Effect of biopolymer chitosan on manganese immobilization improvement by microbial­induced carbonate precipitation.

Microbially induced carbonate precipitation (MICP), as an eco-friendly and promising technology that can transform free metal ions into stable precipitation, has been extensively used in remediation of heavy metal contamination. However, its depressed efficiency of heavy metal elimination remains in question due to the inhibition effect of heavy metal toxicity on bacterial activity. In this work, an efficient, low-cost manganese (Mn) elimination strategy by coupling MICP with chitosan biopolymer as an additive with reduced treatment time was suggested, optimized, and implemented. The influences of chitosan at different concentrations (0.01, 0.05, 0.10, 0.15 and 0.30 %, w/v) on bacterial growth, enzyme activity, Mn removal efficiency and microstructure properties of the resulting precipitation were investigated. Results showed that Mn content was reduced by 94.5 % within 12 h with 0.15 % chitosan addition through adsorption and biomineralization as MnCO3 (at an initial Mn concentration of 3 mM), demonstrating a two-thirds decrease in remediation time compared to the chitosan-absent system, whereas maximum urease activity increased by ∼50 %. Microstructure analyses indicated that the mineralized precipitates were spherical-shaped MnCO3, and a smaller size and more uniform distribution of MnCO3 is obtained by the regulation of abundant amino and hydroxyl groups in chitosan. These results demonstrate that chitosan accelerates nucleation and tunes the growth of MnCO3 by providing nucleation sites for mineral formation and alleviating the toxicity of metal ions, which has the potential to upgrade MICP process in a sustainable and effective manner. This work provides a reference for further understanding of the biomineralization regulation mechanism, and gives a new perspective into the application of biopolymer-intensified strategies of MICP technology in heavy metal contamination.

A case report of Klebsiella aerogenes-caused lumbar spine infection identified by metagenome next-generation sequencing.

BACKGROUND: The early clinical diagnosis of spinal infections in elderly patients with recessive or atypical symptoms is difficult. Klebsiella aerogenes is a common opportunistic bacterium that can infect the respiratory tract, urinary tract, and even the central nervous system. However, whether it can infect the lumbar spine has not been previously described. CASE PRESENTATION: In this paper, we report the case of a 69-year-old female patient with osteoporosis who was initially diagnosed with hemolytic anemia. Later, she was diagnosed with K. aerogenes infection of the lumbar spine based on imaging combined with blood culture and metagenome next-generation sequencing (mNGS) detection. After precise medication, the lumbar degeneration was improved. CONCLUSIONS: Bacterial infection should therefore be considered in cases of lumbar degenerative disease in middle-aged and elderly patients.

A novel aptasensor based on DNA hydrogel for sensitive visual detection of ochratoxin A.

A novel visual detection mode is proposed to improve the detection sensitivity for the determination of ochratoxin A (OTA). The mode is based on aptamer recognition and the signal amplification of rolling circle amplification (RCA) products self-assembled DNA hydrogel. Moreover, gold nanoparticles (AuNPs) were directly assembled inside the DNA hydrogel by adjusting the padlock probe sequences to achieve a stronger binding force between the DNA hydrogel and AuNPs; this avoids the need for modification of AuNPs with DNA sequences. In the presence of OTA, DNA hydrogel is formed. With higher concentrations of OTA, a larger amount of DNA hydrogel is formed. When AuNPs are added to the DNA hydrogel, AuNPs can be enclosed inside the DNA hydrogel. With more DNA hydrogel, there is less AuNPs in the supernatant. Thus, the absorbance of the supernatant is anti-correlated with the concentration of OTA. After optimization of the experimental conditions, the change in the absorbance of the supernatant was linearly correlated with the concentration of OTA, in the range 0.05 to 10 ng/mL; the limit of detection was 0.005 ng/mL. The good specificity of the developed biosensor was confirmed in the presence of other mycotoxins that are coexistent with or analogues of OTA. By comparing the developed method with the ELISA method, the accuracy and stability of this new method were also verified, with good performance obtained in real samples. Diagram of the principle of the colorimetric aptasensor for OTA detection based on rolling circle amplification product self-assembled DNA hydrogel.

