Automatic Microfluidic Harmonized RAA-CRISPR Diagnostic System for Rapid and Accurate Identification of Bacterial Respiratory Tract Infections.

Respiratory tract infections (RTIs) pose a grave threat to human health, with bacterial pathogens being the primary culprits behind severe illness and mortality. In response to the pressing issue, we developed a centrifugal microfluidic chip integrated with a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats (CRISPR) system to achieve rapid detection of respiratory pathogens. The limitations of conventional two-step CRISPR-mediated systems were effectively addressed by employing the all-in-one RAA-CRISPR detection method, thereby enhancing the accuracy and sensitivity of bacterial detection. Moreover, the integration of a centrifugal microfluidic chip led to reduced sample consumption and significantly improved the detection throughput, enabling the simultaneous detection of multiple respiratory pathogens. Furthermore, the incorporation of Chelex-100 in the sample pretreatment enabled a sample-to-answer capability. This pivotal addition facilitated the deployment of the system in real clinical sample testing, enabling the accurate detection of 12 common respiratory bacteria within a set of 60 clinical samples. The system offers rapid and reliable results that are crucial for clinical diagnosis, enabling healthcare professionals to administer timely and accurate treatment interventions to patients.

Screening and Application of DNA Aptamers for Heparin-Binding Protein.

Rapid detection of heparin-binding protein (HBP) is essential for timely intervention in sepsis cases. Current detection techniques are usually antibody-based immunological methods, which have certain problems, such as complexity and slow detection, and fall short in meeting the urgency of clinical needs. The application of an aptamer can address these concerns well. In this study, HBP-specific DNA aptamers were screened first. Among which, Apt-01, Apt-02, and Apt-13 had a high affinity for HBP, exhibiting impressive KD values of 3.42, 1.44, and 1.04 nmol/L, respectively. Then, the aptamer of HBP and its partially complementary primer probe were combined to form double-stranded DNA (dsDNA) and synthesize a circular DNA template. The template is complementary to the primer probe, but due to the presence of dsDNA, ExoIII cleaves C2-13 as an RCA primer probe, rendering the template unable to recognize the primer probe and preventing the RCA reaction from proceeding. When the target is present, it competes with the adapter for recognition and releases C2-13, exposing its 3' end. After initiating the RCA at room temperature and reacting with SYBR GreenII at 37 °C for 20 min, fluorescence changes can be observed and quantitatively analyzed at a 530 nm wavelength, achieving quantitative biological analysis. Apt-01 was used to develop a fluorescent biosensor for HBP detection, which exhibited a good linear range (0.01 nmol/L to 10 nmol/L) and detection limit (0.0056 nmol/L). This advancement holds the potential to lay a solid groundwork for pioneering sensitive and specific methods for HBP detection and to significantly enhance the diagnostic processes for sepsis.

High-throughput identification of meat ingredients in adulterated foods based on centrifugal integrated purification-CRISPR array.

Rapid, accurate, and sensitive analytical methods for the detection of food fraud are now an urgent requirement in the global food industry to ensure food quality. In response to this demand, a centrifugal integrated purification-CRISPR array for meat adulteration (CIPAM) was established. In detail, CIPAM system combines microneedles for DNA extraction and RAA-CRISPR/Cas12a integrated into a centrifugal microfluidic chip for the detection of meat adulteration. The RAA-CRISPR/Cas12a reaction reagents were pre-embedded into the different reaction chambers on the microfluidic chip to achieve the streamline of operations, markedly simplifying the detection process. The whole reaction was completed within 30 min with a detection limit of 0.1 % (w/w) in pig, chicken, duck, and lamb products. Referring to the results of the standard method, CIPAM system achieved 100 % accuracy. The automatic multiplex detection process implemented in the developed CIPAM system met the needs of food regulatory authorities.

How Much Good I Can Afford.

This essay describes the author's experience balancing the different necessities in medical practice, in particular writing perfect patient notes vs establishing a face-to-face connection.

