Parallel genetic adaptation of Bacillus subtilis to different plant species.

Plant growth-promoting rhizobacteria benefit plants by stimulating their growth or protecting them against phytopathogens. Rhizobacteria must colonize and persist on plant roots to exert their benefits. However, little is known regarding the processes by which rhizobacteria adapt to different plant species, or behave under alternating host plant regimes. Here, we used experimental evolution and whole-population whole-genome sequencing to analyse how Bacillus subtilis evolves on Arabidopsis thaliana and tomato seedlings, and under an alternating host plant regime, in a static hydroponic setup. We observed parallel evolution across multiple levels of biological organization in all conditions, which was greatest for the two heterogeneous, multi-resource, spatially structured environments at the genetic level. Species-specific adaptation at the genetic level was also observed, possibly caused by the selection stress imposed by different host plants. Furthermore, a trade-off between motility and biofilm development was supported by mutational changes in motility- and biofilm-related genes. Finally, we identified several condition-specific and common targeted genes in different environments by comparing three different B. subtilis biofilm adaptation settings. The results demonstrate a common evolutionary pattern when B. subtilis is adapting to the plant rhizosphere in similar conditions, and reveal differences in genetic mechanisms between different host plants. These findings will likely support strain improvements for sustainable agriculture.

Treponema denticola Induces Interleukin-36γ Expression in Human Oral Gingival Keratinocytes via the Parallel Activation of NF-κB and Mitogen-Activated Protein Kinase Pathways.

The oral epithelial barrier acts as both a physical barrier to the abundant oral microbiome and a sentry for the immune system that, in health, constrains the accumulation of the polymicrobial plaque biofilm. The immune homeostasis during gingivitis that is largely protective becomes dysregulated, unproductive, and destructive to gingival tissue as periodontal disease progresses to periodontitis. The progression to periodontitis is associated with the dysbiosis of the oral microbiome, with increasing prevalences and abundances of periodontal pathogens such as Treponema denticola. Despite the association of T. denticola with a chronic inflammatory disease, relatively little is known about gingival epithelial cell responses to T. denticola infection. Here, we characterized the transcriptome of gingival keratinocytes following T. denticola challenge and identified interleukin-36γ (IL-36γ) as the most differentially expressed cytokine. IL-36γ expression is regulated by p65 NF-κB and the activation of both the Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways downstream of Toll-like receptor 2 (TLR2). Finally, we demonstrate for the first time that mitogen- and stress-activated kinase 1 (MSK1) contributes to IL-36γ expression and may link the activation of MAPK and NF-κB signaling. These findings suggest that the interactions of T. denticola with the gingival epithelium lead to elevated IL-36γ expression, which may be a critical inducer and amplifier of gingival inflammation and subsequent alveolar bone loss.

Experimental evolution of Bacillus subtilis on Arabidopsis thaliana roots reveals fast adaptation and improved root colonization.

Bacillus subtilis is known to promote plant growth and protect plants against disease. B. subtilis rapidly adapts to Arabidopsis thaliana root colonization, as evidenced by improved root colonizers already after 12 consecutive transfers between seedlings in a hydroponic setup. Re-sequencing of single evolved isolates and endpoint populations revealed mutations in genes related to different bacterial traits, in accordance with evolved isolates displaying increased root colonization associated with robust biofilm formation in response to the plant polysaccharide xylan and impaired motility. Interestingly, evolved isolates suffered a fitness disadvantage in a non-selective environment, demonstrating an evolutionary cost of adaptation to the plant root. Finally, increased root colonization by an evolved isolate was also demonstrated in the presence of resident soil microbes. Our findings highlight how a plant growth-promoting rhizobacterium rapidly adapts to an ecologically relevant environment and reveal evolutionary consequences that are fundamental to consider when evolving strains for biocontrol purposes.

Diversification of Bacillus subtilis during experimental evolution on Arabidopsis thaliana and the complementarity in root colonization of evolved subpopulations.

