. The topological edge soliton is a hybrid edge state that is both topologically protected and immune to defects or disorders, and a localized bound state that is diffraction-free, owing to the self-balance of diffraction by nonlinearity. Topological edge solitons hold great potential for on-chip optical functional device fabrication. In this report, we present the discovery of vector valley Hall edge (VHE) solitons in type-II Dirac photonic lattices, formed by breaking lattice inversion symmetry with distortion operations. The distorted lattice features a two-layer domain wall that supports both in-phase and out-of-phase VHE states, appearing in two different band gaps. Superposing soliton envelopes onto VHE states generates bright-bright and bright-dipole vector VHE solitons. The propagation dynamics of such vector solitons reveal a periodic change in their profiles, accompanied by the energy periodically transferring between the layers of the domain wall. The reported vector VHE solitons are found to be metastable.

Association of the L3MBTL3 rs1125970 and rs4897367 Gene Polymorphisms With Coronary Heart Disease Susceptibility in the Chinese Population: A Case-Control Study.

ABSTRACT: Coronary heart disease (CHD) is a prevalent heart disease with high incidence and mortality rates worldwide, and its pathogenesis is related to genetic factors. L3MBTL3 has been reported to be potentially linked to CHD susceptibility. This study aims to explore the correlation between L3MBTL3 single nucleotide polymorphisms (SNPs) and CHD risk in the Chinese population. Three SNPs (rs1125970 A/T, rs4897367 T/C, and rs2068957 A/G) in L3MBTL3 from 649 patients with CHD and 649 healthy controls were genotyped using the Agena MassARRAY platform. The relationship between SNPs and CHD risk was evaluated by logistic regression analysis. Our study indicated that rs1125970 (TT: odds ratio [OR] = 0.76, P = 0.014) and rs4897367 (TT: OR = 0.74, P = 0.021) were related to a decreased susceptibility to CHD. Stratified analyses showed that rs1125970 could reduce the risk of CHD in males, subjects aged <60 years, with a body mass index <24 kg/m 2 , and nonhypertensive patients. rs4897367 exerted a risk-decreasing influence on CHD in nondiabetic patients. In the haplotype analysis, individuals with the T rs4897367 A rs2068957 haplotype were less likely to develop CHD (OR = 0.74, P = 0.024). In summary, L3MBTL3 rs1125970 and rs4897367 were significantly correlated with a decreased susceptibility to CHD in the Chinese population.

Permeability enhancement of Kv1.2 potassium channel by a terahertz electromagnetic field.

As biomolecules vibrate and rotate in the terahertz band, the biological effects of terahertz electromagnetic fields have drawn considerable attention from the physiological and medical communities. Ion channels are the basis of biological electrical signals, so studying the effect of terahertz electromagnetic fields on ion channels is significant. In this paper, the effect of a terahertz electromagnetic field with three different frequencies, 6, 15, and 25 THz, on the Kv1.2 potassium ion channel was investigated by molecular dynamics simulations. The results show that an electromagnetic field with a 15 THz frequency can significantly enhance the permeability of the Kv1.2 potassium ion channel, which is 1.7 times higher than without an applied electric field. By analyzing the behavior of water molecules, it is found that the electromagnetic field with the 15 THz frequency shortens the duration of frozen and relaxation processes when potassium ions pass through the channel, increases the proportion of the direct knock-on mode, and, thus, enhances the permeability of the Kv1.2 potassium ion channel.

Effect of THz Waves of Different Orientations on K+ Permeation Efficiency in the KcsA Channel.