Immunomagnetic Separation Combined with RCA-CRISPR/Cas12a for the Detection of Salmonella typhimurium on a Figure-Actuated Microfluidic Biosensor.

A figure-actuated microfluidic biosensor was developed for the rapid and sensitive detection of Salmonella typhimurium using immunomagnetic separation to separate target bacteria and rolling circle amplification (RCA) combined with CRISPR/Cas12a to amplify the detection signal. The magnetic nanoparticles (MNPs) modified with the capture antibodies (MNPs@Ab1) and RCA primer linked with recognized antibodies (primer@Ab2) were first used to react with S. typhimurium, resulting in the formation of MNPs@Ab1-S. typhimurium-primer@Ab2 complexes. Then, the RCA and CRISPR/Cas12a reagents were successively pumped into the chamber and incubated at the appropriate conditions. With the help of a 3D-printed signal detector, the fluorescence signal was collected and analyzed using the smartphone APP for the determination of bacterial concentration. This biosensor exhibited a wide linear range for the detection of S. typhimurium with a low limit of detection of 1.93 × 102 CFU/mL and a mean recovery of about 106% in the spiked milk sample.

Microfluidic Biosensor Integrated with Signal Transduction and Enhancement Mechanism for Ultrasensitive Noncompetitive Assay of Multiple Mycotoxins.

To achieve high-throughput ultrasensitive detection of mycotoxins in food, a functional DNA-guided transition-state CRISPR/Cas12a microfluidic biosensor (named FTMB) was successfully constructed. The signal transduction CRISPR/Cas12a strategy in FTMB has utilized DNA sequences with a specific recognition function and activators to form trigger switches. Meanwhile, the transition-state CRISPR/Cas12a system was constructed by adjusting the composition ratio of crRNA and activator to achieve a high response for low concentrations of target mycotoxins. On the other hand, the signal enhancement of FTMB has efficiently integrated the signal output of quantum dots (QDs) with the fluorescence enhancement effect of photonic crystals (PCs). The construction of universal QDs for the CRISPR/Cas12a system and PC films matching the photonic bandgap produced a significant signal enhancement by a factor of 45.6. Overall, FTMB exhibited a wide analytic range (10-5-101 ng·mL-1), low detection of limit (fg·mL-1), short detection period (∼40 min), high specificity, good precision (coefficients of variation <5%), and satisfactory practical sample analysis capacity (the consistency with HPLC at 88.76%-109.99%). It would provide a new and reliable solution for the rapid detection of multiple small molecules in the fields of clinical diagnosis and food safety.

Synthesizing Submicron Polyelectrolyte Capsules to Boost Enzyme Immobilization and Enhance Enzyme-Based Immunoassays.

Polyelectrolyte capsules (PCs) exhibit attractive superiorities in enzyme immobilization, including providing a capacious microenvironment for enzyme conformational freedom, highly effective mass transfer, and protecting enzymes from the external environment. Herein, we provide the first systemic evaluation of submicron PCs (SPCs, 500 nm) for enzyme immobilization. The catalytic kinetics results show that SPC encapsulation affected the affinities of enzymes and substrates but significantly enhanced their catalytic activity. The stability test indicates that SPC-encapsulated horseradish peroxidase (HRP) exhibits ultrahigh resistance to external harsh conditions and has a longer storage life than that of soluble HRP. The proposed encapsulation strategy enables 7.73-, 2.22-, and 11.66-fold relative activities when working at a pH as low as 3, at a NaCl concentration as high as 500 mM, and at a trypsin concentration as high as 10 mg/mL. We find that SPC encapsulation accelerates the cascade reaction efficiency of HRP and glucose oxidase. Owing to SPCs enhancing the catalytic activity of the loaded enzymes, we established an amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Escherichia coli O157:H7 using HRP-loaded SPCs. The detection sensitivity of SPC-improved ELISA was found to be 280 times greater than that of conventional HRP-based ELISA. Altogether, we provide an elaborate evaluation of 500 nm SPCs on enzyme immobilization and its application in the ultrasensitive detection of foodborne pathogens. This evaluation provides evidence to reveal the potential advantage of SPCs on enzyme immobilization for enzyme-based immunoassays. It has excellent biological activity and strong stability and broadens the application prospect in urine, soy sauce, sewage, and other special samples.