The soil bacterium Bacillus subtilis is known to suppress pathogens as well as promote plant growth. However, in order to fully exploit the potential as natural fertilizer, we need a better understanding of the interactions between B. subtilis and plants. Here, B. subtilis was examined for root colonization through experimental evolution on Arabidopsis thaliana. The populations evolved rapidly, improved in root colonization and diversified into three distinct morphotypes. In order to better understand the adaptation that had taken place, single evolved isolates from the final transfer were randomly selected for further characterization, revealing changes in growth and pellicle formation in medium supplemented with plant polysaccharides. Intriguingly, certain evolved isolates showed improved root colonization only on the plant species they evolved on, but not on another plant species, namely tomato, suggesting A. thaliana specific adaption paths. Finally, the mix performed better than the sum of its constituents in monoculture, which was demonstrated to be caused by complementarity effects. Our results suggest that genetic diversification occurs in an ecological relevant setting on plant roots and proves to be a stable strategy for root colonization.

Palliative care provision at a tertiary cancer center during a global pandemic.

COVID-19 was first reported in Wuhan, China, in December 2019; it rapidly spread around the world and was declared a global pandemic by the World Health Organization in March 2020. The palliative care program at the Princess Margaret Cancer Centre, Toronto, Canada, provides comprehensive care to patients with advanced cancer and their families, through services including an acute palliative care unit, an inpatient consultation service, and an ambulatory palliative care clinic. In the face of a global pandemic, palliative care teams are uniquely placed to support patients with cancer who also have COVID-19. This may include managing severe symptoms such as dyspnea and agitation, as well as guiding advance care planning and goals of care conversations. In tandem, there is a need for palliative care teams to continue to provide care to patients with advanced cancer who are COVID-negative but who are at higher risk of infection and adverse outcomes related to COVID-19. This paper highlights the unique challenges faced by a palliative care team in terms of scaling up services in response to a global pandemic while simultaneously providing ongoing support to their patients with advanced cancer at a tertiary cancer center.

Molecular Aspects of Plant Growth Promotion and Protection by Bacillus subtilis.

Bacillus subtilis is one of the most widely studied plant growth-promoting rhizobacteria. It is able to promote plant growth as well as control plant pathogens through diverse mechanisms, including the improvement of nutrient availability and alteration of phytohormone homeostasis as well as the production of antimicrobials and triggering induced systemic resistance, respectively. Even though its benefits for crop production have been recognized and studied extensively under laboratory conditions, the success of its application in fields varies immensely. It is widely accepted that agricultural application of B. subtilis often fails because the bacteria are not able to persist in the rhizosphere. Bacterial colonization of plant roots is a crucial step in the interaction between microbe and plant and seems, therefore, to be of great importance for its growth promotion and biocontrol effects. A successful root colonization depends thereby on both bacterial traits, motility and biofilm formation, as well as on a signal interplay with the plant. This review addresses current knowledge about plant-microbial interactions of the B. subtilis species, including the various mechanisms for supporting plant growth as well as the necessity for the establishment of the relationship.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

Challenges of Providing Palliative and End-of-Life Care to Refugee Claimants in Canada: A Case Report.

Refugees are a highly vulnerable marginalized population with unique medical and psychosocial needs. Unfortunately, the Interim Federal Health Program (IFHP) in Canada partially covers the medical needs of refugee claimants but does not include hospice or home-based palliative care. This report describes the case of a refugee claimant cancer patient who was admitted to an inpatient tertiary cancer center medical oncology ward in Ontario, Canada, for ∼11 months due to insufficient community-based palliative care resources available for patients covered by the IFHP. This case report highlights the differences in palliative care coverage between the provincial health care coverage, Ontario Health Insurance Plan, and federal health care coverage for refugees, IFHP, from a practical point of view, how this can affect the palliative care available for patients and their families, and the impact on the Canadian health care system.

Network Analysis of Substituted Bullvalenes.

Substituted bullvalenes are dynamic shape-shifting molecules that exist within complex reaction networks. Herein, we report the synthesis of di- and trisubstituted bullvalenes and investigate their dynamic properties. Trisubstituted bullvalenes share a common major isomer which shows kinetic metastability. A survey of the thermodynamic and kinetic landscapes through computational analysis together with kinetic simulation provides a map of the internal dynamics of these systems.

