Bacterial Flagellum-Drug Nanoconjugates for Carrier-Free Immunochemotherapy.

The combination of immunotherapy and chemotherapy to ablate tumors has attracted substantial attention due to the ability to simultaneously elicit antitumor immune responses and trigger direct tumor cell death. However, conventional combinational strategies mainly focus on the employment of drug carriers to deliver immunomodulators, chemotherapeutics, or their combinations, always suffering from complicated preparation and carrier-relevant side effects. Here, the fabrication of bacterial flagellum-drug nanoconjugates (FDNCs) for carrier-free immunochemotherapy is described. FDNCs are simply prepared by attaching chemotherapeutics to amine residues of flagellin through an acid-sensitive and traceless cis-aconityl linker. By virtue of native nanofibrous structure and immunogenicity, bacterial flagella not only show long-term tumor retention and highly efficient cell internalization, but also provoke robust systemic antitumor immune responses. Meanwhile, conjugated chemotherapeutics exhibit an acid-mediated release profile and durable intratumoral exposure, which can induce potent tumor cell inhibition via direct killing. More importantly, this combination is able to augment immunoactivation effects associated with chemotherapy-enabled immunogenic tumor cell death to further enhance antitumor efficacy. By leveraging the innate response of the immune system to pathogens, the conjugation of therapeutic agents with self-adjuvant bacterial flagella provides an alternative approach to develop carrier-free nanotherapeutics for tumor immunochemotherapy.

The association between polyunsaturated fatty acids and periodontitis: NHANES 2011-2014 and Mendelian randomisation analysis.

BACKGROUND: We aimed to explore the association and potential causality between polyunsaturated fatty acids concentrations and the risk of periodontal disease. MATERIALS AND METHODS: Data were collected from the 2011-2014 National Health and Nutrition Examination Survey (NHANES). Weighted logistic regression analysis and restricted cubic spline (RCS) analysis were used to analyse the associations of the concentrations of omega-3 and omega-6 fatty acids and the omega-6/omega-3 fatty acids ratio with the risk of periodontitis. E-value and propensity score matching (PSM) analyses were used for sensitivity analyses. In addition, two-sample Mendelian randomisation (MR) analyses were performed to assess the potential causal impact of the concentrations of those fatty acids on periodontitis risk. RESULTS: A total of 2462 participants from the NHANES were included. Logistic regression analysis revealed that high omega-3 fatty acids levels were negatively associated with the risk of developing periodontitis (P < 0.05), while the omega-6/omega-3 fatty acids ratio was positively associated with the risk of developing periodontitis (P < 0.05). There was no significant association between omega-6 concentrations and the risk of periodontitis. The findings mentioned above were confirmed by analysis following a 1:1 PSM. Furthermore, MR examination of the two samples indicated no possible causal link between the risk of periodontitis and the concentrations of omega-3 or omega-6 fatty acids or the ratio of omega-6 to omega-3 fatty acids (P > 0.05). CONCLUSION: Although omega-3 fatty acids and the omega-6/omega-3 fatty acids ratio were associated with the risk of periodontitis in cross-sectional studies, the MR results did not support a causal relationship between them. Therefore, there is no indication that an increase in the omega-3 fatty acids concentration or a decrease in the omega-6/omega-3 fatty acids ratio may be beneficial for preventing periodontitis.

Using whole exome sequencing to identify susceptibility genes associated with nonsyndromic cleft lip with or without cleft palate.

Cleft lip and palate is a common congenital birth defect in humans. Its incidence rate in China is as high as 1.82%, and is now a frequent deformity observed among the Chinese population; moreover, it varies across regions. Although the etiology of nonsyndromic cleft lip with or without cleft palate (NSCL/P) has been widely investigated, the results are inconsistent. The specific genes and mechanisms responsible for NSCL/P have not been fully understood. Whole exome sequencing (WES) is a new strategy for studying pathogenic genes. WES studies on NSCL/P have not been conducted in East China. Therefore, the aim of this study was to screen candidate genes of NSCL/P in East China using WES and analyze the temporal and spatial expressions of the candidate genes during embryonic palatal development. WES was performed in 30 children with NSCL/P from East China to screen candidate genes. A bioinformatics analysis was performed using commercially available software. Variants detected by WES were validated by immunohistochemistry and western blotting. After WES, 506,144 single-nucleotide variant sites were found. The results of database comparison, functional analysis, and mass spectrometry revealed that only the laminin alpha 5 (LAMA5) gene (site: rs145192286) was associated with NSCL/P. Immunohistochemistry results showed that LAMA5 expression in the medial edge epithelium changed with formation, lifting, and contact during palatogenesis. Almost no LAMA5 expression was detected in the palatal mesenchyme or after palatal fusion. Western blotting and immunohistochemistry results showed consistent trends. In conclusion, the WES results shows that the mutation at the site (rs145192286) of LAMA5 is associated with NSCL/P. The temporal and spatial expressions of LAMA5 during palatal development further demonstrate the involvement of this gene. Therefore, we speculate that LAMA5 is a new candidate pathogenic gene of NSCL/P. The identification of new pathogenic genes would help elucidate the pathogenesis of NSCL/P and provide a scientific basis for the prenatal diagnosis, prevention, and treatment of NSCL/P.

