Physical Cues in the Microenvironment Regulate Stemness-Dependent Homing of Breast Cancer Cells.

Tissue-specific microenvironmental factors contribute to the targeting preferences of metastatic cancers. However, the physical attributes of the premetastatic microenvironment are not yet fully characterized. In this research, we develop a transwell-based alginate hydrogel (TAH) model to study how permeability, stiffness, and roughness of a hanging alginate hydrogel regulate breast cancer cell homing. In this model, a layer of physically characterized alginate hydrogel is formed at the bottom of a transwell insert, which is placed into a matching culture well with an adherent monolayer of breast cancer cells. We found that breast cancer cells dissociate from the monolayer and home to the TAH for continual growth. The process is facilitated by the presence of rich serum in the upper chamber, the increased stiffness of the gel, as well as its surface roughness. This model is able to support the homing ability of MCF-7 and MDA-MB-231 cells drifting across the vertical distance in the culture medium. Cells homing to the TAH display stemness phenotype morphologically and biochemically. Taken together, these findings suggest that permeability, stiffness, and roughness are important physical factors to regulate breast cancer homing to a premetastatic microenvironment.

Characterization and application of a common epitope recognized by a broad-spectrum C4 monoclonal antibody against capsid proteins of plant potyviruses.

A broad-spectrum monoclonal antibody (C4 MAb) against the capsid proteins (CPs) of plant potyviruses has been generated in previous studies. To clarify which epitope is recognized by this MAb, epitope mapping was performed via phage display library screening and amino acid substitution analysis. Subsequently, a 12-residue epitope in the core region of potyvirus CPs was identified and termed the C4 epitope (WxMMDGxxQxxY/F). This epitope contains tryptophan and tyrosine residues that are crucial for reacting with C4 MAb. The CP of Odontoglossum ringspot tobamovirus (ORSV) separately fused with the C4 epitope of Konjak mosaic potyvirus (KoMV), Zantedeschia mild mosaic potyvirus (ZaMMV), or Dasheen mosaic potyvirus (DsMV) was expressed in a bacterial system and purified. The results of indirect ELISA and Western blotting demonstrated that the C4 epitope of KoMV (Ko) fused to ORSV CP showed the strongest binding affinity to C4 MAb among the three viral epitope tags examined. The binding affinity between Ko tag (WTMMDGEEQIEY) and C4 MAb was determined. To examine the applicability of the Ko tag in planta, GFP and ORSV CP were transiently expressed in Nicotiana benthamiana, and both Ko-tagged proteins were specifically detected using C4 MAb. The Ko tag did not affect the silencing suppressor function of Tomato bushy stunt tombusvirus P19 in N. benthamiana. Furthermore, Ko-tagged EGFP could be successfully expressed, specifically detected and subsequently immunoprecipitated using C4 MAb in a mammalian cell system. Thus, the present study identified a common C4 epitope of potyviruses recognized by the broad-spectrum C4 and PTY 1 MAbs, and the results indicated that the newly designed Ko tag is suitable for application in bacterial, plant, and mammalian cell systems.

Cytochrome c biogenesis in Campylobacter jejuni requires cytochrome c6 (CccA; Cj1153) to maintain apocytochrome cysteine thiols in a reduced state for haem attachment.

The microaerophilic food-borne pathogen Campylobacter jejuni uses complex cytochrome-rich respiratory chains for growth and host colonisation. Cytochrome c biogenesis requires haem ligation to reduced apocytochrome cysteines, catalysed by the cytochrome c synthase, CcsBA. While ccsBA could not be deleted, we showed that the thiol reductase DsbD and the CcsX homologue Cj1207 are involved in, but not essential for, cytochromes c biogenesis. Mutant phenotypic analyses and biochemical studies with purified proteins revealed that the mono-haem c-type cytochromes Cj1153 (CccA) and Cj1020 (CccB) and the di-haem Cj0037 (CccC) are electron donors to the cb-oxidase (CcoNOQP), with CccC being more efficient than CccA. Remarkably, cccA deletion or site-directed mutagenesis resulted in an almost complete loss of all other c-type cytochromes. Cytochrome c structural and biogenesis genes were still transcribed in the cccA deletion mutant and the quinol oxidase genes (cioAB) were up-regulated. Cytochrome c production could be rescued in this mutant by growth with exogenous dithiothreitol or L-cysteine, suggesting that in the absence of CccA, apocytochrome c haem binding motifs become oxidised, preventing haem attachment. Our results identify CccA, the most abundant periplasmic c-type cytochrome in C. jejuni, as a novel and unexpected protein required for cytochrome c biogenesis in this pathogen.

