An roGFP2-Based Bacterial Bioreporter for Redox Sensing of Plant Surfaces.

The reduction-oxidation (redox) environment of the phytobiome (i.e., the plant-microbe interface) can strongly influence the outcome of the interaction between microbial pathogens, commensals, and their host. We describe a noninvasive method using a bacterial bioreporter that responds to reactive oxygen species and redox-active chemicals to compare microenvironments perceived by microbes during their initial encounter of the plant surface. A redox-sensitive variant of green fluorescent protein (roGFP2), responsive to changes in intracellular levels of reduced and oxidized glutathione, was expressed under the constitutive SP6 and fruR promoters in the epiphytic bacterium Pantoea eucalypti 299R (Pe299R/roGFP2). Analyses of Pe299R/roGFP2 cells by ratiometric fluorometry showed concentration-dependent responses to several redox active chemicals, including hydrogen peroxide (H2O2), dithiothreitol (DTT), and menadione. Changes in intracellular redox were detected within 5 min of addition of the chemical to Pe299R/roGFP2 cells, with approximate detection limits of 25 and 6 µM for oxidation by H2O2 and menadione, respectively, and 10 µM for reduction by DTT. Caffeic acid, chlorogenic acid, and ascorbic acid mitigated the H2O2-induced oxidation of the roGFP2 bioreporter. Aqueous washes of peach and rose flower petals from young blossoms created a lower redox state in the roGFP2 bioreporter than washes from fully mature blossoms. The bioreporter also detected differences in surface washes from peach fruit at different stages of maturity and between wounded and nonwounded sites. The Pe299R/roGFP2 reporter rapidly assesses differences in redox microenvironments and provides a noninvasive tool that may complement traditional redox-sensitive chromophores and chemical analyses of cell extracts.

Correction: Bacterial detection and identification from human synovial fluids on an integrated microfluidic system.

Correction for 'Bacterial detection and identification from human synovial fluids on an integrated microfluidic system' by Ting-Hang Liu et al., Analyst, 2019, 144, 1210-1222.

Bacterial detection and identification from human synovial fluids on an integrated microfluidic system.

Periprosthetic joint infections (PJIs) are among the most severe complications emerging from prosthetic joint replacement surgeries. In order to possess a rapid means of diagnosing PJIs, an integrated microfluidic system was developed herein for detecting and identifying bacteria in human synovial fluid (HSF). The entire molecular diagnostic process, including (1) sample treatment, (2) bacterial isolation, (3) bacterial lysis, (4) nucleic acid amplification (via polymerase chain reaction (PCR)), and (5) optical detection, could be automated on a single chip. First, N-acetyl-l-cysteine was used to decrease the viscosity of HSF samples and consequently enhance bacterial isolation with vancomycin-coated nano-magnetic beads. Then, a universal 16S ribosomal ribonucleic acid PCR primer set and four species-specific primer sets were used for PCR-based detection and identification of four common bacteria previously associated with PJIs, including Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli, and Acinetobacter baumannii. With this approach, the limit of detection was as low as 100 colony forming units (CFUs) per milliliter (or 20 CFUs per reaction), which is suitable for clinical diagnostics and for making informed decisions regarding post-operative antibiotic administration. More importantly, bacterial detection and identification data could be acquired within 90 minutes, representing a significant improvement over traditional culture-based methods (3-7 days). The developed microfluidic system may therefore serve as a promising tool for rapid diagnosis of PJIs.

Development of a protocol to solidify native and artificial oil bodies for long-term storage at room temperature.

BACKGROUND: Oil bodies isolated from sesame seeds coalesced to form large oil drops when they were solidified in a drying process commonly used for food products. The aim of this study was to develop a protocol to solidify oil bodies for long-term storage at room temperature. RESULTS: On the basis of testing several excipients, the coalescence of oil bodies could be effectively prevented when they were combined with mannitol. Sizes of oil bodies appeared similar under a light microscope before and after powderisation in combination with 70% or more mannitol. Artificial oil bodies were successfully generated with sesame oil, phospholipid and recombinant sesame caleosin. Following the developed protocol, native and artificial oil bodies were stably solidified in tablets. Both native and artificial oil bodies dissolved from the tablets remained stable after an accelerated stress test under a condition of 75% humidity at 40 °C for 4 months. CONCLUSION: A protocol was successfully developed for the solidification of native and artificial oil bodies in stable powder and tablet forms. This successful protocol is very likely to expedite the utilisation of artificial oil bodies in their potential applications.

An integrated microfluidic platform for detection of ovarian clear cell carcinoma mRNA biomarker FXYD2.

