Light-induced formation of dimeric LHCII

It emerges from numerous experiments that LHCII, the major photosynthetic antenna complex of plants, can appear not only in the trimeric or monomeric states but also as a dimer. We address the problem whether the dimeric form of the complex is just a simple intermediate element of the trimer–monomer transformation or if it can also be a physiologically relevant molecular organization form? Dimers of LHCII were analyzed with application of native electrophoresis, time-resolved fluorescence spectroscopy, and fluorescence correlation spectroscopy. The results reveal the appearance of two types of LHCII dimers: one formed by the dissociation of one monomer from the trimeric structure and the other formed by association of monomers into a distinctively different molecular organizational form, characterized by a high rate of chlorophyll excitation quenching. The hypothetical structure of such an energy quencher is proposed. The high light-induced LHCII dimerization is discussed as a potential element of the photoprotective response in plants

Measuring, in solution, multiple-fluorophore labeling by combining Fluorescence Correlation Spectroscopy and photobleaching

Determining the number of fluorescent entities that are coupled to a given molecule (DNA, protein, etc.) is a key point of numerous biological studies, especially those based on a single molecule approach. Reliable methods are important, in this context, not only to characterize the labeling process, but also to quantify interactions, for instance within molecular complexes. We combined Fluorescence Correlation Spectroscopy (FCS) and photobleaching experiments to measure the effective number of molecules and the molecular brightness as a function of the total fluorescence count rate on solutions of cDNA (containing a few percent of C bases labeled with Alexa Fluor 647). Here, photobleaching is used as a control parameter to vary the experimental outputs (brightness and number of molecules). Assuming a Poissonian distribution of the number of fluorescent labels per cDNA, the FCS-photobleaching data could be easily fit to yield the mean number of fluorescent labels per cDNA strand (@ 2). This number could not be determined solely on the basis of the cDNA brightness, because of both the statistical distribution of the number of fluorescent labels and their unknown brightness when incorporated in cDNA. The statistical distribution of the number of fluorophores labeling cDNA was confirmed by analyzing the photon count distribution (with the cumulant method), which showed clearly that the brightness of cDNA strands varies from one molecule to the other.Comment: 38 pages (avec les figures

Reversible Fluorescence Photoswitching in DNA

[Image: see text] We describe the engineering of reversible fluorescence photoswitching in DNA with high-density substitution, and its applications in advanced fluorescence microscopy methods. High-density labeling of DNA with cyanine dyes can be achieved by polymerase chain reaction using a modified DNA polymerase that has been evolved to efficiently incorporate Cy3- and Cy5-labeled cytosine base analogues into double-stranded DNA. The resulting biopolymer, “CyDNA”, displays hundreds of fluorophores per DNA strand and is strongly colored and highly fluorescent, although previous observations suggest that fluorescence quenching at such high density might be a concern, especially for Cy5. Herein, we first investigate the mechanisms of fluorescence quenching in CyDNA and we suggest that two different mechanisms, aggregate formation and resonance energy transfer, are responsible for fluorescence quenching at high labeling densities. Moreover, we have been able to re-engineer CyDNA into a reversible fluorescence photoswitchable biopolymer by using the properties of the Cy3–Cy5 pair. This novel biopolymer constitutes a new class of photoactive DNA-based nanomaterial and is of great interest for advanced microscopy applications. We show that reversible fluorescence photoswitching in CyDNA can be exploited in optical lock-in detection imaging. It also lays the foundations for improved and sequence-specific super-resolution fluorescence microscopy of DNA

Provider and lay perspectives on intra-uterine contraception: a global review

BACKGROUND: Intra-uterine contraception (IUC) involves the use of an intra-uterine device (IUD), a highly effective, long-acting, reversible contraceptive method. Historically, the popularity of IUC has waxed and waned across different world regions, due to policy choices and shifts in public opinion. However, despite its advantages and cost-effectiveness for programmes, IUC's contribution to contraceptive prevalence is currently negligible in many countries. This paper presents the results of a systematic review of the global literature on provider and lay perspectives on IUC. It aims to shed light on the reasons for low use of IUC and reflect on potential opportunities for the method's promotion. METHODS: A systematic search of the literature was conducted in four peer-reviewed journals and four electronic databases (MEDLINE, EMBASE, POPLINE, and Global Health). Screening resulted in the inclusion of 68 relevant publications. RESULTS: Most included studies were conducted in areas where IUD use is moderate or low. Findings are similar across these areas. Many providers have low or uneven levels of knowledge on IUC and limited training. Many wrongly believe that IUC entails serious side effects such as pelvic inflammatory disease (PID), and are reluctant to provide it to entire eligible categories, such as HIV-positive women. There is particular resistance to providing IUC to teenagers and nulliparae. Provider opinions may be more favourable towards the hormonal IUD. Some health-care providers choose IUC for themselves. Many members of the public have low knowledge and unfounded misconceptions about IUC, such as the fear of infertility. Some are concerned about the insertion and removal processes, and about its effect on menses. However, users of IUC are generally satisfied and report a number of benefits. Peers and providers exert a strong influence on women's attitudes. CONCLUSION: Both providers and lay people have inaccurate knowledge and misconceptions about IUC, which contribute to explaining its low use. However, many reported concerns and fears could be alleviated through correct information. Concerted efforts to train providers, combined with demand creation initiatives, could therefore boost the method's popularity. Further research is needed on provider and lay perspectives on IUDs in low- and middle-income countries

