A protocol for registration and correction of multicolour STED superresolution images.

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.

Efficacy of grass pollen sublingual immunotherapy for three consecutive seasons and after cessation of treatment: the ECRIT study.

BACKGROUND: Data supporting a carry-over effect with sublingual immunotherapy (SLIT) are scarce. This randomized, double-blind, placebo-controlled study evaluated the efficacy, carry-over effect and safety of grass pollen SLIT using co-seasonal treatment. METHODS: Patients (7.9-64.7 years) with grass pollen allergy received ultra-rush titration with increasing doses (30, 90, 150 and 300 IR) of a 5-grass pollen mixture every 20 min at the start of the pollen seasons, followed by 300 IR daily until the end of the pollen seasons. A baseline season (no SLIT) was followed by three consecutive treatment seasons and one follow-up season. Symptoms, medication and adverse events were documented and specific immunoglobulin (Ig)E and IgG(4) measured. RESULTS: Data were analysed for 183 of the 213 randomized patients. Mean treatment duration varied between seasons (81.8-92.7 days). Combined scores (symptoms and medication) improved progressively across treatment seasons (up to 44.7% improvement for SLIT compared with baseline) and fluctuated between -11.3% and -14.8% for placebo (P < 0.05). Similar changes were observed for symptom scores, with a successive decrease of 39.7% (SLIT) and fluctuations between +13.6% and -1.51% for placebo (P < 0.05). Combined score (P = 0.0508) and symptom score improvements (P = 0.0144) with SLIT continued during follow up. Increases in specific IgG(4) observed in the first season were sustained for SLIT vs placebo throughout treatment (P = 0.0001). Titration and daily SLIT were well tolerated. No serious systemic or anaphylactic reactions were reported. CONCLUSIONS: Seasonal SLIT with ultra-rush titration is well tolerated and effective from the first treatment season onwards. These data indicate a carry-over effect of seasonal SLIT.

Efficacy of grass pollen sublingual immunotherapy for three consecutive seasons and after cessation of treatment: the ECRIT study.

BACKGROUND: Data supporting a carry-over effect with sublingual immunotherapy (SLIT) are scarce. This randomized, double-blind, placebo-controlled study evaluated the efficacy, carry-over effect and safety of grass pollen SLIT using co-seasonal treatment. METHODS: Patients (7.9-64.7 years) with grass pollen allergy received ultra-rush titration with increasing doses (30, 90, 150 and 300 IR) of a 5-grass pollen mixture every 20 min at the start of the pollen seasons, followed by 300 IR daily until the end of the pollen seasons. A baseline season (no SLIT) was followed by three consecutive treatment seasons and one follow-up season. Symptoms, medication and adverse events were documented and specific immunoglobulin (Ig)E and IgG(4) measured. RESULTS: Data were analysed for 183 of the 213 randomized patients. Mean treatment duration varied between seasons (81.8-92.7 days). Combined scores (symptoms and medication) improved progressively across treatment seasons (up to 44.7% improvement for SLIT compared with baseline) and fluctuated between -11.3% and -14.8% for placebo (P < 0.05). Similar changes were observed for symptom scores, with a successive decrease of 39.7% (SLIT) and fluctuations between +13.6% and -1.51% for placebo (P < 0.05). Combined score (P = 0.0508) and symptom score improvements (P = 0.0144) with SLIT continued during follow up. Increases in specific IgG(4) observed in the first season were sustained for SLIT vs placebo throughout treatment (P = 0.0001). Titration and daily SLIT were well tolerated. No serious systemic or anaphylactic reactions were reported. CONCLUSIONS: Seasonal SLIT with ultra-rush titration is well tolerated and effective from the first treatment season onwards. These data indicate a carry-over effect of seasonal SLIT.

Safety of two cluster schedules for subcutaneous immunotherapy in allergic rhinitis or asthma patients sensitized to inhalant allergens.