Local Electric Field Induced by Atomic-Level Donor-Acceptor Couple of O Vacancies and Mn Atoms Enables Efficient Hybrid Capacitive Deionization.

Transition metal oxides suffer from slow salt removal rate (SRR) due to inferior ions diffusion ability in hybrid capacitive deionization (HCDI). Local electric field (LEF) can efficiently improve the ions diffusion kinetics in thin electrodes for electrochemical energy storage. Nevertheless, it is still a challenge to facilitate the ions diffusion in bulk electrodes with high loading mass for HCDI. Herein, this work delicately constructs a LEF via engineering atomic-level donor (O vacancies)-acceptor (Mn atoms) couples, which significantly facilitates the ions diffusion and then enables a high-performance HCDI. The LEF boosts an extended accelerated ions diffusion channel at the particle surface and interparticle space, resulting in both remarkably enhanced SRR and salt removal capacity. Convincingly, the theoretical calculations demonstrate that electron-enriched Mn atoms center coupled with an electron-depleted O vacancies center is formed due to the electron back-donation from O vacancies to adjacent Mn centers. The resulted LEF efficiently reduce the ions diffusion energy barrier. This work sheds light on the effect of atomic-level LEF on improving ions diffusion kinetics at high loading mass application and paves the way for the design of transition metal oxides toward high-performance HCDI applications.

Internal Electric Field Induced by Superexchange Interaction on Mn4+ -O2- -Ni2+ Unit Enables Highly Efficient Hybrid Capacitive Deionization.

Internal electric field (IEF) construction is an innovative strategy to regulate the electronic structure of electrode materials to promote charge transfer processes. Despite the wide use of IEF in various applications, the underlying mechanism of its formation in an asymmetric TM-O-TM unit still remains poorly understood. Herein, the essential principles for the IEF construction at electron occupancy state level and explore its effect on hybrid capacitive deionization (HCDI) performance is systematically investigated. By triggering a charge separation in Ni-MnO2 via superexchange interactions in a coordination structure unit of Mn4+ -O2- -Ni2+ , the formation of an IEF that can enhance charge transfer during the HCDI process is demonstrated. Experimental and theoretical results confirm the electrons transfer from O 2p orbital to TM (Ni2+ and Mn4+ ) eg orbital via superexchange interactions in the basic Mn4+ -O2- -Ni2+ coordination unit. As a result of the charge redistribution, the IEF endows Ni-MnO2 with superior electron and ion transfer property. This work presents a unique material design strategy that activates the electrochemical performance, and provides insights into the formation mechanism of IEF in an asymmetric TM-O-TM unit, which has potential applications in the construction of other innovative materials.

Effect of Plasticization on Stretching Stability of Poly(Vinyl Alcohol) Films: A Case Study Using Glycerol and Water.

Adding small molecular plasticizers is the most common route to tailor the stretchability of poly(vinyl alcohol) (PVA). However, how the plasticization along with the nature of the plasticizer governs the structural homogeneity during stretching remains an open question to answer. Herein, two representative plasticizers, glycerol (GLY) and water, are chosen to endow the PVA films with ductility. It is found that large strain cavitations cause obvious stress whitening in the PVA/H2 O films; on the contrary, most of the PVA/GLY films maintain transparent undergoing tensile deformation. Through a combination of experimental inspections and molecular dynamic simulation, it is revealed that partial water molecules that behave as free water will aggregate into microdomains, which serve as mechanical defects responsible for yielding voids. Whereas, the GLY plasticizer homogeneously disperses at a molecular level and interacts with PVA chains through strong hydrogen bonds. More interestingly, it is illustrated that the dispersion and bound states of plasticizers are closely related to the mechanical character of the plasticized PVA films. These findings offer new insight into the working mechanism of plasticization on the structural stability during stretching, and guide the design of PVA/plasticizer system to obtain excellent comprehensive mechanics.

p38MAPK silencing attenuates scar proliferation after cleft palate repair surgery in rats via MRTF-A/SRF pathway.

