Wash-Free and High-Contrast Imaging of Single-Nucleotide Variation in Single Cells Using Transcription-Amplified Light-Up RNA Aptamer.

Single-nucleotide variation (SNV) imaging can indicate cellular heterogeneity and spatial pattern, but it remains challenging to produce high-gain signal while also yielding single-nucleotide resolution. Herein, we developed a light-up strategy for visualizing SNVs based on transcription amplification, enabling wash-free and high-contrast imaging of SNVs inside cells. The discrimination of SNVs is achieved by ligase-assisted transcription reaction. Employing a light-up RNA aptamer as a reporter eliminates nonspecific probe binding and the washing process and contributes to a 2-fold improvement of signal gain compared to that using the fluorescence in situ hybridization (FISH) method. The method allowed us to precisely quantify drug-resistant strains in the bacteria mixture and identify drug-resistant Salmonella enterica (S. enterica) isolated from poultry farm. Using this approach, we explored the colonization features of drug-resistant and drug-sensitive S. enterica in the mice intestinal tract and screened the prebiotics for Salmonella colonization inhibition. The SNV imaging method promises for the interrogation of genotypes in physiological and pathological states at the single-cell level.

CRISPR/Cas14a-Based Isothermal Amplification for Profiling Plant MicroRNAs.

MicroRNAs (miRNAs) play key roles in biological processes in plants, such as stress resistance, yet can hardly be quantified by an enzyme-involved terminal polymerization process due to their 2'-O-methyl modifications at the 3'-terminal. Herein, we proposed a CRISPR/Cas14a-based rolling circle amplification (termed Cas14R) assay, allowing reverse transcription-free and demethylation-free detection of plant miRNAs with single-nucleotide resolution. The employment of target-templated rolling circle amplification circumvents the extension of the unaccessible 2'-O-methyl group at the 3'-terminal. Particularly, the activated Cas14a confers the trans-cleavage activity for identifying target single-stranded DNA sequences without the necessity of the protospacer adjacent motif, generalizing the detection of miRNA sequences and the integration of different isothermal amplification techniques. Ultimately, the Cas14R assay has been applied to profile miR156a to evaluate the ripeness process of banana, indicating its feasibility in analyzing the roles of miRNAs in biological processes of plants.

Epstein-Barr virus activates F-box protein FBXO2 to limit viral infectivity by targeting glycoprotein B for degradation.

Epstein-Barr virus (EBV) is a human cancer-related virus closely associated with lymphoid and epithelial malignancies, and EBV glycoprotein B (gB) plays an essential role in viral entry into both B cells and epithelial cells by promoting cell-cell fusion. EBV gB is exclusively modified with high-mannose-linked N-glycans and primarily localizes to the endoplasmic reticulum (ER) with low levels on the plasma membrane (PM). However, the mechanism through which gB is regulated within host cells is largely unknown. Here, we report the identification of F-box only protein 2 (FBXO2), an SCF ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans and attenuates EBV infectivity by targeting N-glycosylated gB for degradation. gB possesses seven N-glycosylation sites, and FBXO2 directly binds to these high-mannose moieties through its sugar-binding domain. The interaction promotes the degradation of glycosylated gB via the ubiquitin-proteasome pathway. Depletion of FBXO2 not only stabilizes gB but also promotes its transport from the ER to the PM, resulting in enhanced membrane fusion and viral entry. FBXO2 is expressed in epithelial cells but not B cells, and EBV infection up-regulates FBXO2 levels. In summary, our findings highlight the significance of high-mannose modification of gB and reveal a novel host defense mechanism involving glycoprotein homeostasis regulation.

Blood Urea Nitrogen to Serum Albumin Ratio as A New Prognostic Indicator in Critically Ill Patients with Diabetic Ketoacidosis: A Retrospective Cohort Study.