Green One-Pot Syntheses of 2-Sulfoximidoyl-3,6-dibromo Indoles Using N-Br Sulfoximines as Both Brominating and Sulfoximinating Reagents.

A green one-pot 2,3,6-trifunctionalization of N-alkyl/aryl indoles was achieved by adding three equivalents of N-Br sulfoximine to the indole solution. A variety of 2-sulfoximidoyl-3,6-dibromo indoles were prepared with 38-94% yields using N-Br sulfoximines as both brominating and sulfoximinating reagents. Based on the results of controlled experiments, we propose that a radical substitution involving 3,6-dibromination and 2-sulfoximination occurs in the reaction process. This is first time that 2,3,6-trifunctionalization of indole in one pot has been achieved.

Investigation of pectolytic and PR genes expression, quality and phytochemical contents in organic and non-organic table grapes at harvest and during storage.

The demand for organic table grapes is increasing worldwide. However, comprehensive information of quality parameters and phytochemical compounds in organically grown fruit remain unclear. Furthermore, table grapes are perishable and postharvest quality retention and waste prevention is very important. In this study we have compared the differences between organic and non-organic table grapes in terms of phytochemical compounds and quality parameters as well as the changes in the expression levels of pathogen related and lytic genes during storage. Organic fruit showed higher levels of phenolics, flavonoids, caffeic acid, hydrogen peroxide, protein content, antioxidant and anti-stress enzymes and total antioxidant activities at harvest and during storage. Although, the expression levels of polygalactronases, pectin methyl esterase, chitinase and glucanase genes was lower in organically grown table grapes at harvest, but the expression of all these genes was significantly increased during cold storage. After 60 days of cold storage the expression levels of pectin methyl esterase, chitinase and glucanase genes was significantly higher than the conventionally grown grape berries in organic ones. The highest expression of polygalacturonase was recorded in organic samples after 30 days of storage. There was no significant difference between the two types of table grapes for decay extension and tissue deterioration rate. The results of this study indicate that due to higher levels of phytochemicals and antioxidant compounds the organic table grapes have a higher nutritional quality. Furthermore, the increase in PR and pectolytic genes expression levels is enough for decreasing the fruit susceptibility to decay pathogens and enhancing the postharvest life of organic grapes.

Untargeted metabolomics reveals the combination effects and mechanisms of Huangqi-fuzi herb-pair against doxorubicin-induced cardiotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Qifu decoction (QFD) is a famous traditional Chinese medicine (TCM) composed of Astragali Radix (HuangQi) and Aconiti Lateralis Radix Praeparaia (Fuzi), which can alleviate doxorubicin (DOX)-induced cardiotoxicity (DIC). However, its protective mechanism remains obscured. AIM OF THE STUDY: The present study aimed to uncover the cardioprotective mechanism and the synergistic effect of QFD against DIC in mice. MATERIALS AND METHODS: The cardioprotective activity of QFD against DIC was assessed by electrocardiogram, serum biochemical assays and histopathology. Mass spectrometry-based metabolomic approach was conducted to elucidate the preventive mechanisms of QFD, HuangQi decoction (HQD), and Fuzi decoction (FZD) against DIC. QFD, HQD, FZD-targeted metabolic pathways were identified and compared to investigate the synergistic mechanism of QFD by computational systems analysis. Quantitative real-time PCR (qRT-PCR) was further employed to validate the key metabolic pathways at the level of the gene. RESULTS: The electrocardiogram combined with the biochemical analysis and histopathology showed that the protection effects were sorted as QFD > HQD ≈ FZD. A total of 41 metabolites contributing to DIC were identified in the mice serum, among which 32, 12 and 10 metabolites were significantly reverted by QFD, HQD and FZD, respectively. Metabolic pathway analysis revealed that DOX perturbed 12 metabolic pathways, and QFD, HQD, and FZD-treated groups could significantly reverse 12, 7 and 6 metabolic pathways of these 12 metabolic pathways. Metabolic pathway and qRT-PCR revealed that QFD could protect DIC mainly by regulating energy metabolism, amino acids metabolism, arachidonic acid metabolism and glycerophospholipid metabolism, and HQD and FZD mutually reinforced each other. CONCLUSION: These evidences revealed that QFD was a promising drug candidate for DIC by maintaining metabolic homeostasis. Meanwhile, this work provided a useful approach for evaluating the efficacy and the synergistic effects of TCMs against cardiomyopathy.

