A label-free ratiometric homogeneous electrochemical aptasensor based on dual catalytic hairpin self-assembly for rapid and sensitive detection of ochratoxin A in food.

The food contamination with Ochratoxin A (OTA) has highlighted the need to create precise, sensitive, and convenient techniques. Herein, we proposed a label-free and immobilization-free ratiometric homogeneous electrochemical aptasensor based on dual catalytic hairpin self-assembly (CHA) for OTA detection. Methylene blue (MB) and ferrocene (Fc) in solution were utilized as label-free signaling molecules, generating a response signal (IMB) and a reference signal (IFc), respectively. The ratio of IMB/IFc was utilized as a measure to quantify OTA. Dual CHA was exploited to increase the ratiometric signal and enhance the amplification efficiency. This aptasensor achieved trace-level detection for OTA over a linear range of lower concentrations (1.0 × 10-3 ng/mL-1.0 × 103 ng/mL) with LOD of 92 fg/mL. The aptasensor was successfully applied to detect OTA in cereal and wine, with comparable results of HPLC-MS/MS. This strategy provided a viable platform for rapid, sensitive, and accurate detection of OTA in food.

A high molecular weight polymalate is synthesized by the whole genome duplicated strain Aureobasidium melanogenum OUC.

Polymalate (PMA) produced by the whole genome duplicated strain Aureobasidium melanogenum OUC had a high molecular weight (Mw) of 3.9 × 105 Da while the Mw of PMA produced by A. melanogenum ATCC62921 was 3.8 × 104 Da. Therefore, the purified PMA produced by A. melanogenum OUC could form hydrogel and film and the precipitated Ca2+-PMA looked like noodle whereas the purified PMA produced by A. melanogenum ATCC62921 could not form such a hydrogel and a film and the precipitated PMA was powder-like. The high Mw PMA biosynthesis in A. melanogenum OUC was also controlled by the PMA synthetase. However, it was still unclear why the PMA synthetase in A. melanogenum OUC could catalyze the high Mw PMA biosynthesis. Both removal of two copies of the PKS genes and overexpression of the PYC1 gene, the VGB gene and the CRZ2 gene rendered the new transformant Crz46 to produce 34.6 ± 0.3 g/L of extracellular Ca2+-PMA with Mw of 4.9 × 105 Da while its native A. melanogenum OUC only produced 17.2 ± 0.3 g/L of Ca2+-PMA. During the 10-Liter fermentation, 35.6 ± 1.2 g/L of Ca2+-PMA and 13.9 g/Lof cell mass were produced within 168 h, leading to the yield of 0.36 g/g of glucose and the productivity of 0.21 g/L/h. This was the first time to report that the whole genome duplicated strain A. melanogenum OUC and its engineered mutants could produce the high Mw PMA.

Polymalate (PMA) biosynthesis and its molecular regulation in Aureobasidium spp.

It has been well documented that different strains of Aureobasidium spp. can synthesize and secrete over 30.0 g/L of polymalate (PMA) and the produced PMA has many potential applications in biomaterial, medical and food industries. The substrates for PMA biosynthesis include glucose, xylose, fructose, sucrose and glucose or fructose or xylose or sucrose-containing natural materials from industrial and agricultural wastes. Malate, the only monomer for PMA biosynthesis mainly comes from TCA cycle, cytosolic reduction TCA pathway and the glyoxylate cycle. The PMA synthetase (a NRPS) containing A like domain, T domain and C like domain is responsible for polymerization of malate into PMA molecules by formation of ester bonds between malates. PMA biosynthesis is regulated by the transcriptional activator Crz1 from Ca2+ signaling pathway, the GATA-type transcription factor Gat1 from nitrogen catabolite repression and the GATA-type transcription factor NsdD.

The differences between fungal α-glucan synthase determining pullulan synthesis and that controlling cell wall α-1,3 glucan synthesis.

The fungal α-glucan synthases (Agss) are multi-domain proteins catalyzing biosynthesis of cell wall α-1,3-glucan which determines cell wall integrity or fungal pathogenicity and pullulan which is a maltotriosyl polymer made of α-1,4 and α-1,6 bound glucose units. The Agss family can be divided into 11 groups, some of which lost the original functions due to accumulation of harmful mutations or gene loss. Schizosaccharomyces pombe kept five kinds of Agss in the genome while Aspergillus spp. and Penicillium spp. lost one or two or three kinds of Agss. All the human, animal and plant pathogens kept only one single kind of Ags or only one active Ags for synthesis of cell wall α-1,3-glucan, a virulence factor. While the genus Aureobasidium spp. contained three kinds of Agss, of which only some of the Ags2 was involved in pullulan biosynthesis. Although many Agss contained Big_5 domain, only the Big_5 domain with conserved amino acids LQS from some strains of A. melanogenum could catalyze pullulan biosynthesis. This whole amino acid sequence and phylogenetic differences may cause non-α-1,3-glucan synthesizing activity of some fungal Agss.

A novel PMA synthetase is the key enzyme for polymalate biosynthesis and its gene is regulated by a calcium signaling pathway in Aureobasidium melanogenum ATCC62921.

It has been well known that poly(ß-l-malic acid)(PMA) has many potential applications. However, it is still completely unknown how PMA is biosynthesized in Aureobasidium spp. In this study, it was found that malic acid from TCA cycle was the main source for PMA biosynthesis. Especially, the novel PMA synthetase encoded by the PMAs gene, a non-ribosomal peptide synthetase (NRPS) containing A like domain, T domain and C like domain was the key enzyme for polymerization of malate into PMA. Therefore, abolishment of the PMAs gene encoding the novel PMA synthetase rendered the mutant ΔPMAs-3 totally to lose the ability to synthesize any PMA and complementation of the PMAs gene partially restored PMA biosynthesis, but the mutant could grow normally on the YPD plate and in the PMA medium with CaCO3. The transcriptional activator Crz1 in the Ca2+-signal pathway controlled expression of the PMAs gene and PMA biosynthesis. The complete elucidation of the PMA biosynthesis pathway and its regulation was of significant for a deeper understanding of detailed yeast-like fungal PMA synthesis, metabolic engineering and molecular editing for modifying PMA biosynthesis and its physicochemical properties.

[Characteristic analysis of acute leukemia immunophenotyping in 180 cases by flow cytometry].

To study the acute Leukemia immunophenotype and its diagnosis value, three color direct immunofluorescece staining methods of flow cytometry and immunophenotype of antibody integration system were used for detection of 180 cases of acute leukemia. The results showed that patients with ALL expressed lymphocyte antigen, and 49.4% patients with ALL accompanied myeloid antigen; all patients with AML expressed myeloid antigen, and 43.2% patients with AML accompanied lymphocyte antigen. In conclusion, leukemia immunophenotyping by three-color direct immunofluore staining methods could define some particular types of leukemia with an important value in diagnosis, treatment and predicting prognosis of acute leukemia.