PCR Independent Strategy-Based Biosensors for RNA Detection.

RNA is an important information and functional molecule. It can respond to the regulation of life processes and is also a key molecule in gene expression and regulation. Therefore, RNA detection technology has been widely used in many fields, especially in disease diagnosis, medical research, genetic engineering and other fields. However, the current RT-qPCR for RNA detection is complex, costly and requires the support of professional technicians, resulting in it not having great potential for rapid application in the field. PCR-free techniques are the most attractive alternative. They are a low-cost, simple operation method and do not require the support of large instruments, providing a new concept for the development of new RNA detection methods. This article reviews current PCR-free methods, overviews reported RNA biosensors based on electrochemistry, SPR, microfluidics, nanomaterials and CRISPR, and discusses their challenges and future research prospects in RNA detection.

Nomogram combining pre-operative clinical characteristics and spectral CT parameters for predicting the WHO/ISUP pathological grading in clear cell renal cell carcinoma.

PURPOSE: To develop a novel clinical-spectral-computed tomography (CT) nomogram incorporating clinical characteristics and spectral CT parameters for the preoperative prediction of the WHO/ISUP pathological grade in clear cell renal cell carcinoma (ccRCC). METHODS: Seventy-three ccRCC patients who underwent spectral CT were included in this retrospective analysis from December 2020 to June 2023. The subjects were pathologically divided into low- and high-grade groups (WHO/ISUP 1/2, n = 52 and WHO/ISUP 3/4, n = 21, respectively). Information on clinical characteristics, conventional CT imaging features, and spectral CT parameters was collected. Multivariate logistic regression analyses were conducted to create a nomogram combing clinical data and image data for preoperatively predicting the pathological grade of ccRCC, and the area under the curve (AUC) was utilized to assess the predictive performance of the model. RESULTS: Multivariate logistic regression analyses revealed that age, systemic immune-inflammation index (SII), and the slope of the spectrum curve in the cortex phase (CP-K) were independent predictors for predicting high-grade ccRCC. The clinical-spectral-CT model exhibited high evaluation efficacy, with an AUC of 0.933 (95% confidence interval [CI]: 0.878-0.998; sensitivity: 0.810; specificity: 0.923). The calibration curve revealed that the predicted probability of the clinical-spectral-CT nomogram could better fit the actual probability, with high calibration. The Hosmer-Lemeshow test showed that the model had a good fitness (χ2 = 5.574, p = 0.695). CONCLUSION: The clinical-spectral-CT nomogram has the potential to predict WHO/ISUP grading of ccRCC preoperatively.

Production of a matrix certified reference material for measurement and risk monitoring of clenbuterol in mutton.

A novel matrix certified reference material (CRM) for clenbuterol in mutton (GBW 10216) was developed to assist measurement and risk monitoring of clenbuterol in mutton. The candidate CRM raw samples were obtained by oral administration of clenbuterol and investigating the pharmacokinetics of clenbuterol in sheep. A high-precision isotope dilution coupled with liquid chromatography tandem mass spectrometry (LC-ID-MS/MS) method was established and assigned the value of clenbuterol in mutton powder through combined detection of nine inter-laboratories. The certified value with expanded uncertainty was 21.1 ± 2.2 µg/kg (k = 2, 95% confidence) for clenbuterol in mutton. The prepared matrix CRM was sufficiently homogeneous between and within bottles. The long-term stability of clenbuterol in mutton powder was evaluated for 12 months at -20℃ and short-term stability for 7 days at 4℃ and 50℃. The uncertainties originating from characterization, homogeneity, and stability were systematically analyzed and evaluated. The prepared matrix CRM can be applied for proficiency testing and nationwide risk monitoring programs to guarantee the accuracy and comparability of clenbuterol measurement results in mutton.

Development of matrix certified reference material for accurate determination of docosahexaenoic acid in milk powder.

A novel matrix certified reference material (CRM) of docosahexaenoic acid in milk powder [GBW (E) 100641] was first developed. The CRM candidates was prepared by adding appropriate levels of docosahexaenoic acid to cow's milk, then powder sprayed, lyophilized, mixed, dispensed and sterilized. An optimized acetylchloride-methanol method was proposed and used for the characterization. The CRM characterization was carried out in six laboratories in accordance with ISO Guide 35 requirements. The certified value of CRM was 0.69 mg/g with an uncertainty of 0.08 mg/g (k = 2). The CRM was sufficiently homogeneous between and within bottles and stable up to 6 month at -20℃ and 7 days below 50 â„ƒ. The uncertainty was evaluated by combing the contributions from characterization, homogeneity and stability. Thus, the CRM can be used for quality control and method validation to ensure the accurate and reliable measurements of docosahexaenoic acid in milk for quality monitoring.

