In Situ Biomineralization and Particle Deposition Distinctively Mediate Biofilm Susceptibility to Chlorine.

Microbial biofilms and mineral precipitation commonly co-occur in engineered water systems, such as cooling towers and water purification systems, and both decrease process performance. Microbial biofilms are extremely challenging to control and eradicate. We previously showed that in situ biomineralization and the precipitation and deposition of abiotic particles occur simultaneously in biofilms under oversaturated conditions. Both processes could potentially alter the essential properties of biofilms, including susceptibility to biocides. However, the specific interactions between mineral formation and biofilm processes remain poorly understood. Here we show that the susceptibility of biofilms to chlorination depends specifically on internal transport processes mediated by biomineralization and the accumulation of abiotic mineral deposits. Using injections of the fluorescent tracer Cy5, we show that Pseudomonas aeruginosa biofilms are more permeable to solutes after in situ calcite biomineralization and are less permeable after the deposition of abiotically precipitated calcite particles. We further show that biofilms are more susceptible to chlorine killing after biomineralization and less susceptible after particle deposition. Based on these observations, we found a strong correlation between enhanced solute transport and chlorine killing in biofilms, indicating that biomineralization and particle deposition regulate biofilm susceptibility by altering biocide penetration into the biofilm. The distinct effects of in situ biomineralization and particle deposition on biocide killing highlight the importance of understanding the mechanisms and patterns of biomineralization and scale formation to achieve successful biofilm control.

Spatial patterns of carbonate biomineralization in biofilms.

Microbially catalyzed precipitation of carbonate minerals is an important process in diverse biological, geological, and engineered systems. However, the processes that regulate carbonate biomineralization and their impacts on biofilms are largely unexplored, mainly because of the inability of current methods to directly observe biomineralization within biofilms. Here, we present a method for in situ, real-time imaging of biomineralization in biofilms and use it to show that Pseudomonas aeruginosa biofilms produce morphologically distinct carbonate deposits that substantially modify biofilm structures. The patterns of carbonate biomineralization produced in situ were substantially different from those caused by accumulation of particles produced by abiotic precipitation. Contrary to the common expectation that mineral precipitation should occur at the biofilm surface, we found that biomineralization started at the base of the biofilm. The carbonate deposits grew over time, detaching biofilm-resident cells and deforming the biofilm morphology. These findings indicate that biomineralization is a general regulator of biofilm architecture and properties.

Distribution and frequency of plasmodesmata in relation to photoassimilate pathways and phloem loading in the barley leaf.

Large, intermediate, and small bundles and contiguous tissues of the leaf blade of Hordeum tvulgare L. 'Morex' were examined with the transmission electron microscope to determine their cellular composition and the distribution and frequency of the plasmodesmata between the various cell combinations. Plasmodesmata are abundant at the mesophyll/parenchymatous bundle sheath, parenchymatous bundle sheath/mestome sheath, and mestome sheath/vascular parenchyma cell interfaces. Within the bundles, plasmodesmata are also abundant between vascular parenchyma cells, which occupy most of the interface between the sieve tube-companion cell complexes and the mestome sheath. Other vascular parenchyma cells commonly separate the thick-walled sieve tubes from the sieve tube-companion cell complexes. Plasmodesmatal frequencies between all remaining cell combinations of the vascular tissues are very low, even between the thin-walled sieve tubes and their associated companion cells. Both the sieve tube-companion cell complexes and the thick-walled sieve tubes, which lack companion cells, are virtually isolated symplastically from the rest of the leaf. Data on plamodesmatal frequency between protophloem sieve tubes and other cell types in intermediate and large bundles indicate that they (and their associated companion cells, when present) are also isolated symplastically from the rest of the leaf. Collectively, these data indicate that both phloem loading and unloading in the barley leaf involve apoplastic mechanisms.

Multifunctional polymeric nanoparticles from diverse bioactive agents.

We present a rational approach for assembling diverse bioactive agents, such as DNA, proteins, and drug molecules, into core-shell multifunctional polymeric nanoparticles (PNPs) that can be internalized in human breast cancer cells. Using ring-opening metathesis polymerization (ROMP), block copolymers containing small-molecule drug segments (>50% w/w) and tosylated hexaethylene glycol segments were prepared and assembled into PNPs that allowed for the surface conjugation of single-stranded DNA sequences and/or tumor-targeting antibodies. The resulting antibody-functionalized particles were readily uptaken by breast cancer cells that overexpressed the corresponding antigens.

A novel mechanism for protein delivery: granzyme B undergoes electrostatic exchange from serglycin to target cells.

The molecular interaction of secreted granzyme B-serglycin complexes with target cells remains undefined. Targets exposed to double-labeled granzyme B-serglycin complexes show solely the uptake of granzyme B. An in vitro model demonstrates the exchange of the granzyme from serglycin to immobilized, sulfated glycosaminoglycans. Using a combination of cell binding and internalization assays, granzyme B was found to exchange to sulfated glycosaminoglycans and, depending on the cell type, to higher affinity sites. Apoptosis induced by purified granzyme B and cytotoxic T-cells was diminished in targets with reduced cell surface glycosaminoglycan content. A mechanism of delivery is proposed entailing electrostatic transfer of granzyme B from serglycin to cell surface proteins.

NF-kappaB protects from the lysosomal pathway of cell death.

The programme of gene expression induced by RelA/NF-kappaB transcription factors is critical to the control of cell survival. Ligation of 'death receptors' such as tumor necrosis factor receptor 1 (TNF-R1) triggers apoptosis, as well as NF-kappaB, which counteracts this process by activating the transcription of anti-apoptotic genes. In addition to activating caspases, TNF-R1 stimulation causes the release of cathepsins, most notably cathepsin B, from the lysosome into the cytoplasm where they induce apoptosis. Here we report a mechanism by which NF-kappaB protects cells against TNF-alpha-induced apoptosis: inhibition of the lysosomal pathway of apoptosis. NF-kappaB can protect cells from death after TNF-R1 stimulation, by extinguishing cathepsin B activity in the cytosol. This activity of NF-kappaB is mediated, at least in part, by the upregulation of Serine protease inhibitor 2A (Spi2A), a potent inhibitor of cathepsin B. Indeed, Spi2A can substitute for NF-kappaB in suppressing the induction of cathepsin B activity in the cytosol. Thus, inhibition of cathepsin B by Spi2A is a mechanism by which NF-kappaB protects cells from lysosome-mediated apoptosis.