Cellular hysteresis as a principle to maximize the efficacy of antibiotic therapy.

Antibiotic resistance has become one of the most dramatic threats to global health. While novel treatment options are urgently required, most attempts focus on finding new antibiotic substances. However, their development is costly, and their efficacy is often compromised within short time periods due to the enormous potential of microorganisms for rapid adaptation. Here, we developed a strategy that uses the currently available antibiotics. Our strategy exploits cellular hysteresis, which is the long-lasting, transgenerational change in cellular physiology that is induced by one antibiotic and sensitizes bacteria to another subsequently administered antibiotic. Using evolution experiments, mathematical modeling, genomics, and functional genetic analysis, we demonstrate that sequential treatment protocols with high levels of cellular hysteresis constrain the evolving bacteria by (i) increasing extinction frequencies, (ii) reducing adaptation rates, and (iii) limiting emergence of multidrug resistance. Cellular hysteresis is most effective in fast sequential protocols, in which antibiotics are changed within 12 h or 24 h, in contrast to the less frequent changes in cycling protocols commonly implemented in hospitals. We found that cellular hysteresis imposes specific selective pressure on the bacteria that disfavors resistance mutations. Instead, if bacterial populations survive, hysteresis is countered in two distinct ways, either through a process related to antibiotic tolerance or a mechanism controlled by the previously uncharacterized two-component regulator CpxS. We conclude that cellular hysteresis can be harnessed to optimize antibiotic therapy, to achieve both enhanced bacterial elimination and reduced resistance evolution.

Evaluation Protocol for Review of Method Validation Data by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals Expert Review Panel.

Methods under consideration as part of the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals process are to be evaluated against a set of Standard Method Performance RequirementsSM (SMPRs) via peer review by an expert review panel (ERP). A validation protocol and a checklist have been developed to assist the ERP to evaluate experimental data and to compare multiple candidate methods for each nutrient. Method performance against validation parameters mandated in the SMPRs as well as additional criteria are to be scored, with the method selected by the ERP proceeding to multilaboratory study prior to Final Action approval. These methods are intended to be used by the infant formula industry for the purposes of dispute resolution.

Status of methodology for the determination of fat-soluble vitamins in foods, dietary supplements, and vitamin premixes.

Fat-soluble vitamins (FSVs) include vitamin A, carotenoids, vitamins D, E, and K. New legislation is being introduced in many countries to reinforce regulatory compliance of declared concentrations of vitamins and other micronutrients in food products and dietary supplements. The levels of FSVs are likely to be more closely scrutinized due to their potential health risks associated with overdosing, in particular of vitamin D. However, a proviso of stricter regulatory compliance is that analytical methods must be fit-for-purpose, providing adequate accuracy and precision. Official methods have been published by organizations such as AOAC INTERNATIONAL, European Committee for Standardization, International Dairy Federation, U.S. Pharmacopeia, and International Organization for Standardization. The methods available for foods, dietary supplements, and vitamin premixes are evaluated in this review. In general, these methods show adequate precision for regulatory compliance; however, the field of application has not often been evaluated for a sufficiently large range of food matrixes. Gaps have been noted in the range of published official procedures, particularly for carotenoids and vitamin premixes. The potential of some recent developments in sample preparation and chromatographic techniques were evaluated to provide improved procedures for FSV analysis the future.

Analytical procedures for water-soluble vitamins in foods and dietary supplements: a review.

Water-soluble vitamins include the B-group vitamins and vitamin C. In order to correctly monitor water-soluble vitamin content in fortified foods for compliance monitoring as well as to establish accurate data banks, an accurate and precise analytical method is a prerequisite. For many years microbiological assays have been used for analysis of B vitamins. However they are no longer considered to be the gold standard in vitamins analysis as many studies have shown up their deficiencies. This review describes the current status of analytical methods, including microbiological assays and spectrophotometric, biosensor and chromatographic techniques. In particular it describes the current status of the official methods and highlights some new developments in chromatographic procedures and detection methods. An overview is made of multivitamin extractions and analyses for foods and supplements.