In this work we developed an integrated microfluidic system for automatically detecting the ovarian clear cell carcinoma (OCCC) biomarker FXYD2. Dealing with ascites from ovarian cancer patients, capture of cancer cells, isolation of messenger RNA, and quantitative reverse-transcription polymerase chain reaction were integrated into a single microfluidic chip and carried out on-chip automatically. OCCC is a subtype of ovarian cancer with a high mortality risk, and a high FXYD2 gene expression level was shown to be closely associated with OCCC. The lowest limit of quantification using a benchtop protocol of this system could be as low as 100 copies per sample. By normalizing the expression to a housekeeping gene, GAPDH, a simple cycle threshold ratio index could distinguish high FXYD2 expression cells from the low-expression ones. This developed platform may therefore facilitate future OCCC diagnosis and/or prognosis.

Rapid identification of pathogens responsible for necrotizing fasciitis on an integrated microfluidic system.

Necrotic fasciitis (NF) is a particularly aggressive and serious infection of the fascia that can penetrate into the musculature and internal organs, resulting in death if not treated promptly. In this work, an integrated microfluidic system composed of micropumps, microvalves, and micromixers was used to automate the detection of pathogens associated with NF. The entire molecular diagnostic process, including bacteria isolation, lysis, nucleic acid amplification and optical detection steps, was enacted on this developed system. Mannose binding lectin coated magnetic beads were first used as probes to isolate all bacteria in a sample. In this work, polymerase chain reaction assays featuring primers specific to genes from each of four NF-causing bacteria (Vibrio vulnificus, Aeromonas hydrophila, and methicillin-sensitive and resistant Staphylococcus aureus) were used to rapidly and exclusively verify the presence of the respective bacterial strains, and the limits of detection were experimentally found to be 11, 1960, 14, and 11 400 colony forming units/reaction, respectively; all values reflect improvement over ones reported in literature. This integrated microfluidic chip may then be valuable in expediting diagnosis and optimizing treatment options for those with NF; such diagnostic improvements could ideally diminish the need for amputation and even reduce the morality rate associated with this life-threatening illness.

Resonance assignments and secondary structure of a phytocystatin from Ananas comosus.

A cDNA encoding a cysteine protease inhibitor, cystatin was cloned from pineapple (Ananas comosus L.) stem. This clone was constructed into an expression vector and expressed in E. coli and purified to homogeneous. The recombinant pineapple cystatins (AcCYS) showed effectively inhibitory activity toward cysteine proteases including papain, bromelain, and cathepsin B. In order to unravel its inhibitory action from structural point of view, multidimensional heteronuclear NMR techniques were used to characterize the structure of AcCYS. The full (1)H, (15)N, and (13)C resonance assignments of AcCYS were determined. The secondary structure of AcCYS was identified by using the assigned chemical shift of (1)Hα, (13)Cα, (13)Cß, and (13)CO through the consensus chemical shift index (CSI). The results of CSI analysis suggest 5 ß-strands (residues 45-47, 84-91, 94-104, 106-117, and 123-130) and one α-helix (residues 55-73).

Solution structure of a phytocystatin from Ananas comosus and its molecular interaction with papain.

The structure of a recombinant pineapple cystatin (AcCYS) was determined by NMR with the RMSD of backbone and heavy atoms of twenty lowest energy structures of 0.56 and 1.11 Å, respectively. It reveals an unstructured N-terminal extension and a compact inhibitory domain comprising a four-stranded antiparallel ß-sheet wrapped around a central α-helix. The three structural motifs (G(45), Q(89)XVXG, and W(120)) putatively responsible for the interaction with papain-like proteases are located in one side of AcCYS. Significant chemical shift perturbations in two loop regions, residues 45 to 48 (GIYD) and residues 89 to 91 (QVV), of AcCYS strongly suggest their involvement in the binding to papain, consistent with studies on other members of the cystatin family. However, the highly conserved W120 appears not to be involved in the binding with papain as no chemical shift perturbation was observed. Chemical shift index analysis further indicates that the length of the α-helix is shortened upon association with papain. Collectively, our data suggest that AcCYS undergoes local secondary structural rearrangements when papain is brought into close contact. A molecular model of AcCYS/papain complex is proposed to illustrate the interaction between AcCYS and papain, indicating a complete blockade of the catalytic triad by AcCYS.

Stability of artificial oil bodies constituted with recombinant caleosins.

Caleosin is a unique calcium binding protein anchoring to the surface of seed oil bodies by its central hydrophobic domain composed of an amphiphatic alpha-helix and a proline-knot subdomain. Stable artificial oil bodies were successfully constituted with recombinant caleosin overexpressed in Escherichia coli. The stability of artificial oil bodies was slightly or severely reduced when the amphiphatic alpha-helix or proline-knot subdomain in the hydrophobic domain of caleosin was truncated. Deletion of the entire central hydrophobic domain substantially increased the solubility of the recombinant caleosin, leading to a complete loss of its capability to stabilize these oil bodies. A recombinant protein engineered with the hydrophobic domain of caleosin replaced by that of oleosin, the abundant structural protein of seed oil bodies, could stabilize the artificial oil bodies, in terms of thermo- and structural stability, as effectively as caleosin or oleosin.