BACKGROUND: Subcutaneous immunotherapy (SCIT) usually requires a long titration phase, which can be associated with various adverse events (AEs). OBJECTIVES: It was the aim of this study to determine the safety of 2 cluster regimens for SCIT in patients with allergic rhinitis, with or without mild or moderate allergic asthma, who were sensitized to grass and/or tree pollen, or house dust mites (HDM). PATIENTS AND METHODS: Adult patients were included in a European, open-label, prospective trial. Pollen-allergic patients received grass pollen, grass and olive pollen, or hazel, alder and birch pollen according to a 3-week titration cluster. HDM-allergic patients received HDM extract according to a 2-week titration cluster. The safety of the titration phase was assessed in terms of local and systemic AEs. RESULTS: The safety analysis included 157 patients: 110 received pollen and 47 HDM extract. During the cluster titration, 248 AE episodes were reported in the pollen group and 113 in the HDM group; these were mainly local reactions. Around one third of patients (30.9% pollen and 38.3% HDM) did not experience any AE. In most cases (67.1% of pollen and 71.1% of HDM patients), AEs did not lead to a change in titration schedule. No anaphylactic reaction or other serious life-threatening systemic AEs were reported. Only 2 patients in the HDM group discontinued treatment because of AEs. CONCLUSIONS: Rapid cluster titration was well tolerated in adults with allergic rhinitis, with or without mild to moderate allergic asthma, due to pollen or HDM. This short-titration, high-dose cluster regime may allow better patient compliance and cost savings.

Safety and tolerability of seasonal ultra-rush, high-dose sublingual-swallow immunotherapy in allergic rhinitis to grass and tree pollens: an observational study in 193 children and adolescents.

OBJECTIVE: We conducted a large observational study in 193 children and adolescents with allergic rhinitis due to grass or tree pollens to evaluate the safety and tolerability of an ultrarush high-dose sublingual immunotherapy (SLIT) regimen reaching a maintenance dose of 300 index of reactivity within 90 minutes. METHODS: Children and adolescents aged 5 to 17 years with at least a 1-year medical history of allergic rhinitis with or without mild to moderate asthma due to tree pollens (birch, alder, hazel) or grass pollens (cocksfoot, meadow grass, rye grass, sweet vernal grass, timothy) were recruited. Standardized grass and tree pollen allergen extracts were used for ultrarush titration and subsequent coseasonal maintenance. RESULTS: During ultrarush titration, 60 patients (31%) reported 117 predominantly mild and local adverse events, which resolved within 150 minutes. During the maintenance phase, 562 adverse events were reported; the most frequent local events were oral pruritus, burning sensation, lip or tongue swelling, and gastrointestinal symptoms, and the most frequent systemic events were rhinoconjunctivitis and asthma. There was 1 clinically significant asthma event in an 11-year old boy with known asthma in whom SLIT was resumed after an interval of 4 days. CONCLUSION: Ultrarush titration was safe and well tolerated. Pediatric patients with asthma should be carefully monitored and adequately trained to use their rescue medications.

Repair of specific base pair mismatches formed during meiotic recombination in the yeast Saccharomyces cerevisiae.

Heteroduplexes formed between DNA strands derived from different homologous chromosomes are an intermediate in meiotic crossing over in the yeast Saccharomyces cerevisiae and other eucaryotes. A heteroduplex formed between wild-type and mutant genes will contain a base pair mismatch; failure to repair this mismatch will lead to postmeiotic segregation (PMS). By analyzing the frequency of PMS for various mutant alleles in the yeast HIS4 gene, we showed that C/C mismatches were inefficiently repaired relative to all other point mismatches. These other mismatches (G/G, G/A, T/T, A/A, T/C, C/A, A/A, and T/G) were repaired with approximately the same efficiency. We found that in spores with unrepaired mismatches in heteroduplexes, the nontranscribed strand of the HIS4 gene was more frequently donated than the transcribed strand. In addition, the direction of repair for certain mismatches was nonrandom.

Generic stabilizability for time-delayed feedback control.

