Temperature and Pressure Dependence of the Reaction between Ethyl Radical and Molecular Oxygen: Experiments and Master Equation Simulations.

We have used laser-photolysis - photoionization mass-spectrometry to measure the rate coefficient for the reaction between ethyl radical and molecular oxygen as a function of temperature (190-801 K) and pressure (0.2-6 Torr) under pseudo-first-order conditions ([He] ≫ [O2] ≫ [C2H5•]). Multiple ethyl precursor, photolysis wavelength, reactor material, and coating combinations were used. We reinvestigated the temperature dependence of the title reaction's rate coefficient to resolve inconsistencies in existing data. The current results indicate that some literature values for the rate coefficient may indeed be slightly too large. The experimental work was complemented with master equation simulations. We used the current and some previous rate coefficient measurements to optimize the values of key parameters in the master equation model. After optimization, the model was able to reproduce experimental falloff curves and C2H4 + HO2• yields. We then used the model to perform simulations over wide temperature (200-1500 K) and pressure (10-4-102 bar) ranges and provide the results in PLOG format to facilitate their use in atmospheric and combustion models.

Reproducibility of Neuroretinal Rim Measurements Obtained from High-Density Spectral Domain Optical Coherence Tomography Volume Scans.

Purpose: To compare the reproducibility of two-dimensional (2D) peripapillary retinal nerve fiber layer (RNFL) thickness and three-dimensional (3D) neuroretinal rim measurements using spectral domain optical coherence tomography (SDOCT) in normal and glaucoma subjects. Methods: One eye per subject for 27 normal and 40 glaucoma subjects underwent repeat SDOCT RNFL thickness scans and optic nerve volume scans on the same day. From the volume scan, custom software calculated five neuroretinal rim parameters: 3D minimum distance band (MDB) thickness, 3D MDB area, 3D rim volume, 2D rim area, and 2D rim thickness. Within-subject variance (Sw), coefficient of variation (CV), and intraclass correlation coefficient (ICC) were analyzed. Results: MDB thickness and RNFL thickness have similar reproducibility among normal and glaucoma subjects (eg, global MDB thickness CVs of 2.4% and 3.6%, and global RNFL thickness CVs of 1.3% and 2.2%; P > 0.05 for both comparisons). Reproducibility of MDB thickness was lower in glaucoma patients for the superior and inferior quadrants compared to normal subjects (CVs of 9.6% versus 3.4% and 6.9% versus 2.7%; P < 0.05, respectively). There were no statistically significant differences between both groups for RNFL thickness in the four quadrants. For both patient groups and for all regions, MDB thickness had the lowest CVs among all five neuroretinal rim parameters (eg, global MDB thickness CVs of 2.4% and 3.6% versus 3.0% and 18.9% for the other four neuroretinal rim parameters). Conclusion: Global MDB and global RNFL thickness are similarly reproducible among normal and glaucoma subjects, though MDB thickness for the superior and inferior quadrants is less reproducible among glaucoma subjects.

The GATA factor HANABA TARANU promotes runner formation by regulating axillary bud initiation and outgrowth in cultivated strawberry.

A runner, as an elongated branch, develops from the axillary bud (AXB) in the leaf axil and is crucial for the clonal propagation of cultivated strawberry (Fragaria × ananassa Duch.). Runner formation occurs in at least two steps: AXB initiation and AXB outgrowth. HANABA TARANU (HAN ) encodes a GATA transcription factor that affects AXB initiation in Arabidopsis and promotes branching in grass species, but the underlying mechanism is largely unknown. Here, the function of a strawberry HAN homolog FaHAN in runner formation was characterized. FaHAN transcripts can be detected in the leaf axils. Overexpression (OE) of FaHAN increased the number of runners, mainly by enhancing AXB outgrowth, in strawberry. The expression of the strawberry homolog of BRANCHED1 , a key inhibitor of AXB outgrowth in many plant species, was significantly downregulated in the AXBs of FaHAN -OE lines, whereas the expression of the strawberry homolog of SHOOT MERISTEMLESS, a marker gene for AXB initiation in Arabidopsis, was upregulated. Moreover, several genes of gibberellin biosynthesis and cytokinin signaling pathways were activated, whereas the auxin response pathway genes were repressed. Further assays indicated that FaHAN could be directly activated by FaNAC2, the overexpression of which in strawberry also increased the number of runners. The silencing of FaNAC2 or FaHAN inhibited AXB initiation and led to a higher proportion of dormant AXBs, confirming their roles in the control of runner formation. Taken together, our results revealed a FaNAC2-FaHAN pathway in the control of runner formation and have provided a means to enhance the vegetative propagation of cultivated strawberry.

