Integrating recombinase polymerase amplification with CRISPR/Cas9-initiated nicking-rolling circle amplification in Staphylococcus aureus assay.

We integrate recombinase polymerase amplification (RPA) with CRISPR/Cas9-initiated nicking rolling circle amplification (CRISPR/Cas9-nRCA) for detecting Staphylococcus aureus. This approach utilizes a unique dimeric G-triplex structure, demonstrating firstly enhanced ThT fluorescence for target detection. The proof-of-concept study introduces a new avenue for integrating isothermal amplifications with CRISPR/Cas9 in the fields of pathogen detection and disease diagnosis.

An allosteric palindromic hairpin probe based dual-mode interactive strand displacement amplification enables robust miRNA biosensing.

This study introduces an allosteric palindromic hairpin probe (APHP)-based dual-mode interactive strand displacement amplification (DMI-SDA) system for ultrasensitive detection of microRNA-155. The system achieves exceptional signal amplification and improved signal preservation using dimeric G-triplexes as signal reporters, enabling robust detection of miRNA-155, representing a promising avenue in molecular diagnosis.

Harnessing intermolecular G-quadruplex-based spatial confinement effect for accelerated activation of CRISPR/Cas12a empowers ultra-sensitive detection of PML/RARA fusion genes.

Accurate detection and classification of the three isoforms of PML/RARA genomic fragments are crucial for predicting disease progression, stratifying risk, and administering precise drug therapies in acute promyelocytic leukemia (APL). In this study, we have developed a highly specific nucleic acid detection platform capable of quantifying the long isoform of the three main PML-RARA isoforms at a constant temperature. This platform integrates the strengths of the CRISPR/Cas12a nuclease-based method and the rolling circle amplification (RCA) technique. Notably, the RCA-assisted CRISPR/Cas12a trans-cleavage system incorporates a spatial confinement effect by utilizing intermolecular G-quadruplex structures. This innovative design effectively enhances the local concentration of CRISPR/Cas12a, thereby accelerating its cleaving efficiency towards reporter nucleic acids and enabling the detection of PML/RARA fusion gene expression through spectroscopy. The robust detection of PML/RARA fusion gene from human serum samples validates the reliability and potential of this platform in the screening, diagnosis, and prognosis of APL cases. Our findings present an approach that holds significant potential for the further development of the robust CRISPR/Cas sensor system, offering a rapid and adaptable paradigm for APL diagnosis.

The Symptom Network Structure of Teachers' Burnout and Its Connection to Psychological Capital.

Background: Teacher burnout is characterized by emotional and physical exhaustion resulting from excessive work-related stress. Previous research based on traditional latent variable theory has revealed a significant negative relationship between TB and psychological capital (PsyCap). This study explored the complex symptoms of TB and the contact points of PsyCap in reducing TB using psychometric network analysis. Methods: A total of 3991 teachers completed the burnout subscale of the Professional Quality of Life and Psychological Capital Scale. Results: The results showed that: (a) In the TB network, the core symptoms displayed by teachers due to burnout are difficulty feeling "I am a very caring person", "I am happy", and "I am the person I always wanted to be"; (b) The TB-PsyCap network was closely connected, and the symptoms affected each other. PsyCap affected the TB network through "I feel optimistic and happy almost every day" and "I often feel that there is a future as a teacher"; (c) PsyCap's bridge symptoms had a negative impact on TB, and PsyCap may reduce TB primarily through optimistic, hopeful dimensions. Conclusion and Implications: Psychometric network analysis helps us understand the complex symptoms of TB and the contact points of PsyCap in reducing TB. This study offers valuable insights into the prevention of, and intervention in, burnout within the teaching community.

Fiber Optic SERS Sensor with Silver Nanocubes Attached Based on Evanescent Wave for Detecting Pesticide Residues.

Surface-enhanced Raman scattering (SERS) has great potential in the field of rapid detection of pesticide residues in food. In this paper, a fiber optic SERS sensor excited by evanescent waves was proposed for efficient detection of thiram. Silver nanocubes (Ag NCs) were prepared as SERS active substrates, which had much stronger electromagnetic field intensity than nanospheres under laser excitation due to much more "hot spots". By using the method of electrostatic adsorption and laser induction, Ag NCs were uniformly assembled at the fiber taper waist (FTW) for enhancing the Raman signal. Different from the traditional way of stimulation, evanescent wave excitation greatly increased the interaction area between the excitation and analyte, while reducing the damage of the excited light to the metal nanostructures. The methods proposed in this work have been successfully used to detect the pesticide residues of thiram and showed good detection performance. The detection limits for 4-Mercaptobenzoic acid (4-MBA) and thiram were determined to be 10-9 and 10-8 M, the corresponding enhancement factor could be 1.64 × 105 and 6.38 × 104. Low concentration of thiram was detected in the peels of tomatoes and cucumbers, indicating its feasibility in actual sample detection. The combination of evanescent waves and SERS provides a new direction for the application of SERS sensors, which had great application potential in the field of pesticide residue detection.

