No abstract

Liver transplantation is the last resort for liver failure patients. However, due to the shortage of donor organs, bioengineered liver generated from decellularized whole liver scaffolds and induced pluripotent stem cell (iPSC)-derived hepatocytes (iPSC-Heps) is being studied as an alternative approach to treat liver disease. Nevertheless, there has been no report on both the interaction of iPSC-Heps with a liver extracellular matrix (ECM) and the analysis of recellularized iPSC-Heps into the whole liver scaffolds. In this study, we produced porcine iPSC-Heps, which strongly expressed the hepatic markers α-fetoprotein and albumin and exhibited hepatic functionalities, including glycogen storage, lipid accumulation, low-density lipoprotein uptake, and indocyanine green metabolism. Supplementation of ECM from porcine decellularized liver containing liver-derived growth factors stimulated the albumin expression of porcine iPSC-Heps during differentiation procedures. The iPSC-Heps were reseeded into decellularized liver scaffolds, and the recellularized liver was cultured using a continuous perfusion system. The recellularized liver scaffolds were transplanted into rats for a short term, and the grafts expressed hepatocyte markers and did not rupture. These results provide a foundation for development of bioengineered liver using stem cell and decellularized scaffolds.

Lactacin F is a nonlantibiotic, heat-stable, peptide bacteriocin produced by LactobaciUus johnsonii VPI11088. Molecular analysis of the lactacin F DNA region characterized a small operon that codes for three open reading frames, designated lafA, laJX, and ORFZ. The peptide encoded by lafA, the lactacin F structural gene, was compared with various peptide bacteriocins from lactic acid bacteria, and similarities were identified in the amino and carboxy termini of the propeptides. Site-directed mutagenesis of the LafA precursor at the two glycine residues in positions-1 and-2 defined an essential motif for processing of mature lactacin F. The involvement of the peptides encoded by laiX and ORFZ in bacteriocin expression was investigated by subcloning various fragments from the lactacin F region into the shuttle vector pGKV210. In addition to lafA, expression of laiX is essential to lactacin F activity. The lactacin F operon resembles the genetic organization of lactococcin M. Although no function has been assigned to ORFZ by genetic analysis, both peptide Z and the lactococcin M immunity protein are predicted to be integral membrane proteins with four putative transmembrane segments. Lactacin F activity, defined by bactericidal action on LactobaciUus delbrueckii, is dependent on the expression of two genes, lafA and lagX.

Greater connectivity to stream surface water may result in greater inputs of allochthonous nutrients that could stimulate internal nitrogen (N) and phosphorus (P) cycling in natural, restored, and created riparian wetlands. This study investigated the effects of hydrologic connectivity to stream water on soil nutrient fluxes in plots ( = 20) located among four created and two natural freshwater wetlands of varying hydrology in the Piedmont physiographic province of Virginia. Surface water was slightly deeper; hydrologic inputs of sediment, sediment-N, and ammonium were greater; and soil net ammonification, N mineralization, and N turnover were greater in plots with stream water classified as their primary water source compared with plots with precipitation or groundwater as their primary water source. Soil water-filled pore space, inputs of nitrate, and soil net nitrification, P mineralization, and denitrification enzyme activity (DEA) were similar among plots. Soil ammonification, N mineralization, and N turnover rates increased with the loading rate of ammonium to the soil surface. Phosphorus mineralization and ammonification also increased with sedimentation and sediment-N loading rate. Nitrification flux and DEA were positively associated in these wetlands. In conclusion, hydrologic connectivity to stream water increased allochthonous inputs that stimulated soil N and P cycling and that likely led to greater retention of sediment and nutrients in created and natural wetlands. Our findings suggest that wetland creation and restoration projects should be designed to allow connectivity with stream water if the goal is to optimize the function of water quality improvement in a watershed.