Rapid quantitative detection of Vibrio parahaemolyticus via high-fidelity target-based microfluidic identification.

With the rapid development of logistics, a growing number of pathogenic microorganisms has the means to spread worldwide using food as a carrier; thus, there is an urgent need to develop effective detection strategies to ensure food safety. By combining novel markers identified by pan-genome analysis and a digital recombinase-aided amplification (RAA) detection method based on a microfluidic chip, a strategy of high-fidelity target-based microfluidic identification (HFTMI) has been developed. Herein, a proof-of-concept study of HFTMI for rapid pathogen detection of V. parahaemolyticus was investigated. Specific primers designed for the gene group_41170 identified in the pan-genome analysis showed high sensitivity and a broad spectrum for the detection of V. parahaemolyticus. Different power systems were investigated to increase the partition rate on specifically designed chamber-based digital chips. The performance of HFTMI was greatly improved compared with qPCR. Collectively, this novel HFTMI system provides more reliable guidance for food safety testing.

Pseudomonas aeruginosa Detection Using Conventional PCR and Quantitative Real-Time PCR Based on Species-Specific Novel Gene Targets Identified by Pangenome Analysis.

Mining novel specific molecular targets and establishing efficient identification methods are significant for detecting Pseudomonas aeruginosa, which can enable P. aeruginosa tracing in food and water. Pangenome analysis was used to analyze the whole genomic sequences of 2017 strains (including 1,000 P. aeruginosa strains and 1,017 other common foodborne pathogen strains) downloaded from gene databases to obtain novel species-specific genes, yielding a total of 11 such genes. Four novel target genes, UCBPP-PA14_00095, UCBPP-PA14_03237, UCBPP-PA14_04976, and UCBPP-PA14_03627, were selected for use, which had 100% coverage in the target strain and were not present in nontarget bacteria. PCR primers (PA1, PA2, PA3, and PA4) and qPCR primers (PA12, PA13, PA14, and PA15) were designed based on these target genes to establish detection methods. For the PCR primer set, the minimum detection limit for DNA was 65.4 fg/µl, which was observed for primer set PA2 of the UCBPP-PA14_03237 gene. The detection limit in pure culture without pre-enrichment was 105 colony-forming units (CFU)/ml for primer set PA1, 103 CFU/ml for primer set PA2, and 104 CFU/ml for primer set PA3 and primer set PA4. Then, qPCR standard curves were established based on the novel species-specific targets. The standard curves showed perfect linear correlations, with R 2 values of 0.9901 for primer set PA12, 0.9915 for primer set PA13, 0.9924 for primer set PA14, and 0.9935 for primer set PA15. The minimum detection limit of the real-time PCR (qPCR) assay was 102 CFU/ml for pure cultures of P. aeruginosa. Compared with the endpoint PCR and traditional culture methods, the qPCR assay was more sensitive by one or two orders of magnitude. The feasibility of these methods was satisfactory in terms of sensitivity, specificity, and efficiency after evaluating 29 ready-to-eat vegetable samples and was almost consistent with that of the national standard detection method. The developed assays can be applied for rapid screening and detection of pathogenic P. aeruginosa, providing accurate results to inform effective monitoring measures in order to improve microbiological safety.

Controlled PAH-mediated method with enhanced optical properties for simple, stable immunochromatographic assays.