It takes two to tango: two TatA paralogues and two redox enzyme-specific chaperones are involved in the localization of twin-arginine translocase substrates in Campylobacter jejuni.

The food-borne zoonotic pathogen Campylobacter jejuni has complex electron transport chains required for growth in the host, many of which contain cofactored periplasmic enzymes localized by the twin-arginine translocase (TAT). We report here the identification of two paralogues of the TatA translocase component in C. jejuni strain NCTC 11168, encoded by cj1176c (tatA1) and cj0786 (tatA2). Deletion mutants constructed in either or both of the tatA1 and tatA2 genes displayed distinct growth and enzyme activity phenotypes. For sulphite oxidase (SorAB), the multi-copper oxidase (CueO) and alkaline phosphatase (PhoX), complete dependency on TatA1 for correct periplasmic activity was observed. However, the activities of nitrate reductase (NapA), formate dehydrogenase (FdhA) and trimethylamine N-oxide reductase (TorA) were significantly reduced in the tatA2 mutant. In contrast, the specific rate of fumarate reduction catalysed by the flavoprotein subunit of the methyl menaquinone fumarate reductase (MfrA) was similar in periplasmic fractions of both the tatA1 and the tatA2 mutants and only the deletion of both genes abolished activity. Nevertheless, unprocessed MfrA accumulated in the periplasm of the tatA1 (but not tatA2) mutant, indicating aberrant signal peptide cleavage. Surprisingly, TatA2 lacks two conserved residues (Gln8 and Phe39) known to be essential in Escherichia coli TatA and we suggest it is unable to function correctly in the absence of TatA1. Finally, only two TAT chaperones (FdhM and NapD) are encoded in strain NCTC 11168, which mutant studies confirmed are highly specific for formate dehydrogenase and nitrate reductase assembly, respectively. Thus, other TAT substrates must use general chaperones in their biogenesis.

Tetrathionate stimulated growth of Campylobacter jejuni identifies a new type of bi-functional tetrathionate reductase (TsdA) that is widely distributed in bacteria.

Tetrathionate (S4 O6 (2-) ) is used by some bacteria as an electron acceptor and can be produced in the vertebrate intestinal mucosa from the oxidation of thiosulphate (S2 O3 (2-) ) by reactive oxygen species during inflammation. Surprisingly, growth of the microaerophilic mucosal pathogen Campylobacter jejuni under oxygen-limited conditions was stimulated by tetrathionate, although it does not possess any known type of tetrathionate reductase. Here, we identify a dihaem cytochrome c (C8j_0815; TsdA) as the enzyme responsible. Kinetic studies with purified recombinant C. jejuni TsdA showed it to be a bifunctional tetrathionate reductase/thiosulphate dehydrogenase with a high affinity for tetrathionate. A tsdA null mutant still slowly reduced, but could not grow on, tetrathionate under oxygen limitation, lacked thiosulphate-dependent respiration and failed to convert thiosulphate to tetrathionate microaerobically. A TsdA paralogue (C8j_0040), lacking the unusual His-Cys haem ligation of TsdA, had low thiosulphate dehydrogenase and tetrathionate reductase activities. Our data highlight a hitherto unrecognized capacity of C. jejuni to use tetrathionate and thiosulphate in its energy metabolism, which may promote growth in the host. Moreover, as TsdA represents a new class of tetrathionate reductase that is widely distributed among bacteria, we predict that energy conserving tetrathionate respiration is far more common than currently appreciated.

Performance of electrospun nanofibers for SPE of drugs from aqueous solutions.

A novel extraction technique was reported. The solid phase material, nanofiber, was prepared by electrospinning using polystyrene. Twenty different drugs (10 microg/L in water) were extracted using 1 mg of nanofibers within 5 min. The analytes can be desorpted from the fibers with 50 microL of the methanol and then monitored by LC coupled to a UV detector. Packed-fiber SPE (PFSPE) provide high recoveries (>50%) for some relatively non-polar drugs (log P >1.5) (n-octanol-to-water partition ratio), and relatively low recoveries (9.9-39.8%) for the drugs within the log P window below 1. Experimental optimization of the technique has been carried out using seven representative drugs, edaravone, cinchonine, quinine, voriconazole, chlordiazepoxide, verapamil, and rutonding. Except for edaravone, the maximum yields of seven drugs (0.2 microg/L) from water samples were approximately 100%, and were 33.7-88.2% from human plasma. The advantageous aspect of the technique encompasses high throughput, high sensitivity, simplicity, low cost, and green chemistry.