3D ultrasound measurement of change in carotid plaque volume: a tool for rapid evaluation of new therapies.

BACKGROUND AND PURPOSE: New therapies are being developed that are antiatherosclerotic but that lack intermediate end points, such as changes in plasma lipids, which can be measured to test efficacy. To study such treatments, it will be necessary to directly measure changes in atherosclerosis. The study was designed to determine sample sizes needed to detect effects of treatment using 3D ultrasound (US) measurement of carotid plaque. METHODS: In 38 patients with carotid stenosis >60%, age+/-SD 69.42+/-7.87 years, 15 female, randomly assigned in a double-blind fashion to 80 mg atorvastatin daily (n=17) versus placebo (n=21), we measured 3D plaque volume at baseline and after 3 months by disc segmentation of voxels representing carotid artery plaque, after 3D reconstruction of parallel transverse duplex US scans into volumetric 3D data sets. RESULTS: There were no significant differences in baseline risk factors. The rate of progression was 16.81+/-74.10 mm3 in patients taking placebo versus regression of -90.25+/-85.12 mm3 in patients taking atorvastatin (P<0.0001). CONCLUSIONS: 3D plaque volume measurement can show large effects of therapy on atherosclerosis in 3 months in sample sizes of approximately 20 patients per group. Sample sizes of 22 per group would be sufficient to show an effect size of 25% that of atorvastatin in 6 months. This technology promises to be very useful in evaluation of new therapies.

Antisense suppression of a (+)-delta-cadinene synthase gene in cotton prevents the induction of this defense response gene during bacterial blight infection but not its constitutive expression.

In cotton (Gossypium hirsutum) the enzyme (+)-delta-cadinene synthase (CDNS) catalyzes the first committed step in the biosynthesis of cadinane-type sesquiterpenes, such as gossypol, that provide constitutive and inducible protection against pests and diseases. A cotton cDNA clone encoding CDNS (cdn1-C4) was isolated from developing embryos and functionally characterized. Southern analysis showed that CDNS genes belong to a large multigene family, of which five genomic clones were studied, including three pseudogenes and one gene that may represent another subfamily of CDNS. CDNS expression was shown to be induced in cotton infected with either the bacterial blight or verticillium wilt pathogens. Constructs for the constitutive or seed-specific antisense suppression of cdn1-C4 were introduced into cotton by Agrobacterium-mediated transformation. Gossypol levels were not reduced in the seeds of transformants with either construct, nor was the induction of CDNS expression affected in stems of the constitutive antisense plants infected with Verticillium dahliae Kleb. However, the induction of CDNS mRNA and protein in response to bacterial blight infection of cotyledons was completely blocked in the constitutive antisense plants. These results suggest that cdn1-C4 may be involved specifically in the bacterial blight response and that the CDNS multigene family comprises a complex set of genes differing in their temporal and spatial regulation and responsible for different branches of the cotton sesquiterpene pathway.

Simultaneous quantification of vitamins A, D3 and E in fortified infant formulae by liquid chromatography-mass spectrometry.

A novel method for the simultaneous quantification of Vitamins A, D3 and E in fortified infant formulae has been developed using isocratic normal-phase liquid chromatography with positive atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). Food products were saponified and the vitamins were extracted by solid-phase extraction (SPE) on a Chromabond XTR cartridge. Quantification of Vitamins D3 and E were performed with Vitamin D2 and 5,7-dimethyltocol (DMT) as internal standards (IS), respectively while no IS was used for Vitamin A. Detection of the vitamins was made in the selected ion monitoring (SIM) mode. MS calibration curves were linear between 0.15 and 12 mg/l for Vitamin A, 5-400 microg/l for Vitamin D3 and 0.25-20 mg/l for Vitamin E with regression coefficient r2 > 0.996 and the limits of detection were below 1.4 ng. The repeatability (CV) obtained on a reference dietetic infant formula was 2.3% for Vitamin A, 2.6% for Vitamin E and 5.9% for Vitamin D3. The between-day variations (CV) over 6 days were in the ranges of 2.4-6.9% for the three vitamins. The mean recoveries from a reference infant formula spiked with all three vitamins ranged from 96 to 105% with a relative standard error less than 9%. The applicability of the method was demonstrated by analyzing a set of infant formula and infant cereals; similar results were obtained with the LC-MS method and reference HPLC methods.