Scarring is the primary factor of maxilla growth restriction among people who have undergone cleft palate repair surgery. p38 mitogen-activated protein kinase (p38MAPK) promotes fibrosis in a variety of organs. However, its role in post-surgery scarring on the hard palate has not been fully understood. This study is designed to investigate the role of p38MAPK in scar formation and maxilla growth of rats. We removed the mucosa on the hard palate of rats and applied the p38MAPK silencing adenovirus vector on it two weeks after surgery. Then the scarring tissue and maxilla growth were evaluated by histological and morphological examination. The effect of p38MAPK silencing on scarring-related genes in fibroblasts was also studied. We found that local injection of Ad-p38MAPK-1 in vivo effectively reduces the expression of p38MAPK and scarring-related proteins and weakens the impact of scarring on the width of the hard palate. Mechanistically, p38MAPK silencing inhibits the expression of α-smooth muscle actin (α-SMA) via mediating the production and nuclear localization of myocardin-related transcription factor A (MRTF-A) in fibroblasts. These results reveal a molecular pathway of scar formation involving p38MAPK/MRTF-A stimulation and support targeting p38MAPK as a potentially effective treatment for post-surgery scarring on the hard palate.

Ceramide d18:1/24:1 as a potential biomarker to differentiate obesity subtypes with unfavorable health outcomes.

BACKGROUND: The criteria for metabolically healthy obesity (MHO) and metabolically unhealthy obesity (MUO) remain controversial. This research aimed to identify a potential biomarker to differentiate the subtypes of obesity. METHODS: The study conducted a lipidomic evaluation of ceramide in the serum of 77 Chinese adults who had undergone hyperinsulinemic-euglycemic clamps. These adults were divided into three groups according to the clinical data: normal weight control group (N = 21), MHO (N = 20), and MUO (N = 36). RESULTS: The serum Cer d18:1/24:1 level in the MHO group was lower than that in the MUO group. As the Cer d18:1/24:1 level increased, insulin sensitivity decreased, and the unfavorable parameters increased in parallel. Multivariate logistic regression analysis revealed that serum Cer d18:1/24:1 levels were independently correlated with MUO in obesity. Individuals with higher levels of Cer d18:1/24:1 also had an elevated risk of cardiovascular disease. Most ceramide subtype levels increased in obesity compared to normal-weight individuals, but the levels of serum Cer d18:0/18:0 and Cer d18:1/16:0 decreased in obesity. CONCLUSIONS: The relationships between ceramide subtypes and metabolic profiles might be heterogeneous in populations with different body weights. Cer d18:1/24:1 could be a biomarker that can be used to differentiate MUO from MHO, and to better predict who will develop unfavorable health outcomes among obese individuals. TRIAL REGISTRATION: The First Affiliated Hospital of Nanjing Medical University's Institutional Review Board authorized this study protocol, and all participants provided written informed consent (2014-SR-003) prior to study entry.

Hydrogen-bond-dominated mechanical stretchability in PVA films: from phenomenological to numerical insights.

Hydrogen bonds (H-bonds) in poly(vinyl alcohol) (PVA) play a crucial role in macroscopic mechanical properties, particularly for stretchability. However, there is still some ambiguity about the quantitative dependence of H-bond interactions on the mechanical performance, mainly attributed to the difficulty in the discrimination of various H-bond types. Herein, small molecular chemicals as plasticizers were incorporated into the PVA matrix to tailor the H-bonding interactions. By altering the PVA molecular weight, plasticizer type and loading, both the stretchability and H-bond content were regulated on a large scale. By a combination of DMA, IR spectroscopy, MD simulation and solid-state 13C-NMR, every sort of H-bond in PVA was assigned, and their relative fractions were ascertained quantitatively. After correlating the elongation ratio with the relative fraction of the different types of H-bonding interaction, it was found that all the pairs of elongation vs. intermolecular H-bond content derived from different series of PVA/plasticizer films could be plotted into a master curve and exhibited good linearity, indicating that intermolecular H-bonds dominate the mechanical stretchability in PVA films. Our efforts contribute towards an in-depth understanding of performance optimization induced by H-bond manipulation from empirical, phenomenological aspects to intrinsic, numerical insights.

Rare Diseases in Glycosphingolipid Metabolism.