Nonmetallic SERS-based biosensor for ultrasensitive and reproducible immunoassay of ferritin mediated by magnetic molybdenum disulfide nanoflowers and black phosphorus nanosheets.

To improve the curability of cancer patients, it is essential to propose an early diagnosis technology with ultra-high sensitivity and reliable biocompatibility. Herein, a sophisticated nonmetallic SERS-based immunosensor, comprised by a MoS2 @Fe3O4 nanoflower-based immunoprobe with magnetism and a black phosphorus (BP) nanosheet-based immunosubstrate, was proposed for the specific in-situ monitoring of ferritin (FER). The sandwich immunosensor was endowed with an excellent SERS performance mainly ascribed to a synergistic chemical enhancement as well as an additional electrostatic adsorption effect, achieving a limit of detection down to 7.3 × 10-5 µg/mL. Particularly, all the Raman label, target FER, and anti-FER could be completely degraded within 70 min under visible light irradiation owing to the favorable photocatalytic activities of MoS2 and BP which could be then effectively separated and collected with the assistance of an external magnet. Such a recyclable nonmetallic immunosensor holds great potential and practicality in the clinical screening of cancer.

An adhesive SERS substrate based on a stretched silver nanowire-tape for the in situ multicomponent analysis of pesticide residues.

Two essential factors in powerful surface-enhanced Raman spectroscopy analysis of trace pesticide residues are viz., high sensitivity and efficient sampling. Herein, owing to elastic properties, a stretched Ag nanowire (Ag NW)-tape under the strain of 15% formed a wrinkled structure with periodic microridges and microgrooves, where abundant nanogaps were generated by the aggregated Ag NWs. Compared with the unstretched Ag NW-tape substrate, an appreciable signal enhancement of the modified 4-mercaptobenzoic acid (4-MBA) molecules with a ratio of 2.6 was discerned from the sophisticated SERS substrate due to the electromagnetic enhancement induced by the relatively high density of "hot spots" around the Ag NW aggregates. The as-fabricated Ag NW-tape substrate performed admirably in detecting 4-MBA and demonstrated an enhancement factor of 1.16 × 106. Moreover, for the in situ detection of tetramethylthiuram disulfide, thiabendazole, and their mixture, the relatively high recovery rates of over 88% were favorably realized by the Ag NW-tape substrate with superior sensitivity, distinct flexibility, and adhesiveness. This fascinating SERS substrate, dependent on the flexible and adhesive Ag NW-tape, is promising for application in SERS analysis of trace residues on various practical surfaces.

Construction of automated high-throughput screening method for finding efficient 3-ketosteroid 1,2-dehydrogenating strains.

Dehydrogenation reaction at C1(2) positions is typical and representative of industrial production of steroid drugs. Anti-inflammatory activity can be doubled when the nucleus of the anti-inflammatory steroid hormone drug introduces double bonds at the C1(2) positions. Arthrobacter simplex is currently the most widely studied and used strain for C1(2) dehydrogenation. Therefore, breeding Arthrobacter simplex with high-efficiency dehydrogenation ability is of great significance. In order to obtain high-efficiency strains, the research proposed a new screening strategy based on image process technique: firstly, a color reaction between 2,4-dinitrophenylhydrazine (DNPH) and 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD) was established to characterize the dehydrogenation ability of the strain; secondly, the color data of strains mutated by atmospheric and room temperature plasma (ARTP) in the "color reaction" were automated and analyzed for dehydrogenation ability prediction using optimized support vector machine model. Result showed that the prediction accuracy reached as high as 96% in verification experiments. After a series of mutagenesis, including breaking the bottleneck of a single mutation in ARTP, the dominant strain ARLU-146 was finally obtained from 5168 strains. Its initial conversion rate was 0.8059 g/L/h, with a conversion of 94.41% at 24 h, compared to the original strain ASP which increased the transformation rate by more than 10%. By further process optimization, a high conversion (94.34% within 20 h) with high substrate (85 g/L cortisone acetate) was achieved. According to literature research, it is the highest conversion at this substrate concentration. KEY POINTS: • A high-throughput screening method was developed by using image processing and machine learning technique. • "Mutation bottleneck" of single ARTP mutagenesis was surpassed by complex mutagenesis. • A high substrate (85 g/L CA) and high transformation rate craft (94.34% within 20 h) were built.

Metabolic profile of main organic acids and its regulatory mechanism in solid-state fermentation of Chinese cereal vinegar.