OBJECTIVE: To investigate the predictive value of the blood urea nitrogen to serum albumin ratio for in-hospital and out-of-hospital mortality in critically ill patients with diabetic ketoacidosis. METHODS: Data were obtained from the Medical Information Mart for Intensive Care III (MIMIC III) database, and all eligible participants were categorized into two groups based on the BAR cutoff value. Multiple logistic regression analysis was conducted to determine the association between BAR and in-hospital mortality. The Kaplan-Meier (K-M) analysis was performed to evaluate the predictive performance of BAR. Propensity score matching (PSM) was applied to control confounding factors between the low and high BAR groups. RESULTS: A total of 589 critically ill patients with diabetic ketoacidosis were enrolled. Patients with diabetic ketoacidosis with a higher BAR level were associated with higher in- and out-hospital mortality (all p<0.001). A significant 4-year survival difference was observed between the low and high BAR groups (p<0.0001). After PSM analysis, two PSM groups (202 pairs, n=404) were generated, and similar results were observed in the K-M curve (p<0.0001). DISCUSSION: Elevated BAR levels were associated with an increased risk of in-hospital mortality in critically ill patients with diabetic ketoacidosis, and BAR could serve as an independent prognostic factor in in-hospital and out-of-hospital mortality for patients diagnosed with diabetic ketoacidosis.

Effect of hydroxypropyl distarch phosphate on the retrogradation properties of sterilized pea starch jelly and its possible mechanism.

Due to the high content of amylose in pea starch (PS), PS jelly is prone to retrogradation during storage and its quality reduces subsequently. Hydroxypropyl distarch phosphate (HPDSP) shows a potential inhibitory effect on the retrogradation of starch gel. Based on this, five retrograded PS-HPDSP blends containing 1 %, 2 %, 3 %, 4 % and 5 % (w/w, based on the weight of PS) of HPDSP were prepared, and their long-range, short-range ordered structure and retrogradation properties, and the possible interaction between PS and HPDSP were investigated. The addition of HPDSP significantly reduced the hardness of PS jelly and maintained its springiness during cold storage, and this effect was enhanced with HPDSP dosage being from 1 % to 4 %. The presence of HPDSP destroyed both short-range ordered structure and long-range ordered structure. Rheological results indicated that all the gelatinized samples were typical non-Newtonian fluids with shear-thinning characteristics and HPDSP increased their viscoelasticity in a dose-dependent manner. In conclusion, HPDSP delays the retrogradation of PS jelly mainly by combining with amylose in PS through hydrogen bonds and steric hindrance.

Single-Cell Genotyping of Single-Nucleotide Mutations Using In Situ Allele-Specific Loop-Mediated Isothermal Amplification.

Single-nucleotide mutations (SNMs) in the bacterial genome may cause antibiotic resistance. The visualization of SNMs can indicate antibiotic resistance phenotypes at the single-cell level but remains challenging. Herein, we proposed an in situ allele-specific isothermal amplification proceeded inside cells, allowing us to image bacterial genes with single-nucleotide resolution. The primer for loop-mediated isothermal amplification (LAMP) was designed with artificial mismatch bases to serve as an allele-specific probe, endowing LAMP to specifically amplify genes with SNMs. Due to the high amplification efficiency of LAMP, the method termed AlleLAMP can generate high gain for imaging SNMs and precisely quantify mutated quinolone-resistant Salmonella in bacterial mixture. We utilized AlleLAMP to survey the selection of antibiotic resistance under the preservative stress and found that the mutant quinolone-resistant strain owned a survival advantage over the wild-type quinolone-sensitive strain under the stress of preservatives. AlleLAMP can serve as a single-cell tool for analyzing the relationship between bacterial genotype and phenotype.

Primer-Engineered Transferase Enzyme for One-Pot and Amplified Detection of Cobalt Pollution and Peptide Remover Screening.