Nuclease-assisted target recycling signal amplification strategy for graphene quantum dot-based fluorescent detection of marine biotoxins.

Saxitoxin (STX) is a major marine toxin from shellfish, and it is responsible for paralytic shellfish poisoning (PSP). In this study, a highly sensitive and rapid aptamer assay was developed for STX detection by combining fluorescence resonance energy transfer (FRET) and nuclease-assisted target recycling signal amplification. The aptamer STX-41 conjugated with graphene quantum dots (GQDs) was adsorbed on magnetic reduced graphene oxide (MRGO) to establish a fluorescence quenching system. Then, the binding between STX and aptamer induced the desorption of GQD-aptamer from MRGO and the restoring of fluorescence for the fluorescent determination of STX. The digestion of the target bound aptamer by DNase I could release the target for recycling thus achieving signal amplification. Under the optimized conditions, the aptamer assay showed a wide detection range (0.1-100 ng·mL-1), low detection limit (LOD of 0.035 ng·mL-1), high specificity, good recovery (86.75-94.08% in STX-spiked clam samples) and repeatability (RSD of 4.27-7.34%). Combined with fluorescent detection technology, signal amplification technology, and magnetic separation technology, the proposed method can be used to detect STX in seafood products successfully.

Fluorometric determination of lipopolysaccharides via changes of the graphene oxide-enhanced fluorescence polarization caused by truncated aptamers.

A broad-spectrum ssDNA aptamer containing 80 nucleotides (LA80) and capable of binding to four different sources of lipopolysaccharides (LPSs) was truncated. Two strategies are used to produce truncated aptamers of different length. The results show that LA27, a 27-nt aptamer, retained broad-spectrum capability and has a higher affinity (Kd = 46.2 ± 9.5 nM). A graphene oxide based fluorescence polarization assay (excitation/emission wavelengths: 485/520 nm) was worked out using FAM-labeled LA27. It can detect LPSs from Salmonella entericaserotype typhimurium, Pseudomonas aeruginosa 10 and Escherichia coli 055:B5 with enhanced performance (4.8 to 29-fold improvements) compared to LA80. The assay can be performed within 30 min, and the detection limits are 38.7, 88.0 and 154 ng·mL-1, respectively. Graphical abstract Schematic presentation of the assay: A shorter aptamer, with higher affinity than its original aptamer, was obtained by truncated strategies. This core aptamer lead to release easily and enhance the sensivity of the GO-based fluorescence polarization assay.

Advances in aptasensors for the detection of food contaminants.

Food safety is a global health objective, and foodborne diseases represent a major crisis in health. Techniques that are simple and suitable for fast screening to detect and identify pathogenic factors in the food chain are vital to ensure food safety. At present, a variety of analytical methods have been reported for the detection of pathogenic agents. Whereas the sensitivity of detection and quantification are still important challenges, we expect major advances from new assay formats and synthetic bio-recognition elements, such as aptamers. Owing to the specific folding capability of aptamers in the presence of an analyte, aptasensors have substantially and successfully been exploited for the detection of a wide range of small and large molecules (e.g., toxins, antibiotics, heavy metals, bacteria, viruses) at very low concentrations. Here, we review the use of aptasensors for the development of highly sensitive and affordable detection tools for food analysis.

Metagenomic next-generation sequencing confirmed a case of sporadic human infection with Streptococcus suis in an urban area.

