Over 104-fold amplified upconversion luminescence of lanthanide nanocrystals through optical oscillator-like system

Recently, lanthanide (Ln) luminescent nanocrystals have attracted increasing attention in various fields such as biomedical imaging, lasers, and anticounterfeiting. However, due to the forbidden 4f–4f transition of lanthanide ions, the absorption cross-section and luminescence brightness of lanthanide nanocrystals are limited. To address the challenge, we constructed an optical oscillator-like system to repeatedly simulate lanthanide nanocrystals to enhance the absorption efficiency of lanthanide ions on excitation photons. In this optical system, the upconversion luminescence (UCL) of Tm³⁺ emission of ~450 nm excited by a 980 nm laser can be amplified by a factor beyond 10⁴. The corresponding downshifting luminescence of Tm³⁺ at 1460 nm was enhanced by three orders of magnitude. We also demonstrated that the significant luminescence enhancement in the designed optical oscillator-like system was general for various lanthanide nanocrystals including NaYF₄:Yb³⁺/Ln³⁺, NaErF₄@NaYF₄ and NaYF₄:Yb³⁺/Ln³⁺@NaYF₄:Yb³⁺@NaYF₄ (Ln = Er, Tm, Ho) regardless of the wavelengths of excitation sources (808 and 980 nm). The mechanism study revealed that both elevated laser power in the optical system and multiple excitations on lanthanide nanocrystals were the main reason for the luminescence amplification. Our findings may benefit the future development of low-threshold upconversion and downshifting luminescence of lanthanide nanocrystals and expand their applications.     

Battle for the EF-hands: magnesium-calcium interference in calmodulin.

The ubiquitous Ca(2+)-regulatory protein calmodulin activates target enzymes as a response to submicromolar Ca(2+) increases in a background of millimolar Mg(2+). The potential influence of Mg(2+)/Ca(2+) competition is especially intriguing for the N-terminal domain of the protein which possesses the sites with the lowest Ca(2+) specificity. The interdependence of Ca(2+) and Mg(2+) binding in the N-terminal domain of calmodulin was therefore studied using (43)Ca NMR, (1)H-(15)N NMR, and fluorescent Ca(2+) chelator techniques. The apparent affinity for Ca(2+) was found to be significantly decreased at physiological Mg(2+) levels. At Ca(2+) concentrations of an activated cell the (Ca(2+))(2) state of the N-terminal domain is therefore only weakly populated, indicating that for this domain Ca(2+) binding is intimately associated with binding of target molecules. The data are in good agreement with a two-site model in which each site can bind either Ca(2+) or Mg(2+). The Mg(2+)-Ca(2+) binding interaction is slightly positively allosteric, resulting in a significantly populated (Mg(2+))(1)(Ca(2+))(1) state. The Ca(2+) off-rate from this state is determined to be at least one order of magnitude faster than from the (Ca(2+))(2) state. These two findings indicate that the (Mg(2+))(1)(Ca(2+))(1) state is structurally and/or dynamically different from the (Ca(2+))(2) state. The (43)Ca quadrupolar coupling constant and the (1)H and (15)N chemical shifts of the (Mg(2+))(1)(Ca(2+))(1) state were calculated from titration data. The values of both parameters suggest that the (Mg(2+))(1)(Ca(2+))(1) state has a conformation more similar to the "closed" apo and (Mg(2+))(2) states than to the "open" (Ca(2+))(2) state.     

Reprogramming homing endonuclease specificity through computational design and directed evolution

Homing endonucleases (HEs) can be used to induce targeted genome modification to reduce the fitness of pathogen vectors such as the malaria-transmitting Anopheles gambiae and to correct deleterious mutations in genetic diseases. We describe the creation of an extensive set of HE variants with novel DNA cleavage specificities using an integrated experimental and computational approach. Using computational modeling and an improved selection strategy, which optimizes specificity in addition to activity, we engineered an endonuclease to cleave in a gene associated with Anopheles sterility and another to cleave near a mutation that causes pyruvate kinase deficiency. In the course of this work we observed unanticipated context-dependence between bases which will need to be mechanistically understood for reprogramming of specificity to succeed more generally.     