Facilitating glaucoma diagnosis with intereye neuroretinal rim asymmetry analysis using spectral-domain optical coherence tomography.

Purpose: To determine whether intereye asymmetry of a three-dimensional neuroretinal rim parameter, the minimum distance band, is useful in differentiating normal eyes from those with open-angle glaucoma. Materials and Methods: This is a cross-sectional study of 28 normal subjects and 33 glaucoma subjects. Subjects underwent spectral domain optical coherence tomography imaging of both eyes. From high-density raster scans of the optic nerve head, a custom-designed segmentation algorithm calculated mean minimum distance band neuroretinal rim thickness globally, for four quadrants, and for four sectors. Intereye minimum distance band thickness asymmetry was calculated as the absolute difference in minimum distance band thickness values between the right and left eyes. Results: Increasing global minimum distance band thickness asymmetry was not associated with increasing age or increasing refractive error asymmetry. Glaucoma patients had thinner mean neuroretinal rim thickness values compared to normal patients (209.0 µm vs 306.0 µm [P < 0.001]). Glaucoma subjects had greater intereye thickness asymmetry compared to normal subjects for the global region (51.9 µm vs 17.6 µm [P < 0.001]) as well as for all quadrants and all sectors. For detecting glaucoma, a thickness asymmetry value >28.3 µm in the inferior quadrant yielded the greatest sum of sensitivity (87.9%) and specificity (75.0%). Globally, thickness asymmetry >30.7 µm yielded the greatest sum of sensitivity (66.7%) and specificity (89.3%). Conclusions: This study indicates that intereye neuroretinal rim minimum distance band asymmetry measurements, using high-density spectral domain optical coherence tomography volume scans, may be an objective and quantitative tool for assessing patients suspected of open-angle glaucoma.

Woodland strawberry axillary bud fate is dictated by a crosstalk of environmental and endogenous factors.

Plant architecture is defined by fates and positions of meristematic tissues and has direct consequences on yield potential and environmental adaptation of the plant. In strawberries (Fragaria vesca L. and F. × ananassa Duch.), shoot apical meristems can remain vegetative or differentiate into a terminal inflorescence meristem. Strawberry axillary buds (AXBs) are located in leaf axils and can either remain dormant or follow one of the two possible developmental fates. AXBs can either develop into stolons needed for clonal reproduction or into branch crowns (BCs) that can bear their own terminal inflorescences under favorable conditions. Although AXB fate has direct consequences on yield potential and vegetative propagation of strawberries, the regulation of AXB fate has so far remained obscure. We subjected a number of woodland strawberry (F. vesca L.) natural accessions and transgenic genotypes to different environmental conditions and growth regulator treatments to demonstrate that strawberry AXB fate is regulated either by environmental or endogenous factors, depending on the AXB position on the plant. We confirm that the F. vesca GIBBERELLIN20-oxidase4 (FvGA20ox4) gene is indispensable for stolon development and under tight environmental regulation. Moreover, our data show that apical dominance inhibits the outgrowth of the youngest AXB as BCs, although the effect of apical dominance can be overrun by the activity of FvGA20ox4. Finally, we demonstrate that the FvGA20ox4 is photoperiodically regulated via FvSOC1 (F. vesca SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1) at 18°C, but at higher temperature of 22°C an unidentified FvSOC1-independent pathway promotes stolon development.