Sphingolipidoses is a cluster of genetic rare disorders regarding glycosphingolipid metabolism, classified as lysosomal storage disorders (LSD). Here, we focus on eight inheritable diseases, including GM1 gangliosidosis, GM2 gangliosidosis, Fabry disease, Gaucher's disease, metachromatic leukodystrophy, Krabbe disease, Niemann-Pick disease A and B, and Farber disease. Mostly, pathogenic mutations in the key enzyme are loss-function, resulting in accumulation of substrates and deficiency of products. Thus, cellular overload of substrates causes lipotoxicity, which is deleterious to cellular and organ function. In the terms of clinical manifestations in sphingolipidoses, multiple systems and organs, especially central nervous system (CNS) are usually affected. As for diagnosis strategy, enzymatic activity assay and genetic sequencing are helpful. Up till now, limited treatment approaches have approved for treating sphingolipidoses, with some potential strategies for further evaluation. In general, enzyme replacement therapy (ERT), substrate reduction therapy (SRT), and molecular chaperones are feasible choices for enzyme deficiency disorders, but these therapies are limited to relieve CNS lesions and symptoms due to prevention from blood-brain barrier. Other possible treatments such as gene therapy, bone marrow transplantation (BMT), and hematopoietic stem cell transplantation (HSCT) need further evaluation.

Establishment and operation of glucose clamp technique in mice.

Glucose metabolism plays a central role in energy supply and metabolism regulation in various tissues and organs. Besides, insulin is the sole hormone lowering blood glucose in the body, and islet function and insulin sensitivity are the key steps modulating glucose metabolism. Since the development of glucose clamp technology, it has been recognized as the gold standard for evaluating insulin metabolism. The main categories include hyperinsulinemia-euglycemia clamp, hyperglycemia clamp, and hyperinsulinemia-hypoglycemia clamp. These can be done on either anesthetized mice or conscious and unrestricted mice. This protocol focuses on the establishment and operation of the mouse glucose clamp technique, including preparation of instrument consumables, surgical operations, clamping procedures, and precautions, serving as reference and guidance.

A study of congenital generalized lipodystrophy (CGL) caused by BSCL2 gene mutation.

Congenital generalized lipodystrophy (CGL) is an extremely rare genetic disease mainly characterized by absence of whole-body adipose tissue and metabolic dysfunctions such as insulin resistance, diabetes mellitus, hypertriglyceridemia, hepatic steatosis, and acanthosis nigricans. In this study, we reported a novel case of a young woman patient with CGL. The patient came to the hospital for early-onset lipodystrophy and diabetes. She was 19-year-old with a height of 160 cm, a weight of 46 kg, BMI of 17.9 kg/m2, and a serum leptin level of 0.14 µg/L. Genomic DNA was extracted from blood samples of the patient and her family members, including her mother, father and brother. Genetic analysis revealed compound heterozygous mutations of the BSCL2 gene (c.560A>G and c.565G>T) in the patient. Her father carried a heterozygous mutation (c.565G>T), and her mother carried a heterozygous mutation (c.560A>G) in the BSCL2 gene. The mutant p.Y187C plasmid was transfected into HEK293T cells. The protein expression of SEIPIN and its interaction with glycerol-3-phosphate acyltransferase (GPAT3) were observed to be reduced. In addition, based on primary cultured skin fibroblasts from the patient, SEIPIN protein was decreased, and lipid droplets were much smaller when fatty acid was stimulated compared with those observed from healthy subject controls. However, histone deacetylase inhibitors (HDACis) was found capable of rescuing SEIPIN protein in fibroblasts of the patient. In addition, we further summarized and discussed gene mutations of BSCL2 reported in the current literature. Collectively, these findings have expanded the clinical phenotype and pathogenic gene spectrum of CGL, which might help clinicians to achieve better management of lipodystrophy.

Diagnosis and genetic analysis of a case with glycogen storage disease type V caused by compound heterozygous mutations in the PYGM gene.

Glycogen storage disease type V is an autosomal recessive genetic disorder caused by muscle glycogen phosphorylase (PYGM) deficiency, which is characterized by exercise intolerance, second wind phenomena and high level of serum creatine kinase. In this study, we reported a Chinese young man with glycogen storage disease type V, with lower extremity weakness after exercise, increased creatine kinase, and slight fat infiltration in the posterior group of thigh muscle by magnetic resonance imaging (MRI). The proband had complex heterozygous PYGM disease-causing mutations, including c.308T>C (p.L103P) variant transmitted from the mother and c.260_261delCT (p.S87Ffs*23) from the father, of which the former was a novel PYGM mutation. This study enriched the PYGM pathogenic gene mutation spectrum, contributed to improve clinicians' understanding of glycogen storage disease type V and provided a reference for further genetic study of the disease.