Reprogramming bacteriophage host range: design principles and strategies for engineering receptor binding proteins

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NMR studies of calcium-binding to mutant alpha-spectrin EF-hands

The co-operative calcium binding mechanism of the two C-terminal EF-hands of human alphaII-spectrin has been investigated by site-specific mutagenesis and multi-dimensional NMR spectroscopy. To analyse the calcium binding of each EF-hand independently, two mutant structures (E33A and D69S) of wild type alpha-spectrin were prepared. According to NMR analysis both E33A and D69S were properly folded. The unmutated EF-hand in these mutants remained nearly intact and active in calcium binding, whereas the mutated EF-hand lost its affinity for calcium completely. The apparent calcium binding affinity of the E33A mutant was much lower compared to the D39S mutant (approximately 2470 microM and approximately 240 microM, respectively). When the chemical shift perturbations were followed upon calcium titration, a positive correlation between the D69S mutant and the binding of the first calcium ion to the wild type was revealed. These observations showed that the first EF-hand in spectrin binds the first calcium ion and thereby triggers a conformational change that allows the second calcium ion to bind to the other EF-hand.     

Conformation and structural transitions in the EF-hands of calmodulin

Calcium plays a key role in cellular signal transduction. Calmodulin, a protein binding four calcium ions, is found in all eukaryotic cells and is believed to activate such processes. The calcium binding loop found in this protein, the canonical EF-hand, is also found in a large number of other proteins such as troponins, parvalbumins, calbindins etc. Earlier analysis of the amino acid sequences of these proteins with a view of understanding evolution of protein families and signaling mechanisms have provided extensive evidence for a characteristic double gene duplication event in this family of proteins. These analyses have been extended here to the three dimensional structures and the biophysical properties of the sequence segments of calmodulin EF-hands. The clear evolutionary history that shows up in sequences is not reflected as clearly in the conformation of individual EF-hands, which may be a consequence of the much higher conservation pressure on the structure. Some evidence for the proposed gene duplication is implicit in the apo-holo structural transitions of the EF-hands. The profile of amino acid properties that might be significant for calcium binding, however, clearly reflects the gene duplication. These profiles might also provide insightful information on the calcium affinity of the EF-hand motifs and the nature of amino acid residues that constitute them.     

Structures and luminescent sensors of mixed‐counterions based salen‐type lanthanide coordination polymers

Reactions of N,N′‐bis (salicylidene)‐1,2‐cyclohexanediamine (H₂L) with mixed lanthanide counterions of LnCl₃·6H₂O and Ln (NO₃)₃·6H₂O afford six H₂L lanthanide coordination polymers, e.g. {[Pr(H₂L)₂(NO₃)₂Cl]·2CH₂Cl₂}_n ( 1 ); {[Ln(H₂L)_1.5(NO₃)₃]₂·5CHCl₃·mCH₃OH}_n [Ln = Sm ( 2 ), Eu ( 3 ), Gd ( 4 ), Tb ( 5 ) and Yb ( 6 ); m = 1 ( 2 – 5 ); m = 0 ( 6 )]. X‐ray crystallographic analysis reveals that complex 1 exhibits three‐dimensional diamondoid topologic structure and complexes 2 – 6 are of two‐dimensional structure. Luminescent spectra show that complexes 1 and 6 have characteristic near‐infrared (NIR) emission of praseodymium (III) and ytterbium (III) ions and complexes 2 – 5 emit luminescence in the visible region. Complexes 3 and 6 reveal sensitive luminescence responses to formaldehyde.     

The design and development of new chemical sensors for in vivo monitoring.

The latest workshop of the European Community (EC) Concerted Action on 'Chemical sensors for in vivo monitoring' was held in Nauplion, Greece, in April this year. This fifth workshop focused on 'The design and development of new sensors for in vivo monitoring', and was organized into five sessions: design and development of new sensors; operational considerations; performance of analytical systems; novel sensors/tissue heterogeneity; and infra-red spectroscopy.     

Reprogramming cellular identity for regenerative medicine

Although development leads unidirectionally toward more restricted cell fates, recent work in cellular reprogramming has proven that one cellular identity can strikingly convert into another, promising countless applications in biomedical research and paving the way for modeling diseases with patient-derived stem cells. To date, there has been little discussion of which disease models are likely to be most informative. Here, we review evidence demonstrating that, because environmental influences and epigenetic signatures are largely erased during reprogramming, patient-specific models of diseases with strong genetic bases and high penetrance are likely to prove most informative in the near term. We also discuss the implications of the new reprogramming paradigm in biomedicine and outline how reprogramming of cell identities is enhancing our understanding of cell differentiation and prospects for cellular therapies and in vivo regeneration.     

Molecular mechanism of the calcium-induced conformational change in the spectrin EF-hands.

