[DNA assembly by multi-fragment digestion/ligation and homologous recombination].

To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and Bsa I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into Escherichia coli. After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.

Transplantation of gut microbiota derived from patients with schizophrenia induces schizophrenia-like behaviors and dysregulated brain transcript response in mice.

Schizophrenia (SCZ), as a neurodevelopmental disorder and devastating disease, affects approximately 1% of the world population. Although numerous studies have attempted to elucidate the causes of SCZ occurrence, it is not clearly understood. Recently, the emerging roles of the gut microbiota in a range of brain disorders, including SCZ, have attracted much attention. While the molecular mechanism of gut microbiota in regulating the pathogenesis of SCZ is still lacking. Here, we first confirmed the difference of gut microbiome between SCZ patients and healthy controls, and then, we performed fecal microbiota transplantation (FMT) to clarify the roles of SCZ patients-derived microbiota in a specific pathogen free (SPF) mice model. 16 S rDNA sequencing confirmed that a significant difference of gut microbiome was present between two groups of FMT mice, which has a similar trend with the above human gut microbiome. Furthermore, we found that transplantation of fecal microbiota from SCZ patients into SPF mice was sufficient to induce schizophrenia-like (SCZ-like) symptoms, such as deficits in sociability and hyperactivity. Furthermore, the brains of mice colonized with SCZ microbiota displayed dysregulated transcript response and alternative splicing of SCZ-relevant genes. Moreover, 10 key genes were identified to be correlated with SCZ by an integrative transcriptome data analysis. Finally, 4 key genes were identified to be correlated with the 12 differential genera between two groups of FMT mice. Our results thus demonstrated that the gut microbiome might modify the transcriptomic profile in the brain, thereby modulating social behavior, and our present study can help better understand the link between gut microbiota and SCZ pathogenesis through the gut-brain axis.

A gas pressure and colorimetric signal dual-mode strategy for sensitive detection of spermine using ssDNA-coated Au@Pt nanoparticles as the probe.

The naturally occurring spermine (Spm), as one of the many cationic polyamines, plays a key role in biological processes and is involved in a variety of diseases. The very similar structures among biogenic polyamines present a major challenge to achieve discriminative testing among them. In this contribution, using arbitrary ssDNA-coated Au@PtNPs as the probe, we demonstrated that a dual-mode strategy via a gas pressure and colorimetric signal readout can be used for the sensitive and specific detection of Spm, due to the target-responsive aggregation of the Au@PtNPs leading to the inhibition of the catalyzed gas-generation reaction and the colorimetric change of the Au@PtNP solution. The proposed pressure-based signaling strategy has a detection limit of 9.6 nM, and can be used not only in the laboratory but also in the point-of-care setting. Meanwhile, the colorimetric assay displays the advantage of being easily discerned with the naked eye. Compared with the traditional methods of chromatography and capillary electrophoresis combined with chemical derivatization, the strategy described here would provide a convenient new alternative for the specific detection of Spm in biological samples.

A cost-effective and rapid quantitative test of polymyxin B sulphate with a simple and portable pressure meter readout.

Any signals, if their intensities have a simple functional relationship with analyte concentration, can be applied for analytical purposes. Among them, pressure measurement as a signalling technique has been extensively utilized to develop portable and quantitative bioanalysis, with the advantages of highly sensitive detection of a variety of biomedical targets. In this contribution, it was found that polymyxin B sulphate (PMB) could significantly inhibit the catalytic ability of platinum nanoparticles (PtNPs) prepared in different protocols in the H2O2 breakdown reaction. By employing sodium citrate as the reducing agent to prepare the platinum nanoparticles (C-PtNPs) as an example and monitoring the pressure signal changes of the C-PtNP catalyzed H2O2 breakdown against the reaction time, a sensitive, cost-effective, rapid and reliable analysis method for PMB is established on the basis of the pressure signal readout. The proposed method has a detection limit of 28.6 nM and can selectively detect PMB in both POLY-MxB powder injection and human urine samples, demonstrating its potential in bioanalysis, which would be significant to address biological, clinical and medicinal requirements.

Point-of-Care Testing of Pathogenic Bacteria at the Single-Colony Level via Gas Pressure Readout Using Aptamer-Coated Magnetic CuFe2O4 and Vancomycin-Capped Platinum Nanoparticles.

Pressure measurements are performed everyday with simple devices, and in the field of analytical chemistry the pressure-based signaling strategy offers two important advantages, signal amplification and particular applicability in point-of-care settings. Herein, by using vancomycin (Van)-functionalized platinum nanoparticles (PtNPs@Van) and aptamer-coated magnetic CuFe2O4 nanoprobes dual-recognition units integrated with a catalyzed breakdown of H2O2 for O2 generation, we demonstrated that gas pressure can be used as a readout means for highly sensitive pathogenic bacteria identification and quantification. Using Staphylococcus aureus ( S. aureus) as a test case, integration of the molecular dual-recognition component with the catalyzed gas-generation reaction leads to a significant pressure change (Δ P), and the correlation between the concentration of S. aureus and the Δ P signal was found to be linear from 5.0 to 1.0 × 104 cfu/mL with a detection limit of 1.0 cfu/mL. Other nontarget bacteria show negative results, verifying the high specificity of the present strategy. When employed to assay S. aureus in saliva and milk samples, the approach shows recoveries from 93.3% to 107.1% with relative standard derivation (RSD) less than 8.8%. By the integration of catalyzed gas-generation reaction with the designed molecular recognition event, obviously the pressure-based signaling strategy could facilitate pathogenic bacteria identification and quantification not only in the laboratory but also in point-of-care settings, which could have great potential in the application of food safety and infectious disease diagnosis.

A photoluminescence "switch-on" nanosensor composed of nitrogen and sulphur co-doped carbon dots and gold nanoparticles for discriminative detection of glutathione.

Biological thiols play a key role in biological processes and are involved in a variety of diseases. The discriminative detection of biological thiols is still challenging. In this contribution, a platform, an energy transfer-based quenching system composed of nitrogen and sulphur co-doped carbon dots (N, S-CDs) and gold nanoparticles (AuNPs), was established to discriminate glutathione (GSH) from other competitive biothiols including cysteine (Cys) and homocysteine (Hcy) based on a photoluminescence (PL) "switch-on" signal readout. The presence of GSH can encapsulate AuNPs in priority because of the strong affinity towards AuNPs and the steric hindrance effect of GSH, leaving little chance for the N, S-CDs binding on the surface of AuNPs and thus resulting in the PL recovery of N, S-CDs. Compared with the nitrogen-doped carbon dots (N-CDs), the N, S-CDs can enhance 10 times sensitivity for the designed PL "switch-on" sensing strategy. The proposed method has a detection limit of 3.6 nM and can be successfully applied for the detection of GSH in human serum.