Decorating Bacteria with Triple Immune Nanoactivators Generates Tumor-Resident Living Immunotherapeutics.

An approach of decorating bacteria with triple immune nanoactivators is reported to develop tumor-resident living immunotherapeutics. Under cytocompatible conditions, tumor-specific antigens and checkpoint blocking antibodies are simultaneously conjugated onto bacterial surface and then polydopamine nanoparticles are formed via in situ dopamine polymerization. In addition to serving as a linker, polydopamine with its photothermal effect can repolarize tumor-associated macrophages to a pro-inflammatory phenotype. The linked antigens promote the maturation of dendritic cells and generate tumor-specific immune responses, while the anchored antibodies block immune checkpoints and activate cytotoxic T lymphocytes. Decorated bacteria show spatiotemporal tumor retention and proliferation-dependent drug release, achieving potent antitumor effects in two antigen-overexpressing tumor models. This work provides a versatile platform to prepare multimodal and long-acting therapeutics for cancer immunotherapy.

Stabilization of Cu+ via Strong Electronic Interaction for Selective and Stable CO2 Electroreduction.

Copper oxide-based materials effectively electrocatalyze carbon dioxide reduction (CO2 RR). To comprehend their role and achieve high CO2 RR activity, Cu+ in copper oxides must be stabilized. As an electrocatalyst, Cu2 O nanoparticles were decorated with hexagonal boron nitride (h-BN) nanosheets to stabilize Cu+ . The C2 H4 /CO ratio increased 1.62-fold in the CO2 RR with Cu2 O-BN compared to that with Cu2 O. Experimental and theoretical studies confirmed strong electronic interactions between the two components in Cu2 O-BN, which strengthens the Cu-O bonds. Electrophilic h-BN receives partial electron density from Cu2 O, protecting the Cu-O bonds from electron attack during the CO2 RR and stabilizing the Cu+ species during long-term electrolysis. The well-retained Cu+ species enhanced the C2 product selectivity and improved the stability of Cu2 O-BN. This work offers new insight into the metal-valence-state-dependent selectivity of catalysts, enabling the design of advanced catalysts.

Virtual Surgical Planning and Three-Dimensional Printing to Aid the Anatomical Reduction of an Old Malunited Fracture of the Mandible.

ABSTRACT: Mandible fracture is a common injury in maxillofacial surgery. It causes not only maxillofacial dysfunction but also facial deformities. Malunited fractures of the mandible have been a vast challenge in clinical treatment due to the misalignment of the broken ends and the occurrence of occlusal disorders. This case report describes using virtual surgical planning and three-dimensional printing to treat a patient with malunited fracture of the mandible. Failing to perform mandibular surgery due to severe brain trauma after the car accident, the patient got malunited healing of mandible. The authors applied virtual surgical planning to perform preoperative analysis and surgical design on this patient, three-dimensional printing to fabricate occlusal plate, and models of the preoperative and postoperative mandible to guide the operation. Finally, the authors achieved the reduction and reconstruction of the mandible with satisfactory clinical results.

Preparation and characterization of magnetic molecular imprinted polymers with ionic liquid for the extraction of carbaryl in food.

Magnetic molecular imprinted polymers with ionic liquid used as an auxiliary solvent (IL@MMIPs) for the recognition of the methyl carbamate pesticide carbaryl (CBR) in foodstuff have been synthesized. The properties and application of IL@MMIPs were determined. The kinetic and isotherm adsorption processes were found to follow the pseudo-second-order and the Scatchard models, respectively. The selective experiment showed that the IL@MMIPs exhibited good selectivity to CBR compared to magnetic nonimprinted polymers with IL (IL@MNIPs). By using the IL@MMIPs as an adsorbent for the enrichment of CBR in food samples, the limit of detection (LOD, S/N = 3) and the limit of quantitation (LOQ, S/N = 10) of this method were 3 µg kg-1 and 10 µg kg-1, respectively. Compared with the traditional method, the IL@MMIP method has better recoveries (83.23-99.83%), precision (1.12-2.09%), and stabilization (intraday, 1.08-2.81%; interday, 2.26-3.30%). IL@MMIPs are an ideal adsorbent that could be applied to conveniently detect CBR in complex food, and the proposed method can be considered as a selective and sensitive alternative to traditional methods with affordable cost, avoiding the complex pretreatment procedure. Graphical abstract .

