Mechanistic insights into the adaptive evolvability of spore heat resistance in Bacillus cereus sensu lato.

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.

Evolutionary trade-off between heat shock resistance, growth at high temperature, and virulence expression in Salmonella Typhimurium.

Understanding the evolutionary dynamics of foodborne pathogens throughout our food production chain is of utmost importance. In this study, we reveal that Salmonella Typhimurium can readily and reproducibly acquire vastly increased heat shock resistance upon repeated exposure to heat shock. Counterintuitively, this boost in heat shock resistance was invariantly acquired through loss-of-function mutations in the dnaJ gene, encoding a heat shock protein that acts as a molecular co-chaperone of DnaK and enables its role in protein folding and disaggregation. As a trade-off, however, the acquisition of heat shock resistance inevitably led to attenuated growth at 37°C and higher temperatures. Interestingly, loss of DnaJ also downregulated the activity of the master virulence regulator HilD, thereby lowering the fraction of virulence-expressing cells within the population and attenuating virulence in mice. By connecting heat shock resistance evolution to attenuation of HilD activity, our results confirm the complex interplay between stress resistance and virulence in Salmonella Typhimurium. IMPORTANCE: Bacterial pathogens such as Salmonella Typhimurium are equipped with both stress response and virulence features in order to navigate across a variety of complex inhospitable environments that range from food-processing plants up to the gastrointestinal tract of its animal host. In this context, however, it remains obscure whether and how adaptation to one environment would obstruct fitness in another. In this study, we reveal that severe heat stress counterintuitively, but invariantly, led to the selection of S. Typhimurium mutants that are compromised in the activity of the DnaJ heat shock protein. While these mutants obtained massively increased heat resistance, their virulence became greatly attenuated. Our observations, therefore, reveal a delicate balance between optimal tuning of stress response and virulence features in bacterial pathogens.

The Evolution and Evolvability of Photosystem II.

Photosystem II is the water-oxidizing and O2-evolving enzyme of photosynthesis. How and when this remarkable enzyme arose are fundamental questions in the history of life that have remained difficult to answer. Here, recent advances in our understanding of the origin and evolution of photosystem II are reviewed and discussed in detail. The evolution of photosystem II indicates that water oxidation originated early in the history of life, long before the diversification of cyanobacteria and other major groups of prokaryotes, challenging and transforming current paradigms on the evolution of photosynthesis. We show that photosystem II has remained virtually unchanged for billions of years, and yet the nonstop duplication process of the D1 subunit of photosystem II, which controls photochemistry and catalysis, has enabled the enzyme to become adaptable to variable environmental conditions and even to innovate enzymatic functions beyond water oxidation. We suggest that this evolvability can be harnessed to develop novel light-powered enzymes with the capacity to carry out complex multistep oxidative transformations for sustainable biocatalysis.

The Effect of Sensory Level Versus Motor Level Electrical Stimulation of Pharyngeal Muscles in Acute Stroke Patients with Dysphagia: A Randomized Trial.

Dysphagia is a serious cause of morbidity and mortality in stroke survivors. Electrical stimulation is often included as part of the treatment plan for dysphagia and can be applied at a sensory or motor level intensity. However, evidence to support these different modes of stimulation is lacking. This study compared the effectiveness of sensory and motor level stimulation on post-stroke dysphagia. This is a randomized trial conducted in an inpatient rehabilitation facility. Thirty-one participants who had dysphagia caused by stroke within 6 months prior to enrolment were included. Participants were excluded if they had a contraindication for electrical stimulation, previous stroke, psychiatric disorder, contraindications for modified barium swallow study (MBSS), or pre-morbid dysphagia. Each patient received ten sessions that included 45 min of anterior neck sensory or motor level electrical stimulation in addition to traditional dysphagia therapy. Motor stimulation was administered at an intensity sufficient to produce muscle contractions. Sensory stimulation was defined as the threshold at which the patient feels a tingling sensation on their skin. Swallow functional assessment measure (FAM), dysphagia outcome severity scale (DOSS), national outcome measurement system (NOMS), penetration aspiration scale (PAS), diet change, and the swallowing quality of life questionnaire (SWAL-QOL). Clinical outcomes were analyzed using a Wilcoxon signed-rank test, Mann-Whitney U test, RM ANOVA, or chi-square analysis. There was no significant difference in age, length of stay, or initial swallow FAM between groups. Patients in the sensory group showed significant improvement on swallow FAM, DOSS, and NOMS, while those in the motor group did not (Sensory: Swallow FAM (S = 48, p = 0.01), DOSS (S = 49.5, p = 0.001), NOMS (S = 52.5, p = 0.006); Motor: Swallow FAM (S = 20.5, p = 0.2), DOSS (S = 21, p = 0.05), NOMS (S = 29.5, p = 0.2)). When the groups were combined, there was statistically significant improvement on all measures except the PAS (Swallow FAM (S = 138.5, p = 0.003), DOSS (S = 134.5, p < 0.001), NOMS (S = 164, p = 0.0004)). When comparing motor to sensory NMES, there was no significant difference between groups for Swallow FAM (p = .12), DOSS (p = 0.52), or NOMS (p = 0.41). There was no significant difference in diet change for solid food or liquids among the groups, although 50% more participants in the sensory group saw improvement in diet. This study supports the use of electrical stimulation as part of the treatment plan for post-stroke dysphagia. Sensory-level stimulation was associated with greater improvement on outcome measures compared to motor level stimulation.