Despite their potential for signal amplification in immunochromatographic assays (ICAs) with Au nanoparticles (AuNPs) as probes, metal growth methods are of limited practical applicability given their complex non-specificity and lack of robust growth schemes. Here, we propose a novel method of polyallylamine hydrochloride (PAH)-mediated metal growth for the detection of Escherichia coli O157:H7 by AuNP-ICA. The developed method relies on the highly controlled growth of Cu shells on the AuNP core and allows one to achieve highly enhanced colorimetric signals by controlling PAH as the growth framework. The introduction of PAH eliminates the non-specific adsorption of Cu ions on the nitrocellulose membrane and thus provides maximized and effective signal-to-noise ratios to achieve a detection limit of 9.8 CFU/mL for E. coli O157:H7. Moreover, the newly developed detection method exhibits good reproducibility (coefficient of variation <13%), remarkable stability, and practical applicability. The PAH-mediated signal enhancement system paves the way to the realization of stable metal growth methods based on Au, Ag, and other metals and is well suited for the rapid, stable, and sensitive detection of food-borne pathogens using the AuNP-ICA platform.

Novel multiplex PCR assays for rapid identification of Salmonella serogroups B, C1, C2, D, E, S. enteritidis, and S. typhimurium.

Foodborne illnesses caused by Salmonella represent a significant public health problem worldwide. The aim of this study was to establish multiplex PCR (mPCR) for the rapid identification of Salmonella serogroups B, C1, C2, D, and E as well as for the serovars enteritidis and typhimurium. Employing pan-genome analysis and PCR verification, B-rfbJ, C1-9679, C2-pimB, D-rfbJ, E-rfbC, and four genes (SE18636, SE16574, SE2599, and SE13329) were identified as specific target genes for Salmonella serogroups B, C1, C2, D, E, and S. enteritidis, respectively. Thereafter, three novel mPCR assays (one of 3-mPCR and two of 2-mPCR) were successfully developed to identify these bacteria based on the target genes and another S. typhimurium-specific STM4495 gene. The primers targeting C1-9679, C2-pimB, and E-rfbC genes specific to the serogroups C1, C2, and E, respectively, constituted a 3-mPCR, while the other two 2-mPCRs, respectively, consisting primers specific to serogroup D and S. enteritidis (D-rfbJ and SE16574), and serogroup B and S. typhimurium-specific primers (B-rfbJ and STM4495), were also designed. The specificity of each mPCR was further evaluated by using non-target strains. The detection limits of mPCRs were approximately 103-104 CFU mL-1 in pure culture and 104-105 CFU g-1 in spiked chicken meat. In addition, mPCR assays could correctly detect target Salmonella in food samples. These results suggest that specific targets could be mined efficiently through a pan-genome analysis tool, and the novel mPCR assays developed in this study offer a promising technique for rapid and accurate detection of five serogroups of Salmonella (B, C1, C2, D, and E) and two serovars (S. enteritidis and S. typhimurium).

A microfluidic genoserotyping strategy for fast and objective identification of common Salmonella serotypes isolated from retail food samples in China.

Strict monitoring of Salmonella serotypes is the most effective way to limit the risk of its transmission to humans. In this study, a microfluidic genoserotyping strategy was developed for the rapid and cost-effective detection of 11 common Salmonella serotypes from retail food samples, i.e. Typhimurium, Derby, Indiana, Agona, Rissen, Braenderup, Hadar, Enteritidis, Weltevreden, Meleagridis, and Pomona. The programmable LAMP in the strategy can complete the whole detection process within 40 min and avoid false positive results. The limit of detection was 102 or 103 CFU/mL. Referring to the results of standard culture method, the LAMP-chip was verified with 100% accuracy on testing 688 Salmonella and 22 non-Salmonella strains. This simple, portable and accurate microfluidic genoserotyping strategy is fully adapted to the needs of food regulatory authorities and has wide application prospect in food safety field.

Anything But Typical.

A graduating child neurology resident reflects upon how her first neurology "patient" single-handedly taught her an entire textbook worth of knowledge and became the guiding force that led her to leave general pediatrics.

