Thioflavin-T does not report on electrochemical potential and memory of dormant or germinating bacterial spores.

IMPORTANCE: Bacillus and Clostridium spores cause food spoilage and disease because of spores' dormancy and resistance to microbicides. However, when spores "come back to life" in germination, their resistance properties are lost. Thus, understanding the mechanisms of spore germination could facilitate the development of "germinate to eradicate" strategies. One germination feature is the memory of a pulsed germinant stimulus leading to greater germination following a second pulse. Recent observations of increases in spore binding of the potentiometric dye thioflavin-T early in their germination of spores led to the suggestion that increasing electrochemical potential is how spores "remember" germinant pulses. However, new work finds no increased thioflavin-T binding in the physiological germination of Coatless spores or of intact spores germinating with dodecylamine, even though spore memory is seen in both cases. Thus, using thioflavin-T uptake by germinating spores to assess the involvement of electrochemical potential in memory of germinant exposure, as suggested recently, is questionable.

Identification and characterization of new proteins crucial for bacterial spore resistance and germination.

2Duf, named after the presence of a transmembrane (TM) Duf421 domain and a small Duf1657 domain in its sequence, is likely located in the inner membrane (IM) of spores in some Bacillus species carrying a transposon with an operon termed spoVA 2mob. These spores are known for their extreme resistance to wet heat, and 2Duf is believed to be the primary contributor to this trait. In this study, we found that the absence of YetF or YdfS, both Duf421 domain-containing proteins and found only in wild-type (wt) B. subtilis spores with YetF more abundant, leads to decreased resistance to wet heat and agents that can damage spore core components. The IM phospholipid compositions and core water and calcium-dipicolinic acid levels of YetF-deficient spores are similar to those of wt spores, but the deficiency could be restored by ectopic insertion of yetF, and overexpression of YetF increased wt spore resistance to wet heat. In addition, yetF and ydfS spores have decreased germination rates as individuals and populations with germinant receptor-dependent germinants and increased sensitivity to wet heat during germination, potentially due to damage to IM proteins. These data are consistent with a model in which YetF, YdfS and their homologs modify IM structure to reduce IM permeability and stabilize IM proteins against wet heat damage. Multiple yetF homologs are also present in other spore forming Bacilli and Clostridia, and even some asporogenous Firmicutes, but fewer in asporogenous species. The crystal structure of a YetF tetramer lacking the TM helices has been reported and features two distinct globular subdomains in each monomer. Sequence alignment and structure prediction suggest this fold is likely shared by other Duf421-containing proteins, including 2Duf. We have also identified naturally occurring 2duf homologs in some Bacilli and Clostridia species and in wt Bacillus cereus spores, but not in wt B. subtilis. Notably, the genomic organization around the 2duf gene in most of these species is similar to that in spoVA 2mob, suggesting that one of these species was the source of the genes on this operon in the extremely wet heat resistant spore formers.

A 3D star shot to determine the gantry, collimator, and couch axes positions.

A linear accelerator has three independent axes that are nominally intersecting at the isocenter. Modern treatment techniques require the coincidence of these axes to lie within a 1-mm diameter sphere. A solution to verify this requirement is to wrap a film on a cylindrical surface, align the cylinder to the linac's isocenter and gantry axis, and take multiple exposures of slits, rotating either the gantry, collimator, or couch between exposures. The resulting exposure pattern is the 3D equivalent of the 2D star shot and encodes sufficient information to determine each axis' position in 3D. Moreover, this method uses a single sheet 8"x10" film, a standard film scanner, and a phantom that can be readily built in-house, making a practical solution to this 3D-measurement problem.