Bacillus weihenstephanensis can readily evolve for increased endospore heat resistance without compromising its thermotype.

Proper elimination of bacterial endospores in foods and food processing environment is challenging because of their extreme resistance to various stresses. Often, sporicidal treatments prove insufficient to eradicate the contaminating endospore population as a whole, and might therefore serve as a selection pressure for enhanced endospore resistance. In the sporeforming Bacillus cereus group, Bacillus weihenstephanensis is an important food spoilage organism and potential cereulide producing pathogen, due to its psychrotolerant growth ability at 7 °C. Although the endospores of B. weihenstephanensis are generally less heat resistant compared to their mesophilic or thermotolerant relatives, our data now show that non-emetic B. weihenstephanensis strain LMG 18989T can readily and reproducibly evolve to acquire much enhanced endospore heat resistance. In fact, one of the B. weihenstephanensis mutants from directed evolution by wet heat in this study yielded endospores displaying a > 4-fold increase in D-value at 91 °C compared to the parental strain. Moreover, these mutant endospores retained their superior heat resistance even when sporulation was performed at 10 °C. Interestingly, increased endospore heat resistance did not negatively affect the vegetative growth capacities of the evolved mutants at lower (7 °C) and upper (37 °C) growth temperature boundaries, indicating that the correlation between cardinal growth temperatures and endospore heat resistance which is observed among bacterial sporeformers is not necessarily causal.

Directed evolution by UV-C treatment of Bacillus cereus spores.

Bacterial endospores are exposed to a broad variety of sublethal and lethal stresses in the food production chain. Generally, these stresses will not completely eliminate the existing spore populations, and thus constitute a selection pressure on the spores. One stress that is frequently used in the food production chains to disinfect (food) contact surfaces is UV-C. At a wavelength of 254 nm, UV-C has germicidal properties. The aim of this research is to investigate the impact of UV-C stress on the evolution of endospore recalcitrance and germination in B. cereus. A directed evolution experiment was set up in which B. cereus was repeatedly subjected to a cycle of sporulation, sporicidal UV-C treatment, germination and outgrowth. We show here that three independent lineages of UV-C cycled B. cereus spores reproducibly acquired a 30-fold or higher increase in UV-C resistance at 164 mJ/cm2. Surprisingly, the UV-C resistant spores of the clones isolated from each of the lineages also became significantly more sensitive to wet heat as a normally non-lethal heat treatment at 70 °C for 15 min resulted in an average 1.8 log cfu/mL reduction. From time-lapse phase contrast microscopy analysis, UV-C resistant mutant spores also showed a distinctive heterogeneity in refractility and a severe germination defect compared to the wild type. However, UV-C resistance of the corresponding vegetative cells was not altered. In conclusion, this work shows that UV-C resistance of endospores is an adaptive trait that can readily be improved, although at an apparent cost for heat resistance and germination efficiency. As such, these results provide novel insights in the evolvability of, and correlation between, some endospore properties.

Resolution of severe psychogenic dysphagia with ECT in an elderly patient.

ABSTRACTIn this report we describe an 82-year female with a longstanding anxiety disorder who developed severe psychogenic dysphagia, leading to hospitalization due to failure to thrive. We describe for the first time the use of electroconvulsive therapy (ECT) to successfully manage a patient with pharmacological treatment resistant psychogenic dysphagia.

The androgen-regulated protease TMPRSS2 activates a proteolytic cascade involving components of the tumor microenvironment and promotes prostate cancer metastasis.