Potassium (K) channels show the highest variability and most frequent alterations in expression in many tumor types, and modulation of K+ channels may represent a new window for cancer therapy. In previous work, we found that a terahertz (THz) field incident along the z-axis with a frequency of 51.87 THz increased the ion flux through K+ channels. In practice, it is difficult to ensure that the incident electromagnetic (EM) wave is strictly parallel to the direction of channel ion flow. In this paper, we found by changing the direction of the applied electric field that the EM wave of a specific frequency has the largest ion flux when the incident direction is along the ion flow, and the smallest ion flux when the incident direction is perpendicular to the ion flow, and that overall the EM wave of this frequency enhances the ion flow of the K+ channel. Changes in the direction of the applied field at a specific frequency affect the stability of the φ dihedral angle of the GLY77 residue and alter the ion permeation mechanism in the selectivity filter (SF) region, thus affecting the ion flux. Therefore, this frequency can be used to modulate K+ fluxes by THz waves to cause rapid apoptosis in potassium-overloaded tumor cells. This approach consequently represents an important tool for the treatment of cancer and is expected to be applied in practical therapy.

Effect of Terahertz Electromagnetic Field on the Permeability of Potassium Channel Kv1.2.

In this paper, the influence of external terahertz electromagnetic fields with different frequencies of 4 THz, 10 THz, 15 THz, and 20 THz on the permeability of the Kv1.2 voltage-gated potassium ion channel on the nerve cell membrane was studied using the combined model of the "Constant Electric Field-Ion Imbalance" method by molecular dynamics. We found that although the applied terahertz electric field does not produce strong resonance with the -C=O groups of the conservative sequence T-V-G-Y-G amino acid residue of the selective filter (SF) of the channel, it would affect the stability of the electrostatic bond between potassium ions and the carbonyl group of T-V-G-Y-G of SF, and it would affect the stability of the hydrogen bond between water molecules and oxygen atoms of the hydroxyl group of the 374THR side chain at the SF entrance, changing the potential and occupied states of ions in the SF and the occurrence probability of the permeation mode of ions and resulting in the change in the permeability of the channel. Compared with no external electric field, when the external electric field with 15 THz frequency is applied, the lifetime of the hydrogen bond is reduced by 29%, the probability of the "soft knock on" mode is decreased by 46.9%, and the ion flux of the channel is activated by 67.7%. Our research results support the view that compared to "direct knock-on", "soft knock-on" is a slower permeation mode.

Ultrasensitive and visual detection of human norovirus genotype GII.4 or GII.17 using CRISPR-Cas12a assay.

BACKGROUND: Integrating CRISPR-Cas12a sensors with isothermal signal amplification can be exploited to develop low-cost, disposable, and ultrasensitive assays for the diagnostics of human pathogens. METHODS: RT-RAA-Cas12a-mediated real-time or end-point fluorescent and lateral flow strip (LFS) assays for direct detection of norovirus (NOV) genotype GII.4 or GII.17 were explored. RESULTS: The results showed that our RT-RAA-Cas12a-mediated fluorescent and LFS assay could detect NOV GII.4 or GII.17 by targeting the viral protein 1 gene. Our RT-RAA-Cas12a-mediated fluorescent and LFS assay can specifically detect NOV GII.4 or GII.17 with no cross-reactivity for other related viruses. The low limit of detection could reach 0.1 copies/µL within approximately 30-40 min, and the results were visualized using an ultraviolet light illuminator or on a LFS without complex equipment. In addition, our RT-RAA-Cas12a-mediated fluorescent and LFS assay provided a visual and faster alternative to real-time RT-PCR assay, with 95.7% and 94.3% positive predictive agreement and 100% negative predictive agreement. CONCLUSIONS: Together, our RT-RAA-Cas12a-mediated approach would have a great potential for point-of-care diagnostics of NOV GII.4 and/or GII.17 in resource-limited settings.

Regulation of Ion Permeation of the KcsA Channel by Applied Midinfrared Field.