INTRODUCTION: Streptococcus suis (S. suis) disease is a zoonotic infection caused by invasive S. suis and can lead to meningitis, septic shock, arthritis, and endocarditis. Early treatment is the key to reducing mortality. However, clinical manifestations of most cases are atypical, severely limiting rapid diagnosis and treatment. CASE REPORT: Here, we report a 74-year-old female patient diagnosed with S. suis infection. The main symptoms were hearing loss, lumbago, and scattered ecchymosis of the lower extremities and trunk. Blood non-specific infection indexes were significantly increased and platelets were significantly decreased; however, no pathogens were obtained from routine blood culture. Finally, the S. suis infection was confirmed by metagenomic next-generation sequencing (mNGS) of blood and cerebrospinal fluid. After antibiotic treatment, the limb and trunk scattered ecchymosis and lumbago symptoms were significantly relieved, but the hearing did not recover. CONCLUSIONS: Human infection with S. suis is rare in central cities, and it is easy to misdiagnose, especially in cases with atypical early symptoms. mNGS technology, combined with clinical observation, is helpful to clarify the direction of diagnosis and treatment, which is conducive to patient recovery.

Fluorescence Resonance Energy Transfer Aptasensor of Ochratoxin A Constructed Based on Gold Nanorods and DNA Tetrahedrons.

Ochratoxin A (OTA) contamination of corn has received significant attention due to the wide distribution and high toxicity of OTA. The maximum residue limit standard of OTA in corn has been established by the Chinese Government and other unions. Nanoparticle-based fluorescence resonance energy transfer (FRET) assays are promising methods for the sensitive and fast detection of OTA. However, satisfactory detection sensitivity is commonly achieved with complicated signal amplification processes or specific nanoparticle morphologies, which means that these assays are not conducive to fast detection. This study proposes a simple and novel strategy to improve the sensitivity of FRET aptasensors. In this strategy, a DNA tetrahedron was first used in gold nanorod-based FRET aptasensors. DNA tetrahedron-modified gold nanorods are used as fluorescent acceptors, and Cy5-modified complementary sequences of the OTA aptamer are used as fluorescent donors. The aptamers of OTA are embedded in the DNA tetrahedrons, and FRET occurs when the aptamers hybridize with the Cy5-modified complementary sequences. The aptamer-integrated DNA tetrahedron modified on the surface of gold nanorods acts as an anchor, thus avoiding the crowding and entanglement of aptamers. Due to the competitive combination between the OTA aptamers and complementary sequences, the greater the amount of OTA, the less the amount of Cy5-modified complementary sequences that bind with the aptamers and the less the amount of Cy5 that is quenched. Thus, the fluorescence intensity is positively related to the OTA concentration. In this study, in the concentration range of 0.01-10 ng/mL, the fluorescence intensity was found to be linearly related to the logarithmic concentration of OTA. The limit of detection was calculated to be 0.005 ng/mL. The specificity of the developed biosensor was demonstrated to be efficient. The accuracy and stability of the developed aptasensor were also tested, and the method exhibited good performance in real samples.

Focal adhesion kinase promotes progression and predicts poor clinical outcomes in patients with osteosarcoma.

Osteosarcoma (OS) is a fatal form of musculoskeletal tumor that commonly leads to pulmonary metastatic disease. Traditional therapies such as surgery and chemotherapy are not effective treatment modalities in certain patients with OS; therefore, identifying the molecular mechanism of OS is imperative for the development of novel therapeutics. Previous studies have reported that focal adhesion kinase (FAK) is associated with numerous types of human malignancies. Therefore, in order to investigate the biological function of FAK in OS, the present study examined the expression levels of FAK in OS cell lines, OS tissues and paired normal tissue specimens by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). FAK expression in vitro was blocked using small interfering RNA (siRNA) to observe the invasion, proliferation and apoptosis trends of OS cells. Phosphoinositide-dependent kinase-1 (PDK1), AKT and BRAF protein levels were also evaluated by western blotting to analyze the effects of FAK depletion on the AKT and mitogen-activated protein kinase (MAPK) signaling pathways. A significantly reduced level of FAK mRNA was identified in paired normal tissues compared with OS tissues and cell lines. The invasive capability and proliferative potential of OS cells were suppressed due to the transient in vitro transfection of FAK siRNA. It was also demonstrated that decreased FAK expression facilitated the apoptosis of OS cells, as demonstrated by flow cytometric and western blotting analyses. Decreased FAK expression resulted in the downregulation of phosphorylated (p)-AKT, p-PDK1 and p-BRAF protein levels. Higher FAK expression levels are positively associated with clinicopathological characteristics of advanced Enneking stages (P<0.001) and recurrence (P=0.041) in patients with OS. Collectively, these data demonstrated that FAK is an important diagnostic biomarker for OS, and FAK siRNA therapy may be a potentially promising approach for the treatment of OS.