Shanxi aged vinegar (SAV), a traditional Chinese cereal vinegar, is produced using solid-state fermentation (SSF) technology. Organic acids are the key flavor compounds of vinegar. However, the metabolic mechanism of organic acids during SSF process is still unclear. In this study, metatranscriptomics was used to explore the metabolic profile of main organic acids in SSF. The results show that carbon metabolism is the dominant pathway during fermentation, among which pyruvate metabolism, glycolysis and starch and sucrose metabolism associated with organic acids were the most abundant. The metabolic pathways of acetic acid and lactic acid shift from acetyl-P and pyruvate pathways at early and middle-early stages of fermentation to acetaldehyde and L-lactaldehyde pathways at later stages, respectively, and Lactobacillus and Acetobacter are the predominant microorganisms contributed to them. Temperature and acetic acid are proven to be the environmental factors that regulate the metabolic activity during SSF. This study sheds new lights on metabolism of flavor substances in the spontaneous ecosystems of traditional fermented food.

Five immune-gene-signatures participate in the development and pathogenesis of Kawasaki disease.

OBJECTIVE: To screen for immune genes that play a major role in Kawasaki disease and to investigate the pathogenesis of Kawasaki disease through bioinformatics analysis. METHODS: Kawasaki disease-related datasets GSE18606, GSE68004, and GSE73461 were downloaded from the Gene Expression Omnibus database. Three microarrays were integrated and standardized to include 173 Kawasaki disease samples and 101 normal samples. The samples were analyzed using CIBERSORT to obtain the infiltration of 22 immune cells and analyze the differential immune cells in the samples and correlations. The distribution of the samples was analyzed using principal component analysis (PCA). Immune-related genes were downloaded, extracted from the screened samples and analyzed for differential analysis (different expression genes [DEG]) and weighted gene co-expression network analysis (WGCNA). We constructed coexpression networks, and used the cytohobbe tool in Cytoscape to analyze the coexpression networks and select the immune genes that played a key role in them. RESULTS: Immune cell infiltration analysis showed that B cells naive, T cells CD8, natural killer (NK) cells activated, and so forth were highly expressed in normal samples. T cells CD4 memory activated, monocytes, neutrophils, and so forth were highly expressed in Kawasaki disease samples. PCA results showed a significant difference in the distribution of normal and Kawasaki disease samples. From the screened samples, 97 upregulated and 103 downregulated immune-related genes were extracted. WGCNA analysis of DEG yielded 10 gene modules, of which the three most relevant to Kawasaki disease were red, yellow, and gray modules. They were associated with cytokine regulation, T-cell activation, presentation of T-cell receptor signaling pathways, and NK cell-mediated cytotoxicity. CXCL8, CCL5, CCR7, CXCR3, and CCR1 were identified as key genes by constructing a coexpression network. CONCLUSION: Our study shows that we can distinguish normal samples from Kawasaki disease samples based on the infiltration of immune cells, and that CXCL8, CCL5, CCR7, CXCR3, and CCR1 may play important roles in the development of Kawasaki disease.

Development of optimal steam explosion pretreatment and highly effective cell factory for bioconversion of grain vinegar residue to butanol.

BACKGROUND: The industrial vinegar residue (VR) from solid-state fermentation, mainly cereals and their bran, will be a potential feedstock for future biofuels because of their low cost and easy availability. However, utilization of VR for butanol production has not been as much optimized as other sources of lignocellulose, which mainly stem from two key elements: (i) high biomass recalcitrance to enzymatic sugar release; (ii) lacking of suitable industrial biobutanol production strain. Though steam explosion has been proved effective for bio-refinery, few studies report SE for VR pretreatment. Much of the relevant knowledge remains unknown. Meanwhile, recent efforts on rational metabolic engineering approaches to increase butanol production in Clostridium strain are quite limited. In this study, we assessed the impact of SE pretreatment, enzymatic hydrolysis kinetics, overall sugar recovery and applied atmospheric and room temperature plasma (ARTP) mutant method for the Clostridium strain development to solve the long-standing problem. RESULTS: SE pretreatment was first performed. At the optimal condition, 29.47% of glucan, 71.62% of xylan and 22.21% of arabinan were depolymerized and obtained in the water extraction. In the sequential enzymatic hydrolysis process, enzymatic hydrolysis rate was increased by 13-fold compared to the VR without pretreatment and 19.60 g glucose, 15.21 g xylose and 5.63 g arabinose can be obtained after the two-step treatment from 100 g VR. Porous properties analysis indicated that steam explosion can effectively generate holes with diameter within 10-20 nm. Statistical analysis proved that enzymatic hydrolysis rate of VR followed the Pseudop-second-order kinetics equation and the relationship between SE severity and enzymatic hydrolysis rate can be well revealed by Boltzmann model. Finally, a superior inhibitor-tolerant strain, Clostridium acetobutylicum Tust-001, was generated with ARTP treatment. The water extraction and enzymolysis liquid gathered were successfully fermented, resulting in butanol titer of 7.98 g/L and 12.59 g/L of ABE. CONCLUSIONS: SE proved to be quite effective for VR due to high fermentable sugar recovery and enzymatic hydrolysate fermentability. Inverse strategy employing ARTP and repetitive domestication for strain breeding is quite feasible, providing us with a new tool for solving the problem in the biofuel fields.