Extensive consumption of cobalt in the chemical field such as for battery materials, alloy, pigments, and dyes has aggravated the pollution of cobalt both in food and the environment, and assays for its on-site monitoring are urgently demanded. Herein, we utilized enzyme dependence on metal cofactors to develop terminal transferase (TdT) as a recognition element, achieving a one-pot sensitive and specific assay for detecting cobalt pollution. We engineered a 3'-OH terminus primer to improve the discrimination capacity of TdT for Co2+ from other bivalent cations. The TdT extension reaction amplified the recognition of Co2+ and yielded a limit of detection of 0.99 µM for Co2+ detection. Then, the TdT-based assay was designed to precisely detect cobalt in food and agricultural soil samples. By end-measurement of fluorescence using a microplate reader, the multiplexing assay enabled the rapid screening of the peptide remover for cobalt pollution. The TdT-based assay can be a promising tool for cobalt pollution monitoring and control.

DNAzyme-templated exponential isothermal amplification for sensitive detection of lead pollution and high-throughput screening of microbial biosorbents.

Very low concentrations of lead (Pb2+) pollution can have far-reaching adverse impacts on human health, due to the cumulative toxicity of Pb2+. Herein, we report a DNAzyme-templated exponential isothermal amplification strategy (termed DNAzymee) for the ultrasensitive detection of Pb2+ pollution and the high-throughput screening of microbial biosorbents to remove Pb2+ pollution. DNAzyme can specifically recognize Pb2+, and this recognition event can be amplified by the subsequent exponential isothermal amplification reaction (EXPAR) and monitored by a G-quadruplex specific dye. The proposed design showed a low limit of detection (95 pM) and could identify Pb2+ pollution in different real samples with high precision. In particular, the proposed assay was used to screen Pb2+ biosorbents, and the results showed that Leuconostoc mesenteroides is a promising microbial biosorbent for removing Pb2+ pollution. Thus, the DNAzymee assay can serve as a platform to monitor lead pollution in the environment and screen efficient biosorbents for the control of lead pollution.

Consecutive Kummell's Disease Combined with Parkinson's Disease and Experienced Internal Fixation Failure: A Case Report and Literature Review.

BACKGROUND: The continuous occurrence of Kummell's disease is extremely rare in clinical practice, and its treatment is difficult. The study aimed to present a rare case of consecutive Kummell's disease combined with Parkinson's disease (PD) and experienced internal fixation failure. CASE PRESENTATION: A 69-year-old female patient had a history of PD for 10 years, and was treated by posterior decompression, fixation, and fusion because of Kummell's disease of T12 with neurological damage. The patient's back pain and lower limb pain were significantly improved after surgery. Twenty-two months later, the patient was rehospitalized for Kummell's disease of L4 with neuropathic pain of left lower extremity. She received almost identical surgical procedures as T12 lesion, and the difference was no L4 vertebroplasty preformed due to the fact that the L4 vertebrae collapse was not obvious, the intravertebral vacuum cleft (IVC) range was small, and the pedicle screw fixation strength was high. The pain symptoms were significantly relieved after operation. Unfortunately, there was a complication of internal fixation failure that occurred a month later, and a revision operation was carried out. CONCLUSION: Osteoporosis combined with PD may lead patients to become prone to consecutive Kummell's disease, and patients are prone to experience failure of internal fixation. Bone cement filling of vertebral IVC and effective support of anterior vertebral column are very important procedures to ensure the clinical efficacy of treating Kummell's disease.

Label-Free Detection of Transgenic Crops Using an Isothermal Amplification Reporting CRISPR/Cas12 Assay.

Current tools for detecting transgenic crops, such as polymerase chain reaction (PCR), require professional equipment and complex operation. Herein, we introduce a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system to analyze transgenes by designing an isothermal amplification to serve as the amplified reporter, allowing an isothermal and label-free detection of transgenic crops. The use of Cas12a allowed direct and specific recognition of transgenes. To enhance the sensitivity of the assay, we used rolling circle amplification (RCA) to monitor the recognition of transgenes by designing the RCA primer as the cleavage substrate of Cas12a. The presence of transgenes can be detected by monitoring the G-quadruplex in RCA amplicon using a G-quadruplex binding dye, N-methyl mesoporphyrin IX (NMM). We termed the assay as isoCRISPR and showed that the assay allowed distinguishing transgenic corn cultivars ("Bt11" and "MON89034") from nontransgenic corn cultivars ("yellow", "shenyu", "xianyu", and "jingke"). The isoCRISPR assay will enrich the toolbox for transgenic crop identification and broaden the application of CRISPR/Cas in food authenticity and safety.