Ion transport molecules are involved in many physiological and pathological processes and are considered potential targets for cancer treatment. In the large family of ion transport molecules, potassium (K) ion channels, as surface-expressed proteins, show the highest variability and most frequent expression changes in many tumor types. The key to exploring the permeation of K+ through potassium channels lies in the conserved sequence TVGYG, which is common in the selectivity filter (SF) region of all potassium channels. We found that the K+ flux significantly increased with the help of a specific frequency terahertz electromagnetic wave (51.87 THz) in the KcsA channel using a molecular dynamics combined model through the combined simulation of the constant electric field method and ion imbalance method. This frequency has the strongest absorption peak in the infrared spectrum of -C=O groups in the SF region. With the applied electric field of 51.87 THz, the Y78 residue at the S1 site of the SF has a smaller vibration amplitude and a more stable structure, which enables the K+ to bind closely with the carbonyl oxygen atoms in the SF and realize ion conduction in a more efficient direct Coulomb knock-on.

Valley Hall edge solitons in a photonic graphene.

We predict the existence and study properties of the valley Hall edge solitons in a composite photonic graphene with a domain wall between two honeycomb lattices with broken inversion symmetry. Inversion symmetry in our system is broken due to detuning introduced into constituent sublattices of the honeycomb structure. We show that nonlinear valley Hall edge states with sufficiently high amplitude bifurcating from the linear valley Hall edge state supported by the domain wall, can split into sets of bright spots due to development of the modulational instability, and that such an instability is a precursor for the formation of topological bright valley Hall edge solitons localized due to nonlinear self-action and travelling along the domain wall over large distances. Topological protection of the valley Hall edge solitons is demonstrated by modeling their passage through sharp corners of the Ω-shaped domain wall.

Genotyping and zoonotic potential of Cryptosporidium and Enterocytozoon bieneusi in pigs transported across regions in China.

Cryptosporidium spp. and Enterocytozoon bieneusi are common and important enteric parasites that can infect humans and animals, causing diarrhoea and systemic diseases. The objectives of the present study were to examine the prevalence and genetic variations of Cryptosporidium and E. bieneusi in pigs transferred from northeastern China to Ningbo city in Zhejiang Province. Cryptosporidium spp. was detected in 0.9% (2/216) of these samples and belonged to the zoonotic species Cryptosporidium parvum. A high E. bieneusi infection rate (25.0%, 54/216) was observed in this study, with 7 possible novel ITS genotypes (JLNB-1 to JLNB-7) and 10 known genotypes (EbpA, CM11, H, CM6, pigEBITS1, EbpC, CS-4, pigEBITS5, CHS5, and Henan-Ⅳ) identified, and zoonotic EbpA was the dominant genotype. Genotypes H and pigEBITS1 were reported for the first time in pigs in China. Phylogenetic analysis indicated that all the genotypes found in these samples belonged to zoonotic group 1. These findings indicated the potential threat of Cryptosporidium and E. bieneusi to humans or the environment during cross-regional transportation. An effective management control system should be built to avoid parasitic transmission as well as other animal diseases while travelling across different regions. In further studies, attention should be given to the transmission routes and the role of pigs as a potential source of human Cryptosporidium and E. bieneusi infections in China.

A SYBR Green I-based real-time polymerase chain reaction assay for detection and quantification of canine bufavirus.

Canine bufavirus (CBuV) was first discovered in puppies in Italy in 2016, and subsequent studies have reported its possible relationship with acute enteritis. Currently, there is no specific and quantitative detection method for CBuV. This study examined the conserved NS1 gene and used a pair of specific primers to establish a direct SYBR Green I-based real-time quantitative polymerase chain reaction (qPCR) method for the detection and quantification of CBuV. In the sensitivity experiment, the detection limit of SYBR Green I-based real-time qPCR was 4.676 × 101 copies/µL and that of conventional PCR (cPCR) was 4.676 × 103 copies/µL. Furthermore, the qPCR method did not detect other viruses in dogs, indicating good specificity. The intra-assay coefficient of variation was 0.07-0.55% and the inter-assay coefficient of variation was 0.03-0.11%, indicating good repeatability. In clinical sample testing, the detection rate of qPCR was 5.0% (6/120), higher than that of cPCR (2.5%, 3/120). In addition, the samples that tested CBuV-positive in this experiment were all from dogs with acute enteritis. In summary, the SYBR Green I-based qPCR method established in this study has good sensitivity, specificity, and reproducibility for clinical sample detection and can also assist in future research on CBuV.