Structure-Function Mapping Using a Three-Dimensional Neuroretinal Rim Parameter Derived From Spectral Domain Optical Coherence Tomography Volume Scans.

Purpose: To assess the structure-function relationship in glaucoma using Humphrey visual field (HVF) perimetry and a three-dimensional neuroretinal rim parameter derived from spectral domain optical coherence tomography (SD-OCT) volume scans. Methods: Structure-function correlation was analyzed globally and regionally (four quadrants and four sectors). Structural data included peripapillary retinal nerve fiber layer (RNFL) thickness and minimum distance band (MDB) neuroretinal rim thickness, defined as the shortest distance between the inner cup surface and the outer retinal pigment epithelium/Bruch's membrane complex. Logarithmic regression analyses were performed and Pearson correlation coefficients determined to assess relationship strength. Results: The study consisted of 102 open-angle glaucoma patients and 58 healthy subjects. The Pearson correlation coefficient for global MDB thickness (R = 0.585) was higher than for global RNFL thickness (R = 0.492), but the difference was not statistically significant (P = 0.18). The correlation coefficients for regional MDB thicknesses and corresponding HVF sensitivities were higher than those for regional RNFL thicknesses and HVF in six out of eight regions (P = 0.08 to 0.47). In the remaining two out of eight regions, the correlation coefficients were higher for RNFL thickness than for MDB thickness (P = 0.15 to 0.20). Conclusions: Three-dimensional MDB neuroretinal rim thickness relates to visual function as strongly as the most commonly used SD-OCT parameter for glaucoma, two-dimensional peripapillary RNFL thickness. Translational Relevance: This paper illustrates the potential for 3D OCT algorithms to improve in vivo imaging in glaucoma.

Peripheral Leakage on Ultra-Widefield Fluorescein Angiography in Patients With Inherited Retinal Degeneration.

Purpose: This report illustrates that peripheral vascular leakage on ultra-widefield fluorescein angiography (FA) can occur in patients with inherited retinal degeneration (IRD) without evidence of a separate cause of leakage. Methods: We searched the electronic medical records of the Massachusetts Eye and Ear Infirmary from 2010 to 2019 for patients with an IRD diagnosis and examination with an ultra-widefield FA. Images from FAs were evaluated in masked fashion by 2 retina specialists. Documentation of an evaluation for alternative causes of vascular leakage was recorded, as well as results from electroretinography, Goldmann perimetry, and genetic testing. Results: A total of 305 patients with an IRD diagnosis and FA procedure code were identified. Of these, 26 patients had both a clinical diagnosis of IRD and ultra-widefield FA on detailed medical-record review. Three patients had FA to evaluate a Coats-like response and were excluded. Of the remaining 23, 4 patients (17%) had significant peripheral leakage on FA. Of these, 1 had pericentral retinitis pigmentosa (for which the genetic cause of disease was undefined), 1 had Refsum disease with confirmed biallelic PHYH mutations, 1 had a CRB1-associated macular dystrophy, and 1 had CERKL-associated macular dystrophy. There was no evidence of ocular inflammation from history, examination, or laboratory testing to account for the FA findings. Of the 19 patients without significant leakage, 4 had minimal leakage and 15 had no peripheral leakage. Conclusions: Peripheral retinal vascular leakage can be seen on ultra-widefield FA in patients with IRD that is likely due to the IRD disease process itself rather than to an additional, distinct eye condition.

Artifact Rates for 2D Retinal Nerve Fiber Layer Thickness Versus 3D Neuroretinal Rim Thickness Using Spectral-Domain Optical Coherence Tomography.