Magnetic Separation-Based Multiple SELEX for Effectively Selecting Aptamers against Saxitoxin, Domoic Acid, and Tetrodotoxin.

In this study, a novel magnetic separation-based multiple systematic evolution of ligands by exponential enrichment (SELEX) was applied to select aptamers simultaneously against three kinds of marine biotoxins, including domoic acid (DA), saxitoxin (STX), and tetrodotoxin (TTX). Magnetic reduced graphene oxide (MRGO) was prepared to adsorb unbound ssDNAs and simplify the separation step. In the multiple SELEX, after the initial twelve rounds of selection against mixed targets and the subsequent four respective rounds of selection against each single target, the three resulting ssDNA pools were cloned, sequenced, and analyzed. Several aptamer candidates were selected and subjected to the binding affinity and specificity test. Finally, DA-06 ( Kd = 62.07 ± 19.97 nM), TTX-07 ( Kd = 44.12 ± 15.38 nM), and STX-41 ( Kd = 61.44 ± 23.18 nM) showed high affinity and good specificity for DA, TTX, and STX, respectively. They were also applied to detect and quantify DA, TTX, and STX successfully. The other two multitarget aptamers, DA-01 and TTX-27, were also obtained, which can bind with either DA or TTX. These aptamers provide alternative recognition molecules to antibodies for biosensor applications.

An enhanced chemiluminescence resonance energy transfer aptasensor based on rolling circle amplification and WS2 nanosheet for Staphylococcus aureus detection.

A chemiluminescence resonance energy transfer aptasensor was fabricated for the detection of Staphylococcus aureus (S. aureus) with Co2+ enhanced N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functional flowerlike gold nanoparticles (Co2+/ABEI-AuNFs) as donor and WS2 nanosheet as acceptor. In the presence of S. aureus, rolling circle amplification (RCA) can be started. Partially complementary sequence of RCA product functional ABEI-AuNFs (cDNA-ABEI-AuNFs) were then annealed to multiple sites of the RCA product to form duplex complex. This complex is less adsorbed onto the WS2 nanosheet, thus attenuating the quenching of ABEI-AuNFs chemiluminescence by WS2 nanosheet. In the absence of target S. aureus (and hence the absence of RCA and duplex formation), the free cDNA-ABEI-AuNFs is completely adsorbed onto the WS2 nanosheet and chemiluminescence quenching ensues. Under optimal conditions, the logarithmic correlation between the concentration of S. aureus and the CL signal was found to be linear within the range of 50 cfu/mL to 1.5 × 105 cfu/mL (R2 = 0.9913). The limits of detection of the developed method were found to be 15 cfu/mL for S. aureus. The selectivity and the capability of the biosensor in meat samples were also studied. Therefore, this simple and easy operation method can be used to detect S. aureus with high sensitivity and specificity.

A chemiluminescent aptasensor based on rolling circle amplification and Co2+/N-(aminobutyl)-N-(ethylisoluminol) functional flowerlike gold nanoparticles for Salmonella typhimurium detection.

