A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell

A fluorescence correlation spectroscopy (FCS) system based on two independent measurement volumes is presented. The optical setup and data acquisition hardware are detailed, as well as a complete protocol to control the location, size and shape of the measurement volumes. A method that allows to monitor independently the excitation and collection efficiency distribution is proposed. Finally, a few examples of measurements that exploit the two spots in static and/or scanning schemes, are reported.Comment: Accepted for publication in Review of Scientific Instrumen

Photophysical properties of 5-methylcytosine

International audienceCytosine methylation, which determines the hot spots for DNA photo-damage, is shown to induce a red-shift of the nucleoside absorption spectrum, making the chromophore more vulnerable to solar radiation, and a tenfold increase of the fluorescence lifetime, making excited state reactions more probable. A femtosecond investigation of the excited state deactivation reveals a quite complex mechanism

Clinical implementation of deep learning-based automated left breast simultaneous integrated boost radiotherapy treatment planning.

Automation in radiotherapy treatment planning aims to improve both the quality and the efficiency of the process. The aim of this study was to report on a clinical implementation of a Deep Learning (DL) auto-planning model for left-sided breast cancer. The DL model was developed for left-sided breast simultaneous integrated boost treatments under deep-inspiration breath-hold. Eighty manual dose distributions were revised and used for training. Ten patients were used for model validation. The model was then used to design 17 clinical auto-plans. Manual and auto-plans were scored on a list of clinical goals for both targets and organs-at-risk (OARs). For validation, predicted and mimicked dose (PD and MD, respectively) percent error (PE) was calculated with respect to manual dose. Clinical and validation cohorts were compared in terms of MD only. Median values of both PD and MD validation plans fulfilled the evaluation criteria. PE was < 1% for targets for both PD and MD. PD was well aligned to manual dose while MD left lung mean dose was significantly less (median:5.1 Gy vs 6.1 Gy). The left-anterior-descending artery maximum dose was found out of requirements (median values:+5.9 Gy and + 2.9 Gy, for PD and MD respectively) in three validation cases, while it was reduced for clinical cases (median:-1.9 Gy). No other clinically significant differences were observed between clinical and validation cohorts. Small OAR differences observed during the model validation were not found clinically relevant. The clinical implementation outcomes confirmed the robustness of the model

Curvature-coupling dependence of membrane protein diffusion coefficients

We consider the lateral diffusion of a protein interacting with the curvature of the membrane. The interaction energy is minimized if the particle is at a membrane position with a certain curvature that agrees with the spontaneous curvature of the particle. We employ stochastic simulations that take into account both the thermal fluctuations of the membrane and the diffusive behavior of the particle. In this study we neglect the influence of the particle on the membrane dynamics, thus the membrane dynamics agrees with that of a freely fluctuating membrane. Overall, we find that this curvature-coupling substantially enhances the diffusion coefficient. We compare the ratio of the projected or measured diffusion coefficient and the free intramembrane diffusion coefficient, which is a parameter of the simulations, with analytical results that rely on several approximations. We find that the simulations always lead to a somewhat smaller diffusion coefficient than our analytical approach. A detailed study of the correlations of the forces acting on the particle indicates that the diffusing inclusion tries to follow favorable positions on the membrane, such that forces along the trajectory are on average smaller than they would be for random particle positions.Comment: 16 pages, 8 figure

Molecular spectroscopy: Complexity of excited-state dynamics in DNA

Absorption of ultraviolet light by DNA is known to lead to carcinogenic mutations, but the processes between photon absorption and the photochemical reactions are poorly understood. In their study of the excited-stated dynamics of model DNA helices using femtosecond transient absorption spectroscopy1, Crespo-Hernández et al. observe that the picosecond component of the transient signals recorded for the adenine–thymine oligonucleotide (dA)18(dT)18 is close to that for (dA)18, but quite different from that for (dAdT)9(dAdT)9; from this observation, they conclude that excimer formation limits excitation energy to one strand at a time. Here we use time-resolved fluorescence spectroscopy to probe the excited-state dynamics, which reveals the complexity of these systems and indicates that the interpretation of Crespo-Hernández et al. is an oversimplification. We also comment on the pertinence of separating base stacking and base pairing in excited-state dynamics of double helices and question the authors' assignment of the long-lived signal component found for (dA)18(dT)18 to adenine excimers

Novel Peptide Sequence (“IQ-tag”) with High Affinity for NIR Fluorochromes Allows Protein and Cell Specific Labeling for In Vivo Imaging

Probes that allow site-specific protein labeling have become critical tools for visualizing biological processes.Here we used phage display to identify a novel peptide sequence with nanomolar affinity for near infrared (NIR) (benz)indolium fluorochromes. The developed peptide sequence ("IQ-tag") allows detection of NIR dyes in a wide range of assays including ELISA, flow cytometry, high throughput screens, microscopy, and optical in vivo imaging.The described method is expected to have broad utility in numerous applications, namely site-specific protein imaging, target identification, cell tracking, and drug development