Allergenicity Alleviation of Bee Pollen by Enzymatic Hydrolysis: Regulation in Mice Allergic Mediators, Metabolism, and Gut Microbiota.

Bee pollen as a nutrient-rich functional food has been considered for use as an adjuvant for chronic disease therapy. However, bee pollen can trigger food-borne allergies, causing a great concern to food safety. Our previous study demonstrated that the combined use of cellulase, pectinase and papain can hydrolyze allergens into peptides and amino acids, resulting in reduced allergenicity of bee pollen based on in vitro assays. Herein, we aimed to further explore the mechanisms behind allergenicity alleviation of enzyme-treated bee pollen through a BALB/c mouse model. Results showed that the enzyme-treated bee pollen could mitigate mice scratching frequency, ameliorate histopathological injury, decrease serum IgE level, and regulate bioamine production. Moreover, enzyme-treated bee pollen can modulate metabolic pathways and gut microbiota composition in mice, further supporting the alleviatory allergenicity of enzyme-treated bee pollen. The findings could provide a foundation for further development and utilization of hypoallergenic bee pollen products.

Identification of allergens and allergen hydrolysates by proteomics and metabolomics: A comparative study of natural and enzymolytic bee pollen.

Bee pollen as a plant-derived food is consumed as nutritional/functional supplements by humans. But it might confer foodborne allergenicity in susceptible populations, limiting its extensive application. In this study, five potential allergens including profilin, cystatin, prolamin, expansin, and alcohol dehydrogenase in bee pollen derived from Brassica campestris (BP-Bc), were identified through mass spectrometry-based proteomic analysis. Moreover, different types of enzymes (cellulases, pectases, and papains) serve biological roles in pollen wall breaking and expansion, but also promote allergen release and degradation. Proteomic analysis showed that profilin, cystatin, and alcohol dehydrogenase were significantly reduced in BP-Bc following joint treatment with three enzymes. Metabolomic characterization of potential enzymatic hydrolysates of these significantly-decreased allergens was performed, which showed nine major oligopeptides and six amino acids at significantly higher levels in the enzyme-treated BP-Bc. These findings clarified the culprit responsible for bee pollen allergy and the mechanism of enzymatic desensitization for its further development.

Development and Validation of a Clinical-Based Signature to Predict the 90-Day Functional Outcome for Spontaneous Intracerebral Hemorrhage.

We aimed to develop and validate an objective and easy-to-use model for identifying patients with spontaneous intracerebral hemorrhage (ICH) who have a poor 90-day prognosis. This three-center retrospective study included a large cohort of 1,122 patients with ICH who presented within 6 h of symptom onset [training cohort, n = 835; internal validation cohort, n = 201; external validation cohort (center 2 and 3), n = 86]. We collected the patients' baseline clinical, radiological, and laboratory data as well as the 90-day functional outcomes. Independent risk factors for prognosis were identified through univariate analysis and multivariate logistic regression analysis. A nomogram was developed to visualize the model results while a calibration curve was used to verify whether the predictive performance was satisfactorily consistent with the ideal curve. Finally, we used decision curves to assess the clinical utility of the model. At 90 days, 714 (63.6%) patients had a poor prognosis. Factors associated with prognosis included age, midline shift, intraventricular hemorrhage (IVH), subarachnoid hemorrhage (SAH), hypodensities, ICH volume, perihematomal edema (PHE) volume, temperature, systolic blood pressure, Glasgow Coma Scale (GCS) score, white blood cell (WBC), neutrophil, and neutrophil-lymphocyte ratio (NLR) (p < 0.05). Moreover, age, ICH volume, and GCS were identified as independent risk factors for prognosis. For identifying patients with poor prognosis, the model showed an area under the receiver operating characteristic curve of 0.874, 0.822, and 0.868 in the training cohort, internal validation, and external validation cohorts, respectively. The calibration curve revealed that the nomogram showed satisfactory calibration in the training and validation cohorts. Decision curve analysis showed the clinical utility of the nomogram. Taken together, the nomogram developed in this study could facilitate the individualized outcome prediction in patients with ICH.

Nanocomposites of boronic acid-functionalized magnetic multi-walled carbon nanotubes with flexible branched polymers as a novel desorption/ionization matrix for the capture and direct detection of cis-diol-flavonoid compounds coupled with MALDI-TOF-MS.