Light-up RNA aptamer signaling-CRISPR-Cas13a-based mix-and-read assays for profiling viable pathogenic bacteria.

Viable pathogenic bacteria cause serious human diseases via systemic infections and food poisoning. Herein, we constructed a light-up RNA aptamer signaling-CRISPR-Cas13a assay enabling mix-and-read detection of viable pathogenic bacteria. Directly targeting pathogen RNAs via CRISPR-Cas13a allows precisely discriminating viable bacteria from dead bacteria. We introduced a light-up RNA aptamer, Broccoli, serving as the substate of activated CRISPR-Cas13a to monitor the presence of pathogen RNAs, eliminating the need to use chemically labeled RNA substrate. Sequentially, the assay allows a reverse transcription-free, nucleic acid amplification-free, and label-free quantification of RNA targets and viable pathogenic bacteria. It could detect as low as 10 CFU of Bacillus cereus and precisely quantify viable bacteria with a content ranging from 0% to 100% in 105 CFU total bacteria. The quantification of viable bacteria allows more accurately estimating the ability of B. cereus to spoil food. The RNA assay promises its use in point-of-use detection of viable pathogens and biosafety control.

Temperature-robust and ratiometric G-quadruplex proximate DNAzyme assay for robustly monitoring of uranium pollution and its microbial biosorbents screening.

Uranium pollution in environment and food chain is a serious threat to public security and human health. Herein, we proposed a temperature-robust, ratiometric, and label-free bioassay based on G-quadruplex proximate DNAzyme (G4DNAzyme), accommodating us to precisely monitor uranium pollution and biosorption. The proximity of split G-quadruplex probes was proposed to sense UO22+-activated DNAzyme activity, thus eliminating the use of chemically labeled nucleic acid probes. And the simultaneous monitoring of G-quadruplex and double-stranded structures of DNAzyme probes contributed to a ratiometric and robust detection of UO22+. Particularly, the separation of enzymatic digestion and fluorescence monitoring endued a robust and highly responsive detection of UO22+ upon the temperature of enzymatic digestion process ranged from 18° to 41 °C. Consequently, G4DNAzyme assay allowed a robust, label-free and ratiometric quantification of uranium. We demonstrated the feasibility of G4DNAzyme assay for estimating uranium pollution in water and aquatic product samples. Ultimately, G4DNAzyme assay was adopted to serve as the platform to screen bacterial species and conditions for uranium biosorption, promising its roles in uranium associated biosafety control.

Metal-induced G-quadruplex polymorphism for ratiometric and label-free detection of lead pollution in tea.

Lead pollution are critical concerns for food safety and human health. Herein, a ratiometric metal-induced G-quadruplex polymorphism was introduced to construct aptamer probes, enabling label-free and ratiometric detection of lead in tea, thus is promising for on-site detection of lead pollution. The key feature of the aptamer probe is the synergistic utilization of the dual-wavelength fluorescent signal outputs from a G-quadruplex specific dye and a DNA intercalation dye under a single-wavelength excitation, leading to a more stable and reliable recognition of Pb2+ than that of analyses based on single fluorescent reporter. The aptamer probe allowed to a mix-and-read, rapid, cost-effective detection of Pb2+ with high specificity and accuracy. Pb2+ analysis in tap water and tea exhibited good performance with recovery rates of 92.3%-109.0%. The adoption of ratiometric metal-induced G-quadruplex polymorphism would be a compelling design strategy for constructing robust aptasensor, facilitating the translation of aptamer for food safety control.

Label-free DNAzyme assays for dually amplified and one-pot detection of lead pollution.