UNLABELLED: TMPRSS2 is an androgen-regulated cell-surface serine protease expressed predominantly in prostate epithelium. TMPRSS2 is expressed highly in localized high-grade prostate cancers and in the majority of human prostate cancer metastases. Through the generation of mouse models with a targeted deletion of Tmprss2, we demonstrate that the activity of this protease regulates cancer cell invasion and metastasis to distant organs. By screening combinatorial peptide libraries, we identified a spectrum of TMPRSS2 substrates that include pro-hepatocyte growth factor (HGF). HGF activated by TMPRSS2 promoted c-MET receptor tyrosine kinase signaling, and initiated a proinvasive epithelial-to-mesenchymal transition phenotype. Chemical library screens identified a potent bioavailable TMPRSS2 inhibitor that suppressed prostate cancer metastasis in vivo. Together, these findings provide a mechanistic link between androgen-regulated signaling programs and prostate cancer metastasis that operate via context-dependent interactions with extracellular constituents of the tumor microenvironment. SIGNIFICANCE: The vast majority of prostate cancer deaths are due to metastasis. Loss of TMPRSS2 activity dramatically attenuated the metastatic phenotype through mechanisms involving the HGF-c-MET axis. Therapeutic approaches directed toward inhibiting TMPRSS2 may reduce the incidence or progression of metastasis in patients with prostate cancer.

Laboratory tests and compliance of dermatologic outpatients.

Laboratory tests, including blood tests and urine analysis, are frequently performed in the dermatology outpatient clinic, but doctors often do not consider the cognitive or psychological effect of the examinations. Based on terror management theory, we hypothesized that performing laboratory tests increases the patient's fear of mortality, and therefore has a positive effect on the patient's attitude toward the doctor's recommendations and willingness to accept them. The study employed a single factor between-subjects design, using a questionnaire completed by the patients. One group consisted of patients who had undergone laboratory tests 1 week before the survey, and the other group consisted of patients who had not undergone a laboratory test. Although the differences between two groups were not statistically significant, the patients who had laboratory tests had tendency to show even lower positive attitude toward the doctor's recommendations and less intention to follow the recommendations. In contrast to our hypothesis, performing laboratory tests does not subliminally increase patients' fears or anxieties about their disease or their compliance with doctors' recommendations.

Computational fluid dynamics within bifurcated abdominal aortic stent-grafts.

PURPOSE: To assess the hemodynamic forces on a bifurcated abdominal aortic stent-graft under realistic conditions of flow, blood pressure, and sac pressure. METHODS: Computational fluid dynamics was used to study the temporal and spatial variations in surface pressure and shear through the cardiac cycle on models of bifurcated stent-grafts derived from computed tomography in 4 patients who had previously undergone endovascular repair of abdominal aortic aneurysm (AAA). The trunk, bifurcation, and limbs of the graft were analyzed separately and as parts of a unified whole. Analyses were repeated under varying conditions of sac pressure, reflecting different conditions of perigraft flow and sac diameter change. RESULTS: Pressure-related forces were far larger than flow-related forces in all 3 segments of all 4 cases. The largest forces acted at the bifurcation of the stent-graft. High sac pressures, seen in patients with endoleak or aneurysm dilatation, were associated with reduced transmural pressure and low-pressure-derived forces. CONCLUSION: Several parameters of stent-graft design affect the magnitude and distribution of forces on a bifurcated stent-graft. The forces on a stent-graft are also affected by the pressure within the aneurysm sac, which depends on stent-graft performance.

Proteolytic disassembly is a critical determinant for reovirus oncolysis.

Mammalian ortheoreoviruses are currently being investigated as novel cancer therapeutics, but the cellular mechanisms that regulate susceptibility to reovirus oncolysis remain poorly understood. In this study, we present evidence that virion disassembly is a key determinant of reovirus oncolysis. To penetrate cell membranes and initiate infection, the outermost capsid proteins of reovirus must be proteolyzed to generate a disassembled particle called an infectious subviral particle (ISVP). In fibroblasts, this process is mediated by the endo/lysosomal proteases cathepsins B and L. We have analyzed the early events of infection in reovirus-susceptible and -resistant cells. We find that, in contrast to susceptible glioma cells and Ras-transformed NIH3T3 cells, reovirus-resistant cancer cells and untransformed NIH3T3 cells restrict virion uncoating and subsequent gene expression. Disassembly-restrictive cells support reovirus infection, as in vitro-generated ISVPs establish productive infection, and pretreatment with poly(I:C) does not prevent infection in cancer cells. We find that the level of active cathepsin B and L is increased in tumors and that disassembly-restrictive glioma cells support reovirus oncolysis when grown as a tumor in vivo. Together, these results provide a model in which proteolytic disassembly of reovirus is a critical determinant of susceptibility to reovirus oncolysis.

Phenotypic analysis of mice lacking the Tmprss2-encoded protease.