Novel boronic acid-functionalized magnetic multi-walled carbon nanotubes with flexible branched polymer (Fe3O4@MWCNTs@ε-PL@BA) nanocomposites were fabricated and applied as the desorption/ionization matrix for the MALDI-TOF-MS determination of low molecular weight flavonoids. The prepared nanocomposite was systematically characterized by various techniques. Compared to the traditional organic matrix, the proposed Fe3O4@MWCNTs@ε-PL@BA matrix has excellent ionization efficiency and low-background noise interference due to the MWCNTs unique electron-phonon interaction and the high introduction density of boronic acid functional groups. Good sensitivity and ultra-high salt tolerance of the Fe3O4@MWCNTs@ε-PL@BA-assisted MALDI-TOF-MS were permitted for the determination and quantification of flavonoids in actual samples. Noticeably, the limits of detection (LODs) for the target flavonoids were in the range 17-33 nM. The relative standard deviations (RSDs) of spot-to-spot and sample-to-sample (n = 10) were ≤ 9.8% and ≤ 10.1%, respectively. Furthermore, the wide linear ranges (0.1 - 500 µg/mL) and satisfactory calibration plot coefficients (R2 > 0.99) of flavonoids were achieved by MALDI-TOF-MS with the Fe3O4@MWCNTs@ε-PL@BA matrix. Good recoveries (92-105.5%) were achieved for the target flavonoids in practical food samples. Hence, the prepared Fe3O4@MWCNTs@ε-PL@BA nanocomposites have applications in the selective and efficient capture of target flavonoids active biomolecules coupled with MALDI-TOF-MS determination in actual samples.

Cell changes and differential proteomic analysis during biodegradation of decabromodiphenyl ether (BDE-209) by Pseudomonas aeruginosa.

Decabromodiphenyl ether (BDE-209) is a brominated flame retardant widely used in the world which, being an emerging persistent organic pollutant, poses a great potential hazard to both human health and the ecological environment. Microbial biodegradation has been considered as an effective and environment-friendly technique to remediate BDE-209. Pseudomonas aeruginosa, a Gram-negative bacterium capable of degrading BDE-209, was isolated from PBDEs-contaminated soil. To promote microbial biodegradation of BDE-209 and gain further insight into its mechanism, cell changes and differential proteomic analysis of P. aeruginosa during biodegradation were studied. The results showed that high cell surface hydrophobicity of P. aeruginosa make the bacteria absorb BDE-209 more easily. The increase in cell membrane permeability was caused by the P. aeruginosa responding to BDE-209 stress. IR spectra showed that hydroxyl, amide and CH2 groups in the P. aeruginosa cell surface were involved in the interactions between BDE-209 with P. aeruginosa. The apoptotic-like cell changes and cell surface morphology changes were observed by flow cytometry (FCM) and field emission scanning electron microscopy (FESEM), respectively. Differentially expressed protein was analysed by two-dimensional electrophoresis (2-DE) and 40 protein spots were identified to be different after 5 days biodegradation.

Synthesis and characterization of magnetic mesoporous Fe3O4@mSiO2-DODGA nanoparticles for adsorption of 16 rare earth elements.

In this study, novel magnetic mesoporous Fe3O4@mSiO2-DODGA nanoparticles were prepared for efficiently adsorbing and recycling REEs. Fe3O4@mSiO2-DODGA was characterized by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), vibrating sample magnetometry (VSM), Fourier transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA). The adsorption behavior of Fe3O4@mSiO2-DODGA was investigated by ICP-OES. The results showed that the content of DODGA in the adsorbent was 367 µmol g-1. Fe3O4@mSiO2-DODGA exhibited the highest adsorption rates for 15 REEs, except Tm, in a 2 mol L-1 nitric acid solution. Among these elements, the adsorption rates for Nd, Sm, Eu, Dy, Ho, Yb, Lu, Y and Sc ranged from 85.1% to 100.1%. The desorption rates for all 16 REE ions reached their maximum values when 0.01 mol L-1 EDTA was used as the eluent. The desorption rates for Nd, Ce, Sm, Eu, Ho, Yb, Lu, Y, and Sc were 87.7-99.8%. Fe3O4@mSiO2-DODGA had high stability in 2 mol L-1 HNO3 and could be used five times without significant loss of adsorption capacity. Moreover, these nanoparticles had high selectivity, and their adsorption rate was not affected even in a high-concentration solution of a coexisting ion. Therefore, 8 REE ions (Nd, Sm, Eu, Ho, Yb, Lu, Y, and Sc) were selected for the study of adsorption kinetics and adsorption isotherm experiments. It was demonstrated that the values of Q e (equilibrium adsorption capacity) for Nd, Sm, Eu, Ho, Yb, Lu, Y, and Sc were 14.28-60.80 mg g-1. The adsorption of REEs on Fe3O4@mSiO2-DODGA followed the pseudo-second-order kinetic model, Elovich model and Langmuir isotherm model, which indicated that the adsorption process of Fe3O4@mSiO2-DODGA for REEs comprised single-layer adsorption on a non-uniform surface controlled by chemical adsorption. It was concluded that Fe3O4@mSiO2-DODGA represents a new material for the adsorption of REEs in strongly acidic solutions.