Calcium is a universally employed cytosolic messenger in eukaryotic cells. Most of the proteins that bind signalling calcium are members of the calmodulin superfamily and share two or more helix-loop-helix motifs known as EF-hands. A model, based on structure comparison of different domains and supported by preliminary NMR data, has suggested that EF-hands involved in signal transduction undergo a major conformational change upon calcium binding from a 'closed' to an 'open' state allowing protein-protein interaction. We have determined the solution structures of the EF-hand pair from alpha-spectrin in the absence and in the presence of calcium. The structures are in the closed and open conformation respectively, providing a definite experimental proof for the closed-to-open model. Our results allow formulation of the rules which govern the movement induced by calcium. These rules may be generalized to other EF-hands since the key residues involved are conserved within the calmodulin family.     

Functional role of EF-hands 3 and 4 in membrane-binding of KChIP1

The aim of the present study is to explore whether membrane targeting of K⁺ channel-interacting protein 1 (KChIP1) is associated with its EF-hand motifs and varies with specific phospholipids. Truncated KChIP1, in which the EFhands 3 and 4 were deleted, retained the α-helix structure, indicating that the N-terminal half of KChIP1 could fold appropriately. Compared with wild-type KChIP1, truncated KChIP1 exhibited lower lipid-binding capability. Compared with wild-type KChIP1, increasing membrane permeability by the use of digitonin caused a marked loss of truncated KChIP1, suggesting that intact EF-hands 3 and 4 were crucial for the anchorage of KChIP1 on membrane. KChIP1 showed a higher binding capability with phosphatidylserine (PS) than truncated KChIP1. Unlike that of truncated KChIP1, the binding of wild-type KChIP1 with membrane was enhanced by increasing the PS content. Moreover, the binding of KChIP1 with phospholipid vesicles induced a change in the structure of KChIP1 in the presence of PS. Taken together, our data suggest that EF-hands 3 and 4 of KChIP1 are functionally involved in a specific association with PS on the membrane.     

直接重编程用于心脏再生治疗的研究进展

心脏再生治疗有望改变现有的心血管病治疗局面,直接重编程领域的研究为实现这一目标提供了新的有力工具。直接重编程是近年来广泛应用于细胞修复及器官移植研究的一项技术,可绕过诱导多功能干细胞中间阶段,直接将一种终末分化细胞转化为其他种类的终末分化细胞。总结了直接重编程用于心脏再生治疗的研究进展,探讨直接重编程技术尚存的问题和障碍,并展望其未来在再生医学领域的应用。     

A structure for amplified DNA

We have employed gene transfer to generate cell lines in which a chromosomal region consisting solely of defined DNA sequences has undergone gene amplification. We have analyzed recombinant clones from the amplified array to determine the physical structure of amplified DNA in the cell lines. The amplified DNA we have analyzed consists of a tandem array of at least 20 individual repeating units. The individual units are contiguous, and are joined to one another by homologous recombination between repeated sequences. At first approximation, all homologous recombinations are permitted such that crossing-over may occur between any two repeated sequences. Since individual units contain multiple repeated elements, the array is not a regularly repeating structure. The individual units within the array are heterogeneous, both in size and in sequence content. These observations suggest models of gene amplification which involve multiple cycles of unscheduled DNA replication at a single locus, followed by multiple recombination events which serve to link individual units to one another and ultimately to the chromosome.     

Solution structure and calcium-binding properties of EF-hands 3 and 4 of calsenilin

Calsenilin is a member of the recoverin branch of the EF-hand superfamily that is reported to interact with presenilins, regulate prodynorphin gene expression, modulate voltage-gated Kv4 potassium channel function, and bind to neurotoxins. Calsenilin is a Ca+2-binding protein and plays an important role in calcium signaling. Despite its importance in numerous neurological functions, the structure of this protein has not been reported. In the absence of Ca+2, the protein has limited spectral resolution that increases upon the addition of Ca+2. Here, we describe the three-dimensional solution structure of EF-hands 3 and 4 of calsenilin in the Ca+2-bound form. The Ca+2-bound structure consists of five alpha-helices and one two-stranded antiparallel beta-sheet. The long loop that connects EF hands 3 and 4 is highly disordered in solution. In addition to its structural effects, Ca+2 binding also increases the protein's propensity to dimerize. These changes in structure and oligomerization state induced upon Ca+2 binding may play important roles in molecular recognition during calcium signaling.