Purpose: To compare the rates of clinically significant artifacts for two-dimensional peripapillary retinal nerve fiber layer (RNFL) thickness versus three-dimensional (3D) neuroretinal rim thickness using spectral-domain optical coherence tomography (SD-OCT). Methods: Only one eye per patient was used for analysis of 120 glaucoma patients and 114 normal patients. For RNFL scans and optic nerve scans, 15 artifact types were calculated per B-scan and per eye. Neuroretinal rim tissue was quantified by the minimum distance band (MDB). Global MDB neuroretinal rim thicknesses were calculated before and after manual deletion of B-scans with artifacts and subsequent automated interpolation. A clinically significant artifact was defined as one requiring manual correction or repeat scanning. Results: Among glaucomatous eyes, artifact rates per B-scan were significantly more common in RNFL scans (61.7%, 74 of 120) compared to B-scans in neuroretinal rim volume scans (20.9%, 1423 of 6820) (95% confidence interval [CI], 31.6-50.0; P < 0.0001). For clinically significant artifact rates per eye, optic nerve scans had significantly fewer artifacts (15.8% of glaucomatous eyes, 13.2% of normal eyes) compared to RNFL scans (61.7% of glaucomatous eyes, 25.4% of normal eyes) (glaucoma group: 95% CI, 34.1-57.5, P < 0.0001; normal group: 95% CI, 1.3-23.3, P = 0.03). Conclusions: Compared to the most commonly used RNFL thickness scans, optic nerve volume scans less frequently require manual correction or repeat scanning to obtain accurate measurements. Translational Relevance: This paper illustrates the potential for 3D OCT algorithms to improve in vivo imaging in glaucoma.

Economic and Environmental Impact of Single-use Plastics at a Large Ophthalmology Outpatient Service.

PRéCIS:: The use of disposable tonometer prisms and gonioscopy lenses at a large ophthalmology outpatient service incurs significant financial and environmental waste that may not be justified given the limited data surrounding arguments for their use. PURPOSE: To quantify the economic and environmental burden of single-use plastics from an ophthalmology outpatient service at a large tertiary hospital and describe the relative value and evidence for the safety of disposable versus nondisposable tonometer prisms and gonioscopy lenses. METHODS: The total number of single-use applanation tonometer prisms and gonioscopy lenses used per year at Boston Medical Center (BMC) was estimated, and the average dollars spent and plastic waste generated in kilograms per year were then determined. These values were compared with the total spending and waste that would be produced if the clinic were to use nondisposable tonometer prisms and gonioscopy lenses exclusively. RESULTS: Single-use tonometer prisms cost an average of $70,282 per year and produce ~100.8 kg of plastic waste per year at BMC. Single-use gonioscopy lenses cost ~$9,040 per year and produce 8.8 kg of plastic waste per year at BMC. An excess of $65,185 and 109.6 kg of plastic waste could be avoided each year by only using nondisposable tonometer prisms and gonioscopy lenses at the BMC ophthalmology outpatient service. CONCLUSIONS: Single-use plastics in ophthalmology outpatient services generate significant environmental waste and financial cost compared with nondisposable instruments. This cost may outweigh the benefits of these instruments given the limited data surrounding arguments for their use.

I-FABP Is Higher in People With Chronic HIV Than Elite Controllers, Related to Sugar and Fatty Acid Intake and Inversely Related to Body Fat in People With HIV.

BACKGROUND: Intestinal fatty acid binding protein (I-FABP) has been shown to be a marker of intestinal damage among people living with HIV. We hypothesized that I-FABP would be increased in chronically HIV-infected patents more than elite controllers and would relate to specific nutrient intake and body composition. METHODS: In an observational study, serum I-FABP was measured by enzyme-linked immunosorbent assay. Anthropometric measurements, dual-energy x-ray absorptiometry, and single-slice abdominal computed tomography were obtained to assess body composition, as well as visceral and subcutaneous adipose tissue areas (VAT and SAT). Dietary intake was assessed using 4-day food records. RESULTS: One hundred forty-nine people with chronic HIV (65% male, 47 ± 7 years of age, 54.7% white, and 14 ± 6 years of known HIV), 10 elite controllers (60% male, 53 ± 8 years, 60% white, and 20 ± 7 years of known HIV), and 69 HIV-negative controls (59.4% male, 46 ± 7 years, and 52.2% white) were included in the analysis. I-FABP was significantly higher in HIV progressors relative to HIV-negative controls and elite controllers. In the chronic HIV group, I-FABP was positively associated with dietary intake of added sugar and with saturated fatty acids. I-FABP was inversely associated with body mass index, VAT, and SAT. I-FABP also correlated with MCP-1, CXCL10, sCD163, and lipopolysaccharide (LPS) among all participants. CONCLUSIONS: I-FABP was increased among chronically HIV-infected patients to a greater degree than in elite controllers and was related to nutrient intake and body composition in HIV progressors. Future studies to investigate the role of intestinal damage on nutrient absorption are needed to elucidate the mechanisms of these relationships. TRIAL REGISTRATION IDENTIFIER: NCT00455793.