Lead pollution in water and soil often transfers to food, advocating tools for on-site detection of lead pollution to ensure both environmental and food safety. We proposed a label-free, dually amplified and homogeneous DNAzyme assay for sensitive and one-pot detection of lead pollution. Instead of using chemically modified DNA substrate, a structure-response digestion process was introduced to monitor Pb2+ presence-induced cleavage process of unlabeled substrate, further amplifying the response signals and eliminating the use of labeled DNA probes. The DNAzyme assay allowed to detect Pb2+ as low as 0.12 nM and endued a dynamic range from 0.1 nM to 30 nM. In addition, it can specifically identify Pb2+ among other metal ions. We demonstrated that the DNAzyme assay can precisely detect Pb2+ in tap water, milk and fish. Thus, the DNAzyme assay is promising for on-site monitoring lead pollution risk and ensuring environmental and food safety.

CRISPR-Cas12-Based Rapid Authentication of Halal Food.

The halal food market is globally growing along with the increased risk of adulteration. We proposed an amplification-free and mix-to-read CRISPR-Cas12-based nucleic acid analytical strategy allowing rapid identification and analysis of pork components, thus enriching the toolbox for ensuring halal food authenticity. We designed and optimized guide RNA (gRNA) targeting the pork cytochrome b (Cyt b) gene. gRNA allowed specific identification of the target Cyt b gene from pork components followed by activation of Cas12 protein to abundantly cleave single-stranded DNA probes with terminally labeled fluorophore and quencher groups, thus turning on fluorescence. The presence of the pork Cyt b gene thus can be mix-and-read- and only-one-step-detected, which may indicate the risk of halal food adulteration. The method allowed specific discrimination of pork meat from beef, mutton, and chicken and yielded a detection limit of 2.7 ng/µL of total DNA from pork meat. The reliability of the method was tested using the following processed meat products: halal foods beef luncheon meat and spiced beef and non-halal foods sausage and dried pork slices. The CRISPR-Cas12-based nucleic acid test strategy is promising for rapid food authentication.

G-Quadruplex-Probing CRISPR-Cas12 Assay for Label-Free Analysis of Foodborne Pathogens and Their Colonization In Vivo.

Foodborne pathogen infection is a key issue of food safety. Herein, we developed a label-free assay for Salmonella enterica (S. enterica) detection based on the G-quadruplex-probing CRISPR-Cas12 system (termed G-CRISPR-Cas), allowing highly sensitive detection of S. enterica and investigation of their colonization in chickens. The introduction of the G-quadruplex probe serving as the substrate of Cas 12a realized a label-free analysis for foodborne pathogens. Due to the amplification process induced by loop-mediated isothermal amplification (LAMP), G-CRISPR-Cas assay can detect S. enterica as low as 20 CFU. Specificity for pathogenic gene detection was guaranteed by the dual recognition process via LAMP primers and Cas 12a-guided RNA binding. The G-CRISPR-Cas assay was applied to explore S. enterica colonization in the intestinal tract and organs of chickens and showed the risk of S. enterica infection outside of the intestinal tract. The G-CRISPR-Cas assay is promising for on-site diagnosis of the infection or contamination of foodborne pathogens outside the laboratories, such as abattoirs and markets.

Direct Detection of Foodborne Pathogens via a Proximal DNA Probe-Based CRISPR-Cas12 Assay.

Foodborne pathogens can cause illnesses. Existing tools for detecting foodborne pathogens are typically time-consuming or require complex protocols. Here, we report an assay to directly analyze pathogenic genes based on CRISPR-Cas12. This new test, termed proximal DNA probe-based CRISPR-Cas12 (PPCas12), facilitates the detection of foodborne pathogens without amplification steps. The elimination of the nucleic acid amplification process dramatically reduced the processing time, complexity, and costs in the analysis of foodborne pathogens. The substitution of the frequently used dually labeled DNA reporter with a proximal DNA probe in the PPCas12 assay led to a 4-fold sensitivity enhancement. PPCas12 offered a limit of detection of 619 colony-forming units in the detection of Salmonella enterica (S. enterica) without the nucleic acid amplification process. The specific recognition of genes via PPCas12 allowed distinguishing S. enterica from other foodborne pathogens. The PPCas12 assay was applied in the screening of S. enterica contamination on fresh eggs with high precision. Hence, the new PPCas12 assay will be a valuable tool for on-site monitoring of foodborne pathogens.