Time-delayed feedback control is one of the most successful methods to discover dynamically unstable features of a dynamical system in an experiment. This approach feeds back only terms that depend on the difference between the current output and the output from a fixed time T ago. Thus, any periodic orbit of period T in the feedback-controlled system is also a periodic orbit of the uncontrolled system, independent of any modelling assumptions. It has been an open problem whether this approach can be successful in general, that is, under genericity conditions similar to those in linear control theory (controllability), or if there are fundamental restrictions to time-delayed feedback control. We show that, in principle, there are no restrictions. This paper proves the following: for every periodic orbit satisfying a genericity condition slightly stronger than classical linear controllability, one can find control gains that stabilize this orbit with extended time-delayed feedback control. While the paper's techniques are based on linear stability analysis, they exploit the specific properties of linearizations near autonomous periodic orbits in nonlinear systems, and are, thus, mostly relevant for the analysis of nonlinear experiments.

Variable responses of human microbiomes to dietary supplementation with resistant starch.

BACKGROUND: The fermentation of dietary fiber to various organic acids is a beneficial function provided by the microbiota in the human large intestine. In particular, butyric acid contributes to host health by facilitating maintenance of epithelial integrity, regulating inflammation, and influencing gene expression in colonocytes. We sought to increase the concentration of butyrate in 20 healthy young adults through dietary supplementation with resistant starch (unmodified potato starch-resistant starch (RS) type 2). METHODS: Fecal samples were collected from individuals to characterize butyrate concentration via liquid chromatography and composition of the microbiota via surveys of 16S rRNA-encoding gene sequences from the Illumina MiSeq platform. Random Forest and LEfSe analyses were used to associate responses in butyrate production to features of the microbiota. RESULTS: RS supplementation increased fecal butyrate concentrations in this cohort from 8 to 12 mmol/kg wet feces, but responses varied widely between individuals. Individuals could be categorized into three groups based upon butyrate concentrations before and during RS: enhanced, high, and low (n = 11, 3, and 6, respectively). Fecal butyrate increased by 67 % in the enhanced group (from 9 to 15 mmol/kg), while it remained ≥11 mmol/kg in the high group and ≤8 mmol/kg in the low group. Microbiota analyses revealed that the relative abundance of RS-degrading organisms-Bifidobacterium adolescentis or Ruminococcus bromii-increased from ~2 to 9 % in the enhanced and high groups, but remained at ~1.5 % in the low group. The lack of increase in RS-degrading bacteria in the low group may explain why there was no increase in fecal butyrate in response to RS. The microbiota of individuals in the high group were characterized by an elevated abundance of the butyrogenic microbe Eubacterium rectale (~6 % in high vs. 3 % in enhanced and low groups) throughout the study. CONCLUSIONS: We document the heterogeneous responses in butyrate concentrations upon RS supplementation and identify characteristic of the microbiota that appear to underlie this variation. This study complements and extends other studies that call for personalized approaches to manage beneficial functions provided by gut microbiomes.

Ethical considerations in planning and conducting research on human subjects.

The ethical conduct of research on humans requires more than a caring researcher and a legalistic adherence to federal regulations. It requires good planning and managing of issues accruing from the research relationship--communication, risk and perception of risk, and benefits--to all who have a stake in the relationship and outcome. In 1978 the National Commission for the Protection of Human Subjects in Biomedical and Behavioral Research identified ethical principles that should govern research on humans. Institutional review boards (IRBs) now exist in every organization that receives federal funding for human-subjects research. Researchers have an obligation to study the requirements of their IRB when they plan their research. Further, they must establish appropriate procedures for obtaining informed consent and assessing risk, risk perception, and ways to increase the benefits of research.

Nonlinear dynamics of semiconductor lasers with active optical feedback.