Simultaneous determination of trace rare-earth elements in simulated water samples using ICP-OES with TODGA extraction/back-extraction.

The determination of trace rare-earth elements (REEs) can be used for the assessment of environmental pollution, and is of great significance to the study of toxicity and toxicology in animals and plants. N, N, N', N'-tetraoctyl diglycolamide (TODGA) is an environmental friendly extractant that is highly selective to REEs. In this study, an analytical method was developed for the simultaneous determination of 16 trace REEs in simulated water samples by inductively coupled plasma optical emission spectroscopy (ICP-OES). With this method, TODGA was used as the extractant to perform the liquid-liquid extraction (LLE) sample pretreatment procedure. All 16 REEs were extracted from a 3 M nitric acid medium into an organic phase by a 0.025 M TODGA petroleum ether solution. A 0.03 M ethylenediaminetetraacetic acid disodium salt (EDTA) solution was used for back-extraction to strip the REEs from the organic phase into the aqueous phase. The aqueous phase was concentrated using a vacuum rotary evaporator and the concentration of the 16 REEs was detected by ICP-OES. Under the optimum experimental conditions, the limits of detection (3σ, n = 7) for the REEs ranged from 0.0405 ng mL-1 (Nd) to 0.5038 ng mL-1 (Ho). The relative standard deviations (c = 100 ng mL-1, n = 7) were from 0.5% (Eu) to 4.0% (Tm) with a linear range of 4-1000 ng mL-1 (R2 > 0.999). The recoveries of 16 REEs ranged from 95% to 106%. The LLE-ICP-OES method established in this study has the advantages of simple operation, low detection limits, fast analysis speed and the ability to simultaneously determine 16 REEs, thereby acting as a viable alternative for the simultaneous detection of trace amounts of REEs in water samples.

Effect of copper ion and soil humic acid on biodegradation of decabromodiphenyl ether (BDE-209) by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a good environmental microorganism capable of degrading decabromodiphenyl ether (BDE-209). This paper studied the effect of Cu2+ and humic acid (HA) extracted from e-waste contaminated soils on biodegradation of BDE-209 by P. aeruginosa. The adsorption isotherms of Cu2+ on HA, the crude enzyme activity, cell surface morphology, and biodegradation pathway were also investigated. The results showed that BDE-209 biodegradation by P. aeruginosa was inhibited at Cu2+ concentrations above 5 mg L-1 , but exhibited the best effect at the condition of 40 mg L-1 Cu2+  + 3 g L-1 HA. At the condition of 40 mg L-1 Cu2+  + 3 g L-1 HA, 97.35 ± 2.33% of the initial BDE-209 was degraded after 5 days, debromination efficiency was 72.14 ± 1.89%, crude enzyme activity reached the maximum of 0.519 ± 0.022U g-1 protein, cell surface of P. aeruginosa was smooth with normal short-rod shapes, and biodegradation pathway mainly include debromination, hydroxylation, and cleavage of the diphenyl ether bond. It was suggested that soil HA could eliminate the toxic effect of high Cu2+ concentrations and biodegradation of BDE-209 was improved by synergistic effect of HA and Cu2+ .

Biodegradation of Decabromodiphenyl Ether (BDE-209) by Crude Enzyme Extract from Pseudomonas aeruginosa.

The biodegradation effect and mechanism of decabromodiphenyl ether (BDE-209) by crude enzyme extract from Pseudomonas aeruginosa were investigated. The results demonstrated that crude enzyme extract exhibited obviously higher degradation efficiency and shorter biodegradation time than Pseudomonas aeruginosa itself. Under the optimum conditions of pH 9.0, 35 °C and protein content of 2000 mg/L, 92.77% of the initial BDE-209 (20 mg/L) was degraded after 5 h. A BDE-209 biodegradation pathway was proposed on the basis of the biodegradation products identified by GC-MS analysis. The biodegradation mechanism showed that crude enzyme extract degraded BDE-209 into lower brominated PBDEs and OH-PBDEs through debromination and hydroxylation of the aromatic rings.