Genetic characterization and phylogenetic analysis of duck-derived waterfowl parvovirus in Anhui province, eastern China.

Recently, a novel duck-origin goose parvovirus (N-GPV) was reported to cause short beak and dwarfism syndrome in ducks. In this study, we performed complete genome sequencing and analyzed three different duck-derived parvoviruses that infected different breeds of ducks. Phylogenetic trees based on gene sequences indicated that they were classical goose parvovirus (C-GPV), Muscovy duck parvovirus (MDPV), and N-GPV. Furthermore, potential recombination events were found. These results improve our understanding of the diversity of duck-derived parvoviruses in Anhui province, eastern China, and provide a reference for the prevention of associated diseases.

Antimicrobial activity of eugenol against carbapenem-resistant Klebsiella pneumoniae and its effect on biofilms.

A preliminary study found that eugenol expressed an antibacterial activity against Klebsiella pneumoniae. However, the mechanism of action of eugenol against K. pneumoniae still remains unexplored. The aim of this study was to gain further insight into the antibacterial effect of eugenol against carbapenem-resistant Klebsiella pneumoniae (CRKP) and possible mode of action. Here, minimum inhibitory concentration (MIC) of eugenol against CRKP strains was determined using the agar dilution method. Moreover, variations in intracellular ATP concentration, intracellular pH (pHin), membrane potential and membrane integrity were measured to evaluate the effect of eugenol on cell membrane. Besides, changes in cell structure and biofilm formation of CRKP as well as biofilm-associated cell damage were determined using field emission scanning electron microscope (FESEM), transmission electron microscope (TEM) and confocal laser scanning microscopy (CLSM). Finally, gene expression of biofilm-related biosynthesis was investigated. The results showed that MICs of eugenol against four tested CRKP were 0.2 mg/mL. Eugenol damaged the cell membrane of CRKP, as evidenced by decreased intracellular ATP concentration, reduced pHin and cell membrane hyperpolarization, coupled with enhanced membrane permeability. Furthermore, eugenol compromised cell structure and induced loss of intracellular components of CRKP. Additionally, eugenol inhibited biofilm formation and inactivated biofilm CRKP cells. Finally, eugenol presented strong inhibitory effects on biofilm formation and biofilm-associated gene expression, and inactivated CRKP cells growing in biofilms. These findings suggest that eugenol exhibits antimicrobial effect against CRKP strains and could be potentially used to control CRKP-related infections.

Antimicrobial mechanism of luteolin against Staphylococcus aureus and Listeria monocytogenes and its antibiofilm properties.

Luteolin (LUT) is a naturally occurring compound found in a various of plants. Few recent studies have reported LUT antimicrobial activities against bacterial pathogens, however, the fundamental LUT mediated antimicrobial mechanism has never been elucidated. This study aimed to investigate the antimicrobial activities of LUT and its mode of action against Staphylococcus aureus and Listeria monocytogenes, either as planktonic cells or as biofilms. Here, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of LUT against S. aureus and L. monocytogenes were determined using the broth microdilution method, and the antimicrobial mode of LUT was elucidated by evaluating the variations in both cell membrane integrity and cell morphology. Moreover, the biofilm inhibition was measured by crystal violet staining assay, while its qualitative imaging was achieved by confocal laser scanning microscope and field emission scanning electron microscope. MIC and MBC values of LUT against S. aureus were 16-32 and 32-64 µg/mL, and 32-64 and 64-128 µg/mL for L. monocytogenes. LUT destroyed the cell membrane integrity, as evidenced by a significant increase in the number of non-viable cells, and well-defined variations in cell morphology. Moreover, LUT presented robust inhibitory effects on the biofilm formation, enhanced antibiotics diffusion within biofilms and killed efficiently mono- and dual-species biofilm cells. Overall, LUT demonstrates potent antimicrobial properties on planktonic and biofilm cells, and the biofilm formation, and thus has the potential use as a natural food preservative in foods.