Ictal Hypersalivation and Salivary Gland Enlargement in a Patient With Acquired Frontal Lobe Epilepsy.

Hypersalivation is a well-known ictal semiology of benign Rolandic epilepsy and other childhood epilepsy syndromes. There are also occasional reports of adults with temporal, parietal, or frontal lobe epilepsy in which hypersalivation is a prominent seizure manifestation. Notably lacking are reports linking salivary gland enlargement to ictal hypersalivation. A 33-year-old man with frontal lobe epilepsy due to a ruptured aneurysm presented with focal seizures and facial swelling. The only seizures he had in the past were generalized tonic-clonic seizures. Eight days prior to admission, he started having focal seizures characterized by pronounced hypersalivation, speech arrest, impaired awareness, and left upper extremity posturing or automatism. Seizure frequency increased from five to 30 per day. Four days prior to admission, his face started to swell up, and his family thought he had mumps. Computed tomography (CT) of the head showed encephalomalacia in the inferomedial cortex of the right frontal lobe, the same lesion seen in his old CT images. Maxillofacial CT revealed enlargement of the parotid and submandibular glands. Although electroencephalography (EEG) showed seizure onset in the right frontal region, the initial ictal discharge on the scalp may represent seizure propagation from a focus near the zone of encephalomalacia. After seizure freedom was achieved with antiepileptic drugs, the patient's salivary glands decreased in size and returned to normal.

COVID-19 vaccination willingness among people with multiple sclerosis.

BACKGROUND: Hesitancy to receive COVID-19 vaccination is a major public health concern. COVID-19 vaccine willingness and the factors contributing to willingness in adults with multiple sclerosis (MS) is unknown. We administered an online survey from 1 December 2020 to 7 January 2021 to adults with MS to estimate COVID-19 vaccine willingness among adults with MS. Bivariate analysis with chi-square testing compared categorical variables associated with vaccine willingness. RESULTS: Of 401 respondents, 70.1% were willing to receive an authorized COVID-19 vaccination if it was available to them, 22.7% were unsure, and 7.2% were unwilling. The most frequent concern for those unsure was vaccine safety. Vaccine willingness was associated with increased perceived personal risk of COVID-19 (χ2 = 45.4; p < 0.0001), prior influenza vaccine acceptance (χ2 = 97.6; p < 0.0001), higher educational level (χ2 = 50.2; p < 0.0001), and if respondents discussed or planned to discuss the COVID-19 vaccine with their neurologists (χ2 = 64.3; p < 0.0001). CONCLUSION: While COVID-19 vaccination willingness is high among people with MS, nearly 30% were either unwilling or unsure about being vaccinated. Neurologists should be aware of patient-centered factors associated with COVID-19 vaccine willingness and address COVID-19 vaccine safety concerns in discussions with their vaccine-unsure MS patients.

Quantitative detection of aflatoxin B1 using quantum dots-based immunoassay in a recyclable gravity-driven microfluidic chip.

To achieve rapid and sensitive detection of aflatoxin B1 (AFB1), we developed a polydimethylsiloxane gravity-driven cyclic microfluidic chip using the two-signal mode strategy. The structural design of the chip, together with the two-wavelength quantum dot ratio fluorescence, effectively eliminates the influence of environmental factors, improves the signal stability, and ensures that the final detection result positively correlates with the target concentration. Moreover, the theoretical analysis performed for the established physical model of the three-dimensional reaction interface inside the chip confirmed the improved reaction rate of immune adsorption in the microfluidic strategy. Overall, the method exhibited a wide analytic range (0.2-500 ng mL-1), low detection limit (0.06 ng mL-1), high specificity, good precision (coefficient of variation < 5%), excellent reusability (20 times, 89.1%) and satisfactory practical sample analysis capacity. Furthermore, the reusability and designability of this chip provide a reliable scheme for field detection of AFB1, analysis of other small molecules, and establishment of high-throughput detection systems under different conditions.