Lactic acid bacteria isolated from vacuum-packaged fresh meat stored at 4 degrees C were shown to produce antagonistic substances active against closely related bacteria. Growth medium, pH and growth temperature all affected the production of the inhibitory substances. Ten strains including aciduric Lactobacillus-type organisms, Carnobacterium spp. and Leuconostoc spp. were selected that produced protein-aceous substances that caused inhibition of indicator strains. These were considered to be bacteriocins or bacteriocin-like compounds based on their inactivation with protease, generally narrow spectra of antibacterial activity and bactericidal or bacteriostatic modes of action. Activity was not lost from supernatant fluids as a result of heat treatment at 62 degrees C for 30 min, except for the Leuconostoc strains. Inhibitory spectra of some strains included Enterococcus spp. and Listeria monocytogenes. Some strains were of interest because their inhibitory substances were detected in the supernatant fluid early in the growth cycle. The inhibitory substances differed in characteristics between strains and there is evidence that more than one bacteriocin-like substance may be produced by some strains.

A novel cytidine analog fluorocyclopentenylcytosine (RX-3117; TV-1360) was characterized for its cytotoxicity in a 59-cell line panel and further characterized for cytotoxicity, metabolism and mechanism of action in 15 additional cancer cell lines, including gemcitabine-resistant variants. In both panels sensitivity varied 75-fold (IC50: 0.4- > 30 μM RX-3117). RX-3117 showed a different sensitivity profile compared to cyclopentenyl-cytosine (CPEC) and azacytidine, substrates for uridine-cytidine-kinase (UCK). Dipyridamole, an inhibitor of the equilibrative-nucleoside-transporter protected against RX-3117. Uridine and cytidine protected against RX-3117, but deoxycytidine (substrate for deoxycytidine-kinase [dCK]) not, although it protected against gemcitabine, demonstrating that RX-3117 is a substrate for UCK and not for dCK. UCK activity was abundant in all cell lines, including the gemcitabine-resistant variants. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). RX-3117 was rapidly metabolised to its nucleotides predominantly the triphosphate, which was highest in the most sensitive cells (U937, A2780) and lowest in the least sensitive (CCRF-CEM). RX-3117 did not significantly affect cytidine and uridine nucleotide pools. Incorporation of RX-3117 into RNA and DNA was higher in sensitive A2780 and low in insensitive SW1573 cells. In sensitive U937 cells 1 μM RX-3117 resulted in 90% inhibition of RNA synthesis but 100 μM RX-3117 was required in A2780 and CCRF-CEM cells. RX-3117 at IC50 values did not affect the integrity of RNA. DNA synthesis was completely inhibited in sensitive U937 cells at 1 μM, but in other cells even higher concentrations only resulted in a partial inhibition. At IC50 values RX-3117 downregulated the expression of DNA methyltransferase. In conclusion, RX-3117 showed a completely different sensitivity profile compared to gemcitabine and CPEC, its uptake is transporter dependent and is activated by UCK. RX-3117 is incorporated into RNA and DNA, did not affect RNA integrity, depleted DNA methyltransferase and inhibited RNA and DNA synthesis. Nucleotide formation is related with sensitivity.

No abstract

Carnobacterium piscicola LV17 isolated from vacuum-packed meat produces bacteriocin(s) that is active against closely related lactic acid bacteria, Enterococcus spp., and a strain of Listeria monocytogenes but not against gram-negative bacteria. The bacteriocin has a bactericidal mode of action, is heat resistant, and is stable over a wide range of pH but is inactivated by proteolytic enzymes. Sensitive and resistant cells were shown to adsorb the bacteriocin, but cell death depended on contact of the bacteriocin with the cell membrane. Bacteriocin production is detected early in the growth cycle of the organism in APT broth, but it is not produced in APT broth adjusted to pH 5.5. Bacteriocin production and resistance to the bacteriocin produced are associated with two plasmids of 40 and 49 megadaltons. The possibility that two bacteriocins are produced is indicated because the inhibitory substances of the mutant strains containing either the 40or 49-megadalton plasmids have different antimicrobial spectra.