Bidirectional motivated bimodal isothermal strand displacement amplifier with a table tennis-like movement for the ultrasensitive fluorescent and colorimetric detection of depression-related microRNA.

An increasing number of studies have highlighted the potential of microRNAs (miRNAs) as physiological indicators of major depressive disorder (MDD). Herein, we developed a bidirectional-motivated bimodal isothermal strand displacement amplifier (BB-ISDA) for the ultrasensitive fluorescent and colorimetric detection of MDD-related miRNA-132. In the BB-ISDA system, a pair of functionalized hairpin probes (HP1 and HP2) with nicking recognition sites are designed to recognize target miRNA. The recognition of target miRNA by HP1 (or HP2) generates copious numbers of nicked triggers by HP1 (or HP2)-based ISDA to recognize HP2 (or HP1) by autonomous strand polymerization, cleavage, and displacement, which in turn induces the subsequent generation of copious numbers of nicked G-quadruplex triggers by HP2 (or HP1)-based ISDA to recognize HP1 (or HP2) along a same line. After many cycles, this bidirectional motivated table-tennis-like movement amplifies the fluorescent signal from HP1 and the colorimetric signal from HP2, simultaneously. The dual-signal output pattern was cross-validated for sensing miRNA-132. Each of the detection modal shows the capability for qualitative and quantitative detection of miRNA-132 with high sensitivity and specificity. The adaptability of the bimodal mechanism was verified via the detection of target miRNA-132 from clinical human blood samples. We envision that this BB-ISDA with dual-signal output for accurate and reliable analysis of miRNA is promising for the molecular diagnosis of human mental diseases.

Identification of a 43-kDa outer membrane protein of Fusobacterium necrophorum that exhibits similarity with pore-forming proteins of other Fusobacterium species.

A pair of primers was designed in an attempt to amplify outer membrane protein (OMP) gene of Fusobacterium necrophorum based on nucleotide sequence of the OMP of Fusobacterium nucleatum. Further analysis was performed to characterize its molecular properties and phylogeny in the genus Fusobacterium. We identified a predicated 43kDa outer membrane protein (43K OMP) in F. necrophorum, which showed the same properties as other pore-forming proteins of Gram-negative anaerobic bacteria according to analysis of signal peptide, AT-rich, membrane-spanning region and conserved motifs. The predicated 43K OMP exhibited 70.22%, 62.04%, 56.75%, 58.72%, 51.59%, 31.49% and 50.26% amino acid identity with the OMPs of F. nucleatum, Fusobacterium varium, Fusobacterium ucerans, Fusobacterium periodonticum, Fusobacterium mortiferum, Fusobacterium gonidiaformans and F. necrophorum (hypothetical protein), respectively. 11 common conserved domains and 10 common variable domains were found among the 45 aligned OMPs of Fusobacterium species. Distributions of the conserved and variable domains were highly associated with predicted membrane-spanning regions, cell surface exposed regions and B-cell epitope regions. Phylogenetic analysis revealed the predicated 43K OMP of F. necrophorum was closely related with the OMPs from F. nucleatum and F. periodonticum. These data will increase understanding of pathogenesis and genetic evolution of F. necrophorum.

Metal-binding activity of the soluble recombinant pig metallothionein 1A expressed in Escherichia coli.

Full-length cDNA for the pig metallothionein 1A (pMT1A) gene was synthesized based on the pig MT1A gene sequence in Genbank and cloned into the pMD18-T vector. After sequence analysis and structure prediction, the pMT1A gene was cloned into vector pET-32a (+) containing a His-tag. The recombinant pMT1A (rpMT1A) was expressed in a soluble form using Escherichia coli Rosetta™ (DE3) plysS cells. Western blotting showed that the purified rpMT1A protein bound an anti-His-tag monoclonal antibody. Further investigation revealed that the rpMT1A protein showed high metal-binding activity with the divalent metal ions copper (Cu²âº), zinc (Zn²âº), and cadmium (Cd²âº).