Neoantigen landscape in metastatic nasopharyngeal carcinoma.

Background: Reportedly, nasopharyngeal carcinoma (NPC) patients with MHC I Class aberration are prone to poor survival outcomes, which indicates that the deficiency of tumor neoantigens might represent a mechanism of immune surveillance escape in NPC. Methods: To clearly delineate the landscape of neoantigens in NPC, we performed DNA and RNA sequencing on paired primary tumor, regional lymph node metastasis and distant metastasis samples from 26 patients. Neoantigens were predicted using pVACseq pipeline. Subtype prediction model was built using random forest algorithm. Results: Portraying the landscape of neoantigens in NPC for the first time, we found that the neoantigen load of NPC was above average compared to that of other cancers in The Cancer Genome Atlas program. While the quantity and quality of neoantigens were similar among primary tumor, regional lymph node metastasis and distant metastasis samples, neoantigen depletion was more severe in metastatic sites than in primary tumors. Upon tracking the clonality change of neoantigens, we found that neoantigen reduction occurred during metastasis. Building a subtype prediction model based on reported data, we observed that subtype I lacked T cells and suffered from severe neoantigen depletion, subtype II highly expressed immune checkpoint molecules and suffered from the least neoantigen depletion, and subtype III was heterogenous. Conclusions: These results indicate that neoantigens are conducive to the guidance of clinical treatment, and personalized therapeutic vaccines for NPC deserve deeper basic and clinical investigations to make them feasible in the future.

Dual Triple Helix-Aptamer Probes for Mix-and-Read Detecting Antibiotics in Fish and Milk.

Antibiotic abuse in agricultural products leads to serious food safety issues. To this end, we proposed a mix-and-read and enzyme-free amplified assay for antibiotics based on a dual triple helix-aptamer probe, potentially applicable for on-site monitoring of antibiotic residues. A dual triple helix-aptamer probe can leverage the response toward target molecules without enzyme-based amplification, rendering it sensitive and robust for profiling target molecules. The proposed assay allowed mix-and-read detection of chloramphenicol with a detection limit of 0.18 nM. Besides, it accommodated for specifically resolving chloramphenicol among other antibiotics. Chloramphenicol residual in aquatic products in fish and milk can be precisely determined. Thus, the aptamer probe deems to enrich the toolbox for managing antibiotic use.

Ratiometric-enhanced G-Quadruplex Probes for Amplified and Mix-to-Read Detection of Mercury Pollution in Aquatic Products.

Mercury, as a global toxic pollutant, is easy to be accumulated in aquatic products and poses a great threat to human health. In this work, we proposed a mix-to-read, label-free, and robust assay for detecting mercury pollution in aquatic products by engineering a ratiometric-enhanced G-quadruplex probe. The transformation from the G-quadruplex to a hairpin-like structure allows us to confer a ratiometric and leveraged response to Hg2+, amplifying the signal-to-background ratio for Hg2+ detection. Hg2+ response was further improved by screening parallel- and antiparallel-, single-, and multiple-stranded G-quadruplex structures. Compared to the common aptamer probes, the ratiometric-enhanced G-quadruplex probe increased the sensitivity for Hg2+ detection by 4.7 times. This proposed sensing system allowed a simple and one-tube homogenous detection of Hg2+ at room temperature using a single unlabeled DNA sequence. Its application for Hg2+ detection in fish and shrimp conferred satisfactory recovery rates ranging from 98.5 to 105.9%. The label-free and mix-to-read assay is promising for the onsite detection of mercury pollution and facilitating food safety of aquatic products.