An in-depth theoretical as well as experimental analysis of the nonlinear dynamics in semiconductor lasers with active optical feedback is presented. Use of a monolithically integrated multisection device of submillimeter total length provides access to the short-cavity regime. By introducing an amplifier section as a special feature, phase and strength of the feedback can be separately tuned. In this way, the number of modes involved in the laser action can be adjusted. We predict and observe specific dynamical scenarios. Bifurcations mediate various transitions in the device output, from single-mode steadystate to self-pulsation and between different kinds of self-pulsations, reaching eventually chaotic behavior in the multimode limit.

Will the new code help researchers to be more ethical?

A code of ethics has 2 largely incompatible objectives: to set forth enforceable minimal standards of conduct and to teach about or invoke ethical conduct. The section of the new American Psychological Association code dealing with research ethics achieves the former to some degree. However, it neither provides needed education in the ethics of research nor states where the reader might turn for such information. The code is particularly deficient in the following areas: privacy and confidentiality; institutional review boards; deception; debriefing; data sharing; and research on marginal populations, on children and adolescents, and in organizational contexts. Suggestions are offered for providing a bibliographic resource, in hard copy and on-line, that would stimulate independent interest, scholarship, education, and research on research ethics.

The effects of pulsed fluid irrigation on bony ingrowth in porous polyethylene implants.

Twelve adult dogs were used to evaluate the effect of pulsed isotonic saline irrigation on bony ingrowth. The distal supracondylar ridge of the knee was transected. Six dogs had the area irrigated with a pulsed lavage system for 15 sec and six dogs did not. A porous piece of polyethylene was then stapled to the cancellous surface. Three of each were evaluated histologically at 3 weeks and 6 weeks. The pulsed irrigation significantly decreased the depth of bone penetration into the sections of polyethylene.

Systematic experimental exploration of bifurcations with noninvasive control.

We present a general method for systematically investigating the dynamics and bifurcations of a physical nonlinear experiment. In particular, we show how the odd-number limitation inherent in popular noninvasive control schemes, such as (Pyragas) time-delayed or washout-filtered feedback control, can be overcome for tracking equilibria or forced periodic orbits in experiments. To demonstrate the use of our noninvasive control, we trace out experimentally the resonance surface of a periodically forced mechanical nonlinear oscillator near the onset of instability, around two saddle-node bifurcations (folds) and a cusp bifurcation.

Medication persistence with long-term, specific grass pollen immunotherapy measured by prescription renewal rates.

OBJECTIVE: We assessed medication persistence using prescription renewal rates for grass pollen specific immunotherapy (SIT) in a representative population of patients in Germany to evaluate whether the perception of superior persistence for the subcutaneous route compared to the sublingual route could be confirmed in clinical practice. METHODS: Individual prescriptions for allergen immunotherapy were extracted from a national prescription database (INSIGHT Health) and followed over 3 years on a per-patient basis. However, patients' medical history and treatment schedules were not available for analysis. Products were identified by the national drug code (PZN number) and grouped to either subcutaneous immunotherapy (SCIT) with natural extract injections, SCIT with modified allergens (allergoids) or sublingual immunotherapy (SLIT) with natural pollen extract solutions. Persistence was defined as at least one prescription of the individual drug in the respective years. RESULTS: A total of 1409 patients started SIT in 2005 (112, 695, and 602 for natural extract SLIT, natural extract SCIT, and allergoid SCIT, respectively). In 2006, 71%, 55%, and 59% of those patients had at least one renewal prescription of natural extract SLIT, natural extract SCIT, and allergoid SCIT, respectively, as well as 51%, 34%, and 39% in 2007. In both years, persistence with natural extract SLIT was significantly higher than with natural extract SCIT (p = 0.0015 for 2006, p = 0.0003 for 2007) and allergoid SCIT (p = 0.0152 for 2006, p = 0.0111 for 2007). There were no significant differences between the two SCIT groups. CONCLUSION: Medication persistence with grass pollen SIT in a representative sample of patients in Germany was similar to published medication persistence in asthma and COPD patients. The sublingual application route shows significantly better persistency than the subcutaneous route with native allergens or allergoids.