Brief Report: Statin Effects on Myocardial Fibrosis Markers in People Living With HIV.

BACKGROUND: In observational studies, patients with HIV have higher levels of soluble ST2 (sST2), galectin-3, and growth differentiation factor-15 (GDF-15) than non-HIV controls. As statins exert pleiotropic immunomodulatory effects that may affect markers of myocardial fibrosis, the objective of the current study is to determine whether biomarkers of myocardial fibrosis reflecting subclinical pathology may be modified by statin therapy in patients with HIV. SETTING AND METHODS: Forty HIV+ men and women participated in a single center 12-month randomized, double-blind placebo-controlled trial of atorvastatin 40 mg every day vs. placebo. At baseline and 12-months, sST2, GDF-15, galectin-3 were measured. RESULTS: The changes in sST2 were -0.310 (-4.195, 2.075) vs. 1.163 (0.624, 4.715) ng/mL, median (interquartile range) atorvastatin vs. placebo (P = 0.04). The change in sST2 was significantly related to changes in monocyte activation marker sCD14 (r = 0.63, P < 0.0001) and MCP (r = 0.52, P = 0.0009), markers of generalized inflammation hs-IL-6 (r = 0.58, P = 0.0002), oxLDL (r = 0.49, P = 0.002), and GDF-15 (r = 0.54, P = 0.0008). CONCLUSIONS: sST2, a member of the IL-1 receptor family and a marker of fibrosis and inflammation increases over time among patients with HIV and this increase is attenuated by statin therapy in HIV. This effect may relate to immunomodulatory mechanisms of statins.

Altered regulation of TERMINAL FLOWER 1 causes the unique vernalisation response in an arctic woodland strawberry accession.

Vernalisation requirement is an agriculturally important trait that postpones the development of cold-sensitive floral organs until the spring. The family Rosaceae includes many agriculturally important fruit and berry crops that suffer from crop losses caused by frost injury to overwintering flower buds. Recently, a vernalisation-requiring accession of the Rosaceae model woodland strawberry (Fragaria vesca) has been identified in northern Norway. Understanding the molecular basis of the vernalisation requirement in this accession would advance the development of strawberry cultivars better adapted to temperate climate. We use gene silencing, gene expression analysis, genetic mapping and population genomics to study the genetic basis of the vernalisation requirement in woodland strawberry. Our results indicate that the woodland strawberry vernalisation requirement is endemic to northern Norwegian population, and mapping data suggest the orthologue of TERMINAL FLOWER1 (FvTFL1) as the causal floral repressor. We demonstrate that exceptionally low temperatures are needed to downregulate FvTFL1 and to make these plants competent to induce flowering at low postvernalisation temperatures in the spring. We show that altered regulation of FvTFL1 in the northern Norwegian woodland strawberry accession postpones flower induction until the spring, allowing plants to avoid winter injuries of flower buds that commonly occur in temperate regions.

Statin Effects to Reduce Hepatosteatosis as Measured by Computed Tomography in Patients With Human Immunodeficiency Virus.

Hepatosteatosis is highly prevalent among patients living with human immunodeficiency virus. In a 1-year, randomized, double-blind trial of atorvastatin or placebo, atorvastatin increased liver/spleen ratio among patients with nonalcoholic fatty liver disease, indicating a reduction in hepatosteatosis. This reduction in hepatosteatosis is associated with reduction in low-density lipoprotein cholesterol with statin therapy.