Negative pressure drainage-assisted irrigation for maxillofacial space infection.

OBJECTIVE: In this study, the clinical effect of negative pressure drainage-assisted irrigation (NPDI) technique was evaluated in treating maxillofacial space infection (MSI) by comparing with traditional technique. METHOD: A prospective study was conducted in 58 patients with MSI. The patients were randomly divided into two groups based on different treatment techniques. Thirty patients receiving NPDI were included in NPDI group, and 28 patients receiving traditional technique were included in traditional group. Case data (gender, age, etiology, concurrent illness, diabetes, involved spaces, preoperative white cell count, airway control method) and clinical effect (postoperative hospital stay, total cost of admission) for the two groups were analyzed. RESULTS: Patients in both groups were cured clinically. There were no significant differences in gender, age, etiology, concurrent illness, diabetes, involved spaces, preoperative white cell count, and airway control method in NPDI group and traditional group (p > .05). The postoperative hospital stay and the total cost of admission in the NPDI group were significantly lower than the traditional group (p < .001). CONCLUSION: Negative pressure drainage-assisted irrigation used in the treatment of MSI can shorten the postoperative hospital stay, reduce the total cost of admission, and show favorably clinical effect. It is a clinically recommended method for MSI.

Rare Diseases Related with Lipoprotein Metabolism.

Rare diseases are gathering increasing attention in last few years, not only for its effects on innovation scientific research, but also for its propounding influence on common diseases. One of the most famous milestones made by Michael Brown and Joseph Goldstein in metabolism field is the discovery of the defective gene in familial hypercholesterolemia, a rare human genetic disease manifested with extreme high level of serum cholesterol (Goldstein JL, Brown MS, Proc Natl Acad Sci USA 70:2804-2808, 1973; Brown MS, Dana SE, Goldstein JL, J Biol Chem 249:789-796, 1974). Follow-up work including decoding the gene function, mapping-related pathways, and screening therapeutic targets are all based on the primary finding (Goldstein JL, Brown MS Arterioscler Thromb Vasc Biol 29:431-438, 2009). A series of succession win the two brilliant scientists the 1985 Nobel Prize, and bring about statins widely used for lipid management and decreasing cardiovascular disease risks. Translating the clinical extreme phenotypes into laboratory bench work has turned out to be the first important step in the paradigm conducting translational and precise medical research. Here we review the main categories of rare disorders related with lipoprotein metabolism, aiming to strengthen the notion that human rare inheritable genetic diseases would be the window to know ourselves better, to treat someone more efficiently, and to lead a healthy life longer. Few rare diseases related with lipoprotein metabolism were clustered into six sections based on changes in lipid profile, namely, hyper- or hypocholesterolemia, hypo- or hyperalphalipoproteinemia, abetalipoproteinemia, hypobetalipoproteinemia, and sphingolipid metabolism diseases. Each section consists of a brief introduction, followed by a summary of well-known disease-causing genes in one table, and supplemented with one or two diseases as example for detailed description. Here we aimed to raise more attention on rare lipoprotein metabolism diseases, calling for more work from basic research and clinical trials.

Label-free and enzyme-free sensitive fluorescent method for detection of viable Escherichia coli O157:H7.

We have developed a label-free, enzyme-free, modification-free and DNA extraction-free fluorescent aptasensing (LEFA) method for detection of E. coli O157:H7 based on G-quadruplex formation using two ingeniously designed hairpin probes (GHP1 and GHP2). In the presence of E. coli O157:H7, it released the single stranded initiation sequence (IS) resulting in the toehold strand displacement between GHP1 and GHP2, which in turn led to the cyclic reuse of the production of DNA assemblies with numerous G-quadruplex structures and initiation sequences. Then these G-quadruplex structures can be recognized quickly by N-methyl mesoporphyrin IX (NMM) resulting in significantly enhanced fluorescence. The LEFA method was successfully implemented for detecting E. coli O157:H7 with a detection limit of 66 CFU/mL in pure culture, 10 CFU/mL and 1 CFU/mL after pre-incubation of the milk and tap water for 4 and 8 h, respectively. Moreover, the strategy could distinguish viable E. coli O157:H7 from dead E. coli O157:H7 and other species of pathogen cells. Furthermore, the whole process of the strategy is accomplished within 100 min. The results indicated that the approach may be used to effectively control potential microbial hazards in human health, food safety, and animal husbandry.