Tmprss2 encodes an androgen-regulated type II transmembrane serine protease (TTSP) expressed highly in normal prostate epithelium and has been implicated in prostate carcinogenesis. Although in vitro studies suggest protease-activated receptor 2 may be a substrate for TMPRSS2, the in vivo biological activities of TMPRSS2 remain unknown. We generated Tmprss2-/- mice by disrupting the serine protease domain through homologous recombination. Compared to wild-type littermates, Tmprss2-/- mice developed normally, survived to adulthood with no differences in protein levels of prostatic secretions, and exhibited no discernible abnormalities in organ histology or function. Loss of TMPRSS2 serine protease activity did not influence fertility, reduce survival, result in prostate hyperplasia or carcinoma, or alter prostatic luminal epithelial cell regrowth following castration and androgen replacement. Lack of an observable phenotype in Tmprss2-/- mice was not due to transcriptional compensation by closely related Tmprss2 homologs. We conclude that the lack of a discernible phenotype in Tmprss2-/- mice suggests functional redundancy involving one or more of the type II transmembrane serine protease family members or other serine proteases. Alternatively, TMPRSS2 may contribute a specialized but nonvital function that is apparent only in the context of stress, disease, or other systemic perturbation.

Biochemical properties of purified human retinol dehydrogenase 12 (RDH12): catalytic efficiency toward retinoids and C9 aldehydes and effects of cellular retinol-binding protein type I (CRBPI) and cellular retinaldehyde-binding protein (CRALBP) on the oxidation and reduction of retinoids.

Retinol dehydrogenase 12 (RDH12) is a novel member of the short-chain dehydrogenase/reductase superfamily of proteins that was recently linked to Leber's congenital amaurosis 3 (LCA). We report the first biochemical characterization of purified human RDH12 and analysis of its expression in human tissues. RDH12 exhibits approximately 2000-fold lower K(m) values for NADP(+) and NADPH than for NAD(+) and NADH and recognizes both retinoids and lipid peroxidation products (C(9) aldehydes) as substrates. The k(cat) values of RDH12 for retinaldehydes and C(9) aldehydes are similar, but the K(m) values are, in general, lower for retinoids. The enzyme exhibits the highest catalytic efficiency for all-trans-retinal (k(cat)/K(m) approximately 900 min(-)(1) microM(-)(1)), followed by 11-cis-retinal (450 min(-)(1) mM(-)(1)) and 9-cis-retinal (100 min(-)(1) mM(-)(1)). Analysis of RDH12 activity toward retinoids in the presence of cellular retinol-binding protein (CRBP) type I or cellular retinaldehyde-binding protein (CRALBP) suggests that RDH12 utilizes the unbound forms of all-trans- and 11-cis-retinoids. As a result, the widely expressed CRBPI, which binds all-trans-retinol with much higher affinity than all-trans-retinaldehyde, restricts the oxidation of all-trans-retinol by RDH12, but has little effect on the reduction of all-trans-retinaldehyde, and CRALBP inhibits the reduction of 11-cis-retinal stronger than the oxidation of 11-cis-retinol, in accord with its higher affinity for 11-cis-retinal. Together, the tissue distribution of RDH12 and its catalytic properties suggest that, in most tissues, RDH12 primarily contributes to the reduction of all-trans-retinaldehyde; however, at saturating concentrations of peroxidic aldehydes in the cells undergoing oxidative stress, for example, photoreceptors, RDH12 might also play a role in detoxification of lipid peroxidation products.

Delayed dark adaptation in 11-cis-retinol dehydrogenase-deficient mice: a role of RDH11 in visual processes in vivo.

The oxidation of 11-cis-retinol to 11-cis-retinal in the retinal pigment epithelium (RPE) represents the final step in a metabolic cycle that culminates in visual pigment regeneration. Retinol dehydrogenase 5 (RDH5) is responsible for a majority of the 11-cis-RDH activity in the RPE, but the formation of 11-cis-retinal in rdh5-/- mice suggests another enzyme(s) is present. We have previously shown that RDH11 is also highly expressed in RPE cells and has dual specificity for both cis- and trans-retinoid substrates. To investigate the role of RDH11 in the retinoid cycle, we generated rdh11-/- and rdh5-/-rdh11-/- mice and examined their electrophysiological responses to various intensities of illumination and during dark adaptation. Retinoid profiles of darkadapted rdh11-/- mice did not show significant differences compared with wild-type mice, whereas an accumulation of cis-esters was detected in rdh5-/- and rdh5-/-rdh11-/- mice. Following light stimulation, 73% more cis-retinyl esters were stored in rdh5-/-rdh11-/- mice compared with rdh5-/- mice. Single-flash ERGs of rdh11-/- showed normal responses under dark- and light-adapted conditions, but exhibited delayed dark adaptation following high bleaching levels. Double knockout mice also had normal ERG responses in dark- and light-adapted conditions, but had a further delay in dark adaptation relative to either rdh11-/- or rdh5-/- mice. Taken together, these results suggest that RDH11 has a measurable role in regenerating the visual pigment by complementing RDH5 as an 11-cis-RDH in RPE cells, and indicate that an additional unidentified enzyme(s) oxidizes 11-cis-retinol or that an alternative pathway contributes to the retinoid cycle.