Serum oxidized low-density lipoprotein decreases in response to statin therapy and relates independently to reductions in coronary plaque in patients with HIV.

OBJECTIVE: Circulating oxidized low-density lipoprotein (oxLDL) levels are elevated in HIV-infected patients and have been associated with atherosclerosis. Statins have been shown to reduce plaque on coronary computed tomography angiography (cCTA) in HIV-infected individuals. Thus, we investigated the effect of statins on serum oxLDL levels and the relationship between changes in oxLDL and coronary atherosclerosis on cCTA in patients with HIV. DESIGN: We previously conducted a 12-month randomized, placebo-controlled trial with atorvastatin in 40 HIV-infected patients on stable antiretroviral therapy with subclinical coronary atherosclerosis and low-density lipoprotein (LDL)-cholesterol less than 130 mg/dl. METHODS: In the current analysis, patients underwent cCTA and measurements of serum oxLDL, sCD14, sCD163, lipoprotein phospholipase-A2, and fasting lipids at baseline and end of the study. RESULTS: Nineteen patients were randomized to atorvastatin and 21 patients to placebo. Serum oxLDL decreased -22.7% (95% CI -28.7 to -16.7) in the atorvastatin group and increased 7.5% (95% CI -3.3 to 18.4) in the placebo group (P < 0.0001). Change in oxLDL significantly correlated with changes in noncalcified plaque volume, total plaque volume, positively remodeled plaque, and low attenuation plaque. The association between changes in oxLDL and noncalcified plaque volume was independent of the baseline 10-year Framingham risk, LDL, CD4 cell count, and viral load. CONCLUSION: Statins lower oxLDL levels in HIV-infected patients, and reductions in oxLDL are related to improvements in coronary atherosclerosis, independent of traditional cardiovascular risk factors. Reductions in oxLDL may be one mechanism through which statins exert beneficial effects on reducing atherosclerosis in HIV-infected individuals.

Divergence of a strain of Pseudomonas aeruginosa during an outbreak of ovine mastitis.

Bacterial infections causing mastitis in sheep can result in severe economic losses for farmers. A large survey of milk samples from ewes with mastitis in Sardinia, Italy, indicated an increasing prevalence of Pseudomonas aeruginosa infections. It has been shown previously that during chronic, biofilm-associated infections P. aeruginosa populations diversify. We report the phenotypic and genomic characterisation of two clonal P. aeruginosa isolates (PSE305 and PSE306) from a mastitis infection outbreak, representing distinct colony morphology variants. In addition to pigment production, PSE305 and PSE306 differed in phenotypic characteristics including biofilm formation, utilisation of various carbon and nitrogen sources, twitching motility. We found higher levels of expression of genes associated with biofilm formation (pelB) and twitching motility (flgD) in PSE305, compared to the biofilm and twitching-defective PSE306. Comparative genomics analysis revealed single nucleotide polymorphisms (SNPs) and minor insertion/deletion variations between PSE305 and PSE306, including a SNP mutation in the pilP gene of PSE306. By introducing a wild-type pilP gene we were able to partially complement the defective twitching motility of PSE306. There were also three larger regions of difference between the two genomes, indicating genomic instability. Hence, we have demonstrated that P. aeruginosa population divergence can occur during an outbreak of mastitis, leading to significant variations in phenotype and genotype, and resembling the behaviour of P. aeruginosa during chronic biofilm-associated infections.

The Fragaria vesca homolog of suppressor of overexpression of constans1 represses flowering and promotes vegetative growth.