Noninvasive techniques for the measurement of isovolumetric bladder pressure.

PURPOSE: A total of 184,000 prostatectomies were performed in the United States in 2000 for the relief of presumed bladder outlet obstruction. However, it has been reported that prostatectomy using current indications fails to bring about symptomatic improvement in approximately one-fourth of patients. Pressure flow studies are currently recognized as the gold standard for the diagnosis of bladder outlet obstruction. However, these studies are associated with a number of disadvantages. They are time consuming, invasive and expensive, and carry some morbidity for the patient. It has been suggested that the use of pressure flow studies should be mandatory before surgery. The invasive nature of this test limits its application, and a variety of noninvasive methods have been suggested to circumvent the need for conventional urodynamics. MATERIALS AND METHODS: We conducted a MEDLINE search of the published literature on the use of noninvasive techniques to measure bladder pressure. RESULTS: Two promising techniques involve the noninvasive measurement of isovolumetric detrusor pressure. The first of these methods uses an external condom catheter and the second an inflatable cuff around the penis. Both of these methods rely on the interruption of urinary flow and the measurement of the bladder pressure transmitted along the fluid column between bladder and site of urethral occlusion. An alternative strategy analyzes flow patterns following compression and release of the urethra during voiding. CONCLUSIONS: Of the methods reported the penile cuff, which is inflated during voiding, or the penile squeeze technique, which infers bladder pressure from flow patterns, would seem the most likely to be clinically useful. A noninvasive measure of bladder pressure, allied to a free flow rate, would give a useful adjunct to the assessment of men with lower urinary tract symptoms.

Installation of a ratchet tooth and pawl to restrict rotation in a cyclodextrin rotaxane.

Eight new [2]rotaxanes have been prepared, incorporating an alpha-cyclodextrin as the rotor, a stilbene as the axle, and trinitrophenyl substituents as capping groups. Strategies have been devised to elaborate these by linking the rotor to the axle, to produce two new [1]rotaxanes. Rotational motion in a selection of these rotaxanes has been investigated through the application of two-dimensional NMR spectroscopy by performing TOCSY, DQF-COSY, ROESY and HMQC experiments. This has shown that a methoxyl group incorporated on the stilbene and a succinamide joining the stilbene and the cyclodextrin behave analogously to a ratchet tooth and pawl, respectively, to restrict rotation.

Measurement of carotid plaque and effect of vitamin therapy for total homocysteine.

BACKGROUND: Many new therapies are being developed that are anti-atherosclerotic, but which do not change clinical parameters such as blood pressure or cholesterol. In order to measure the effects of these therapies it will be necessary to measure atherosclerosis. Elevated levels of plasma total homocysteine (tHcy) are associated with increased risk of stroke and myocardial infarction. Measurement of the effect of vitamin therapy on atherosclerosis has therefore been used as an example of this approach. METHODS: 2-Dimensional measurement of carotid plaque cross-sectional area has been used to measure effects of vitamin therapy in patients whose plaque is progressing despite intensive treatment of traditional risk factors. In clinic patients, addition of vitamin therapy halted progression of atherosclerosis, in samples of 50 patients over 2.5 years. However, in patients randomized to high-dose vs. low-dose vitamins in the Vitamin Intervention for Stroke Prevention trial, no difference in plaque progression was seen between high-dose and low-dose vitamin therapy. New methods have been developed for the measurement of 3-dimensional plaque volume, and for the measurement of plaque surface roughness. RESULTS: The accuracy and reliability of the measurement of plaque volume is 95%. This will permit measurement of effects of new anti-atherosclerotic therapies with much smaller sample sizes, in a much shorter time, than previously available methods such as intima-media thickness. CONCLUSION: Measurement of atherosclerotic plaque volume and roughness will greatly enhance the study of new anti-atherosclerotic therapies.