Spectroscopy of Photosynthetic Pigment-Protein Complex LHCII

Light-harvesting pigment-protein complex of photosystem II is the most abundant membrane protein in the biosphere, comprising more than half chlorophyll molecules. The protein plays a role of photosynthetic antenna, collecting solar radiation and transferring excitations towards the reaction centers, where electric charge separation takes place. Efficient excitation energy capture and transfer requires unique organization of the complex and unique photophysical properties of the accessory pigments: chlorophylls and carotenoids. LHCII is also a place where extremely harmful singlet oxygen may be generated, under strong illumination conditions. Several physical mechanisms have been found in LHCII, operating to protect the photosynthetic apparatus against light-induced damage, including chlorophyll triplet and singlet excitations quenching by carotenoids. In this paper we discuss the results of our recent studies, carried out with the application of several molecular spectroscopy techniques (electronic absorption, fluorescence, resonance Raman and FTIR), designed to investigate molecular mechanisms responsible for regulation of excitation density in LHCII. Among the most interesting findings are the light-induced molecular configuration changes of the LHCII-bound xanthophylls, leading to conformational rearrangements of the protein. These mechanisms are discussed in terms of excessive excitation quenching in the pigment-protein complex subjected to overexcitation. Such an activity seems to represent a vital regulatory process in the photosynthetic apparatus, at the molecular level, protecting plants against photodegradation

Spectroscopy of Photosynthetic Pigment-Protein Complex LHCII

Light-harvesting pigment-protein complex of photosystem II is the most abundant membrane protein in the biosphere, comprising more than half chlorophyll molecules. The protein plays a role of photosynthetic antenna, collecting solar radiation and transferring excitations towards the reaction centers, where electric charge separation takes place. Efficient excitation energy capture and transfer requires unique organization of the complex and unique photophysical properties of the accessory pigments: chlorophylls and carotenoids. LHCII is also a place where extremely harmful singlet oxygen may be generated, under strong illumination conditions. Several physical mechanisms have been found in LHCII, operating to protect the photosynthetic apparatus against light-induced damage, including chlorophyll triplet and singlet excitations quenching by carotenoids. In this paper we discuss the results of our recent studies, carried out with the application of several molecular spectroscopy techniques (electronic absorption, fluorescence, resonance Raman and FTIR), designed to investigate molecular mechanisms responsible for regulation of excitation density in LHCII. Among the most interesting findings are the light-induced molecular configuration changes of the LHCII-bound xanthophylls, leading to conformational rearrangements of the protein. These mechanisms are discussed in terms of excessive excitation quenching in the pigment-protein complex subjected to overexcitation. Such an activity seems to represent a vital regulatory process in the photosynthetic apparatus, at the molecular level, protecting plants against photodegradation

Plasmonic platforms of self-assembled silver nanostructures in application to fluorescence

Fluorescence intensity changes were investigated theoretically and experimentally using self-assembled colloidal structures on silver semitransparent mirrors. Using a simplified quasi-static model and finite element method, we demonstrate that near-field interactions of metallic nanostructures with a continuous metallic surface create conditions that produce enormously enhanced surface plasmon resonances. The results were used to explain the observed enhancements and determine the optimal conditions for the experiment. The theoretical parts of the studies are supported with reports on detailed emission intensity changes which provided multiple fluorescence hot spots with 2–3 orders of enhancements. We study two kinds of the fluorophores: dye molecules and fluorescent nanospheres characterized with similar spectral emission regions. Using a lifetime-resolved fluorescence/reflection confocal microscopy technique, we find that the largest rate for enhancement (~1000-fold) comes from localized areas of silver nanostructures

Fluorescence lifetime of actin in the familial hypertrophic cardiomyopathy transgenic heart

Clinical studies have revealed that the D166V mutation in the ventricular myosin regulatory light chain (RLC) can cause a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). It has been proposed that RLC induced FHC in the heart originates at the level of the myosin cross-bridge due to alterations in the rates of cross-bridge cycling. In this report, we examine whether the environment of an active cross-bridge in cardiac myofibrils from transgenic (Tg) mice is altered by the D166V mutation in RLC. The cross-bridge environment was monitored by tracking the fluorescence lifetime (tau) of Alexa488-phalloidin-labeled actin. The fluorescence lifetime is the average rate of decay of a fluorescent species from the excited state, which strongly depends on various environmental factors. We observed that the lifetime was high when cross-bridges were bound to actin and low when they were dissociated from it. The lifetime was measured every 50 ms from the center half of the I-band during 60 s of rigor, relaxation and contraction of muscle. We found no differences between lifetimes of Tg-WT and Tg-D166V muscle during rigor, relaxation and contraction. The duty ratio expressed as a fraction of time that cross-bridges spend attached to the thin filaments during isometric contraction was similar in Tg-WT and Tg-D166V muscles. Since independent measurements showed a large decrease in the cross-bridge turnover rate in Tg-D166V muscle compared to Tg-WT, the fact that the duty cycle remains constant suggests that the D166V mutation of RLC causes a decrease in the rate of cross-bridge attachment to actin