In the annual long-day plant Arabidopsis thaliana, suppressor of overexpression of constans1 (SOC1) integrates endogenous and environmental signals to promote flowering. We analyzed the function and regulation of the SOC1 homolog (Fragaria vesca [Fv] SOC1) in the perennial short-day plant woodland strawberry (Fragaria vesca). We found that Fv SOC1 overexpression represses flower initiation under inductive short days, whereas its silencing causes continuous flowering in both short days and noninductive long days, similar to mutants in the floral repressor Fv terminal flower1 (Fv TFL1). Molecular analysis of these transgenic lines revealed that Fv SOC1 activates Fv TFL1 in the shoot apex, leading to the repression of flowering in strawberry. In parallel, Fv SOC1 regulates the differentiation of axillary buds to runners or axillary leaf rosettes, probably through the activation of gibberellin biosynthetic genes. We also demonstrated that Fv SOC1 is regulated by photoperiod and Fv flowering locus T1, suggesting that it plays a central role in the photoperiodic control of both generative and vegetative growth in strawberry. In conclusion, we propose that Fv SOC1 is a signaling hub that regulates yearly cycles of vegetative and generative development through separate genetic pathways.

Sub-inhibitory concentrations of some antibiotics can drive diversification of Pseudomonas aeruginosa populations in artificial sputum medium.

BACKGROUND: Pseudomonas aeruginosa populations within the cystic fibrosis lung exhibit extensive phenotypic and genetic diversification. The resultant population diversity is thought to be crucial to the persistence of infection and may underpin the progression of disease. However, because cystic fibrosis lungs represent ecologically complex and hostile environments, the selective forces driving this diversification in vivo remain unclear. We took an experimental evolution approach to test the hypothesis that sub-inhibitory antibiotics can drive diversification of P. aeruginosa populations. Replicate populations of P. aeruginosa LESB58 were cultured for seven days in artificial sputum medium with and without sub-inhibitory concentrations of various clinically relevant antibiotics. We then characterised diversification with respect to 13 phenotypic and genotypic characteristics. RESULTS: We observed that higher population diversity evolved in the presence of azithromycin, ceftazidime or colistin relative to antibiotic-free controls. Divergence occurred due to alterations in antimicrobial susceptibility profiles following exposure to azithromycin, ceftazidime and colistin. Alterations in colony morphology and pyocyanin production were observed following exposure to ceftazidime and colistin only. Diversification was not observed in the presence of meropenem. CONCLUSIONS: Our study indicates that certain antibiotics can promote population diversification when present in sub-inhibitory concentrations. Hence, the choice of antibiotic may have previously unforeseen implications for the development of P. aeruginosa infections in the lungs of cystic fibrosis patients.

Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung.

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests. Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions. An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a > 128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions. The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods. Several in vitro models have been used previously to study P. aeruginosa biofilms. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung. In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa and affect antibiotic susceptibility. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Mutation in TERMINAL FLOWER1 reverses the photoperiodic requirement for flowering in the wild strawberry Fragaria vesca.

Photoperiodic flowering has been extensively studied in the annual short-day and long-day plants rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), whereas less is known about the control of flowering in perennials. In the perennial wild strawberry, Fragaria vesca (Rosaceae), short-day and perpetual flowering long-day accessions occur. Genetic analyses showed that differences in their flowering responses are caused by a single gene, SEASONAL FLOWERING LOCUS, which may encode the F. vesca homolog of TERMINAL FLOWER1 (FvTFL1). We show through high-resolution mapping and transgenic approaches that FvTFL1 is the basis of this change in flowering behavior and demonstrate that FvTFL1 acts as a photoperiodically regulated repressor. In short-day F. vesca, long photoperiods activate FvTFL1 mRNA expression and short days suppress it, promoting flower induction. These seasonal cycles in FvTFL1 mRNA level confer seasonal cycling of vegetative and reproductive development. Mutations in FvTFL1 prevent long-day suppression of flowering, and the early flowering that then occurs under long days is dependent on the F. vesca homolog of FLOWERING LOCUS T. This photoperiodic response mechanism differs from those described in model annual plants. We suggest that this mechanism controls flowering within the perennial growth cycle in F. vesca and demonstrate that a change in a single gene reverses the photoperiodic requirements for flowering.