Oscillations by Minimal Bacterial Suicide Circuits Reveal Hidden Facets of Host-Circuit Physiology

Synthetic biology seeks to enable programmed control of cellular behavior though engineered biological systems. These systems typically consist of synthetic circuits that function inside, and interact with, complex host cells possessing pre-existing metabolic and regulatory networks. Nevertheless, while designing systems, a simple well-defined interface between the synthetic gene circuit and the host is frequently assumed. We describe the generation of robust but unexpected oscillations in the densities of bacterium Escherichia coli populations by simple synthetic suicide circuits containing quorum components and a lysis gene. Contrary to design expectations, oscillations required neither the quorum sensing genes (luxR and luxI) nor known regulatory elements in the PluxI promoter. Instead, oscillations were likely due to density-dependent plasmid amplification that established a population-level negative feedback. A mathematical model based on this mechanism captures the key characteristics of oscillations, and model predictions regarding perturbations to plasmid amplification were experimentally validated. Our results underscore the importance of plasmid copy number and potential impact of “hidden interactions” on the behavior of engineered gene circuits - a major challenge for standardizing biological parts. As synthetic biology grows as a discipline, increasing value may be derived from tools that enable the assessment of parts in their final context

Safety and tolerability of sitagliptin in patients with type 2 diabetes: a pooled analysis

<p>Abstract</p> <p>Background</p> <p>Sitagliptin, a highly selective dipeptidyl peptidase-4 inhibitor, is the first in a new class of oral antihyperglycemic agents (AHAs) for the treatment of patients with type 2 diabetes. Type 2 diabetes is a life-long disease requiring chronic treatment and management. Therefore, robust assessment of the long-term safety and tolerability of newer therapeutic agents is of importance. The purpose of this analysis was to assess the safety and tolerability of sitagliptin by pooling 12 large, double-blind, Phase IIb and III studies up to 2 years in duration. Methods: This analysis included 6139 patients with type 2 diabetes receiving either sitagliptin 100 mg/day (N = 3415) or a comparator agent (placebo or an active comparator) (N = 2724; non-exposed group). The 12 studies from which this pooled population was drawn represent the double-blind, randomized, Phase IIB and III studies that included patients treated with the clinical dose of sitagliptin (100 mg/day) for at least 18 weeks up to 2 years and that were available in a single safety database as of November 2007. These 12 studies assessed sitagliptin as monotherapy, initial combination therapy with metformin, or add-on combination therapy with other oral AHAs (metformin, pioglitazone, sulfonylurea, sulfonylurea + metformin, or metformin + rosiglitazone). Patients in the non-exposed group were taking placebo, pioglitazone, metformin, sulfonylurea, sulfonylurea + metformin, or metformin + rosiglitazone. This safety analysis used patient-level data from each study to evaluate clinical and laboratory adverse experiences.</p> <p>Results</p> <p>For clinical adverse experiences, the incidence rates of adverse experiences overall, serious adverse experiences, and discontinuations due to adverse experiences were similar in the sitagliptin and non-exposed groups. The incidence rates of specific adverse experiences were also generally similar in the two groups, with the exception of an increased incidence rate of hypoglycemia observed in the non-exposed group. The incidence rates of drug-related adverse experiences overall and discontinuations due to drug-related adverse experiences were higher in the non-exposed group, primarily due to the increased incidence rate of hypoglycemia in this group. For cardiac- and ischemia-related adverse experiences (including serious events), there were no meaningful between-group differences. No meaningful differences between groups in laboratory adverse experiences, either summary measures or specific adverse experiences, were observed.</p> <p>Conclusion</p> <p>In patients with type 2 diabetes, sitagliptin 100 mg/day was well tolerated in clinical trials up to 2 years in duration.</p

An Environment-Sensitive Synthetic Microbial Ecosystem

Microbial ecosystems have been widely used in industrial production, but the inter-relationships of organisms within them haven't been completely clarified due to complex composition and structure of natural microbial ecosystems. So it is challenging for ecologists to get deep insights on how ecosystems function and interplay with surrounding environments. But the recent progresses in synthetic biology show that construction of artificial ecosystems where relationships of species are comparatively clear could help us further uncover the meadow of those tiny societies. By using two quorum-sensing signal transduction circuits, this research designed, simulated and constructed a synthetic ecosystem where various population dynamics formed by changing environmental factors. Coherent experimental data and mathematical simulation in our study show that different antibiotics levels and initial cell densities can result in correlated population dynamics such as extinction, obligatory mutualism, facultative mutualism and commensalism. This synthetic ecosystem provides valuable information for addressing questions in ecology and may act as a chassis for construction of more complex microbial ecosystems