A sensitive steady-state chemiluminescent aptasensor based on rolling circle amplification (RCA) was fabricated for the detection of Salmonella typhimurium. The sensor utilized aptamer modified Fe3O4 magnetic nanoparticles (MNPs) as capture probes, aptamer as recognition molecules, and Co2+ enhanced N­(aminobutyl)-N-(ethylisoluminol) (ABEI) functional flowerlike gold nanoparticles (AuNFs) and complementary strand (cDNA) complex (Co2+/ABEI-AuNFs-cDNA) as signal probes. And P-Iodophenol (PIP) was also added to form a typical ABEI- AuNFs-PIP-H2O2 steady-state CL system. By virtue of Fe3O4 MNPs based solid-phase RCA strategy, S. typhimurium can be first captured by the aptamer immobilized on the surface of Fe3O4 MNPs then complex with RCA products to form a sandwich complex. Co2+/ABEI-AuNFs-cDNA signal probes were then assembled on the RCA products to produce and enhance CL signals. Under optimal conditions, the logarithmic correlation between the concentration of S. typhimurium and the CL signal was found to be linear within the range of 32cfumL-1 to 3.2×106cfumL-1 (R2 =0.9921). The limits of detection of the developed method were found to be 10cfumL-1 for S. typhimurium. The method was also used to detect S. typhimurium in real pork samples. The results were compared with those obtained from the plate-counting methods and showed good consistency. Therefore, this detection aptasnesor can be a good candidate for sensitive and selective detection of S. typhimurium with simple and effective operations.

Oxidative stress and spermatogenesis suppression in the testis of cadmium-treated Bombyx mori larvae.

Bombyx mori L. (B. mori) were exposed to cadmium chloride (CdCl2) incorporated in an artificial diet (0, 6.25, 12.5, 25, and 50 mg kg(-1)) throughout the larval stage. Changes in malondialdehyde (MDA) and reduced glutathione (GSH) contents and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), as well as their corresponding messenger RNA (mRNA) levels in the testes of the fifth instar larvae were evaluated. Additionally, spermatozoon deformation in the testes was examined. Upon Cd treatment, the MDA content in the testes was significantly increased in a concentration-dependent manner. Cd-exposed larvae had increased levels of glutathione. Pearson's correlation analysis revealed that SOD and CAT activities were positively correlated (R (2) = 0.605, P = 0.017). The changing trends in the mRNA levels of these enzymes were not always consistent with those of enzymatic activities. Alterations in GSH-Px activities and mRNA levels were positively correlated (R (2) = 0.771, P < 0.01). Morphological analysis revealed that Cd deformed and affected the maturation of spermatozoa. Our results collectively support a relationship between Cd and alterations in the levels of antioxidant enzymes in B. mori testes.

Graphene oxide-assisted non-immobilized SELEX of okdaic acid aptamer and the analytical application of aptasensor.

Okadaic acid (OA) is a low-molecular-weight marine toxin from shellfish that causes abdominal pain, vomiting and diarrhea, i.e., diarrheic shellfish poisoning. In this study, a ssDNA aptamer that specifically binds to OA with high affinity was obtained via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assisted by graphene oxide (GO). This aptamer was then applied to fabricate a novel direct competitive enzyme-linked aptamer assay (ELAA). At the optimized conditions, this ELAA method showed a low detection limit (LOD of 0.01 ng/mL), wide linear range (from 0.025 to 10 ng/mL), good recovery rate (92.86-103.34% in OA-spiked clam samples) and repeatability (RSD of 2.28-4.53%). The proposed method can be used to detect OA in seafood products with high sensitivity and can potentially be adapted for the determination of other small molecular analytes.

A Review of the Methods for Detection of Staphylococcus aureus Enterotoxins.

Food safety has attracted extensive attention around the world, and food-borne diseases have become one of the major threats to health. Staphylococcus aureus is a major food-borne pathogen worldwide and a frequent contaminant of foodstuffs. Staphylococcal enterotoxins (SEs) produced by some S. aureus strains will lead to staphylococcal food poisoning (SFP) outbreaks. The most common symptoms caused by ingestion of SEs within food are nausea, vomiting, diarrhea and cramps. Children will suffer SFP by ingesting as little as 100 ng of SEs, and only a few micrograms of SEs are enough to cause SPF in vulnerable populations. Therefore, it is a great challenge and of urgent need to detect and identify SEs rapidly and accurately for governmental and non-governmental agencies, including the military, public health departments, and health care facilities. Herein, an overview of SE detection has been provided through a comprehensive literature survey.