Nonlinear higher-order polariton topological insulator.

We address the resonant response and bistability of the exciton-polariton corner states in a higher-order nonlinear topological insulator realized with a kagome arrangement of microcavity pillars. Such states are resonantly excited and exist due to the balance between pump and losses, on one hand, and between nonlinearity and dispersion in inhomogeneous potential landscape, on the other hand, for pump energy around eigen-energies of corresponding linear localized modes. Localization of the nonlinear corner states in a higher-order topological insulator can be efficiently controlled by tuning pump energy. We link the mechanism of corner state formation with symmetry of the truncated kagome array. Corner states coexist with densely packed edge states but are well isolated from them in energy. Nonlinear corner states persist even in the presence of perturbations in a corner microcavity pillar.

Simultaneous detection of duck circovirus and novel goose parvovirus via SYBR green I-based duplex real-time polymerase chain reaction analysis.

Beak atrophy and dwarfism syndrome (BADS) is commonly caused by co-infection with duck circovirus (DuCV) and novel goose parvovirus (NGPV). Therefore, concurrent detection of both viruses is important for monitoring and limiting BADS, although such a diagnostic test has not been reported. In this study, we developed a duplex, SYBR Green I-based real-time polymerase chain reaction (PCR) assay to enable the simultaneous detection of DuCV and NGPV. The assay readily distinguished between the two viruses, based on their different melting temperatures (Tm), where the Tm for DuCV was 80 °C and that for NGPV was 84.5 °C. Other non-target duck viruses that were tested did not show melting peaks. The detection limit of the duplex assay was 101 copies/µL for both viruses. This method exhibited high repeatability and reproducibility, and both the inter-assay and intra-assay variation coefficients were <1.6%. Thirty-one fecal samples were collected for clinical testing using real-time PCR analysis, and the results were confirmed using sequencing. The rate of co-infection was 6.5%, which was consistent with the sequencing results. This duplex real-time PCR assay offers advantages over other tests, such as rapid, sensitive, specific, and reliable detection of both viruses in a single sample, which enables the quantitative detection of DuCV and NGPV in clinical samples. Using this test may be instrumental in reducing the incidence of BADS and the associated economic losses in the duck and goose industries.

Establishment of a duplex SYBR green I-based real-time polymerase chain reaction assay for the rapid detection of canine circovirus and canine astrovirus.

The similar clinical characteristics of canine circovirus (CaCV) and canine astrovirus (CaAstV) infections and high frequency of co-infection make diagnosis difficult. In this study, a duplex SYBR Green I-based real-time polymerase chain reaction (PCR) assay was established for the rapid, simultaneous detection of CaCV and CaAstV. Two pairs of specific primers were designed based on the Rep gene of CaCV and the Cap gene of CaAstV. By using the real-time PCR assay method, the two viruses can be distinguished by the difference in melting temperatures, 79 °C and 86 °C for CaCV and CaAstV, respectively. This assay had high specificity, showing no cross-reaction with other common canine viruses, as well as high sensitivity, with minimum detection limits of 9.25 × 101 copies/µL and 6.15 × 101 copies/µL for CaCV and CaAstV, respectively. Based on the mean coefficient of variation, the method had good reproducibility and reliability. In a clinical test of 57 fecal samples, the rates of positive detection by real-time PCR were 14.04% (8/57) and 12.28% (7/57) for CaCV and CaAstV, respectively, and the rate of co-infection was 8.77% (5/57). In conclusion, the newly established duplex SYBR Green I-based real-time PCR assay is sensitive, specific, reliable, and rapid and is an effective tool for the detection of co-infections with CaCV and CaAstV.