Switchable Lanthanide Luminescence for Detection of Biomolecules

Binary probes are oligonucleotide probe pairs that hybridize adjacently to a complementary target nucleic acid. In order to detect this hybridization, the two probes can be modified with, for example, fluorescent molecules, chemically reactive groups or nucleic acid enzymes. The benefit of this kind of binary probe based approach is that the hybridization elicits a detectable signal which is distinguishable from background noise even though unbound probes are not removed by washing before measurement. In addition, the requirement of two simultaneous binding events increases specificity. Similarly to binary oligonucleotide probes, also certain enzymes and fluorescent proteins can be divided into two parts and used in separation-free assays. Split enzyme and fluorescent protein reporters have practical applications among others as tools to investigate protein-protein interactions within living cells. In this study, a novel label technology, switchable lanthanide luminescence, was introduced and used successfully in model assays for nucleic acid and protein detection. This label technology is based on a luminescent lanthanide chelate divided into two inherently non-luminescent moieties, an ion carrier chelate and a light harvesting antenna ligand. These form a highly luminescent complex when brought into close proximity; i.e., the label moieties switch from a dark state to a luminescent state. This kind of mixed lanthanide complex has the same beneficial photophysical properties as the more typical lanthanide chelates and cryptates - sharp emission peaks, long emission lifetime enabling time-resolved measurement, and large Stokes’ shift, which minimize the background signal. Furthermore, the switchable lanthanide luminescence technique enables a homogeneous assay set-up. Here, switchable lanthanide luminescence label technology was first applied to sensitive, homogeneous, single-target nucleic acid and protein assays with picomolar detection limits and high signal to background ratios. Thereafter, a homogeneous four-plex nucleic acid array-based assay was developed. Finally, the label technology was shown to be effective in discrimination of single nucleotide mismatched targets from fully matched targets and the luminescent complex formation was analyzed more thoroughly. In conclusion, this study demonstrates that the switchable lanthanide luminescencebased label technology can be used in various homogeneous bioanalytical assays.Kahta nukleiinihappokoetinta, jotka hybridisoituvat vierekkäin komplementaariseen kohdenukleiinihappoon, kutsutaan kaksoiskoettimiksi. Kohteen tunnistamiseksi kaksoiskoettimet voidaan leimata fluoresoivilla väreillä, kemiallisilla ryhmillä tai entsyymeillä. Kaksoiskoettimien etu verrattuna yhden nukleiinihappokoettimen käyttöön on, että kaksoiskoettimien hybridisaatio kohteeseen aikaansaa mitattavan signaalin muodostumisen, vaikka hybridisoitumattomia koettimia ei erotettaisi pois reaktioseoksesta pesuvaiheella ennen mittausta. Kaksoiskoettimilla on hyvä spesifisyys, sillä mitattava signaali muodostuu vain, kun koettimet hybridisoituvat kohdenukleiinihappoon yhtä aikaa. Myös entsyymejä ja fluoresoivia proteiineja voidaan jakaa kahteen osaan. Niitä voidaan käyttää reporttereina erotusvapaissa määrityksissä. Jaetut entsyymit ja fluoresoivat proteiinireportterit ovat käyttökelpoisia erityisesti soluissa tapahtuvien proteiinien välisten vuorovaikutusten tutkimisessa. Tässä tutkimuksessa esiteltiin uusi leimateknologia, kytkeytyvä lantanidiluminesenssi, jota käytettiin kaksoiskoettimien kanssa. Tässä leimateknologiassa luminoiva lantanidikelaatti on jaettu kahteen ei-luminoivaan osaan, ioninkantajakelaattiin ja valoa keräävään antenniin, jotka kytkeytyvät luminoivaksi kompleksiksi kun osat tuodaan lähelle toisiaan. Tällä kahdesta osasta muodostuvalla luminoivalla lantanidikompleksilla on samat fotofysikaaliset edut kuin tavallisemmin käytetyillä luminoivilla lantanidikelaateilla ja -kryptaateilla: terävät emissiopiikit, pitkä emission elinikä, joka mahdollistaa aikaerotteisen mittaamisen, sekä suuri Stokesin siirtymä. Nämä ominaisuudet pienentävät määrityksen herkkyyttä rajoittavaa taustasignaalia. Kytkeytyvän lantanidiluminesenssin lisäetu on, että niitä hyödyntäen voidaan toteuttaa erotusvapaita määrityksiä. Tässä väitöstutkimuksessa uuden leimateknologian käyttökelpoisuus osoitettiin ensin herkissä erotusvapaissa nukleiinihappo- ja proteiinimäärityksissä, joissa saavutettiin pikomolaarinen herkkyys ja korkea signaalin ja taustan suhde. Seuraavaksi kehitettiin erotusvapaa monianalyyttimääritys neljän kohdenukleiinihapon yhtäaikaiseen havaitsemiseen. Lopuksi uuden leimateknologian osoitettiin soveltuvan myös yhden nukleotidin mutaatioiden erotteluun ja luminoivan kompleksin muodostumista tutkittiin tarkemmin. Loppupäätelmänä voidaan todeta, että tämä tutkimus osoitti kytkeytyvän lantanidiluminesenssin mahdollisuudet erilaisissa erotusvapaissa määrityksissä.Siirretty Doriast

Spacer length, label moiety interchange and probe pair orientation in a homogeneous solid-phase hybridization assay utilizing lanthanide chelate complementation

We have studied parameters affecting DNA hybridization and lanthanide chelate complementation based signal formation in a separation-free solid-phase assay. This binary probe assay system consists of two probes labeled either with a europium carrier chelate or a light harvesting antenna ligand. One probe was immobilized on the microtiter well bottom in spot format while the other probe was free in solution. The probe concentration used in spotting, spacer length, and the choice and orientation of the either 3&acute; or 5&acute;-end immobilized probe had significant impact on signal-to-background (S/B) ratios. The highest ratio was achieved by saturating the spot with the 5&acute;-end immobilized antenna ligand probe separated from the solid support with a 25 nucleotide poly dT spacer. The obtained detection limit of 18 pM for synthetic Pseudomonas aeruginosa heat shock protein groES gene sequence was close to a 20-fold improvement compared to the previous assay. The dynamic range of the assay was three orders of magnitude.</p

Local C-Reactive Protein Expression in Obliterative Lesions and the Bronchial Wall in Posttransplant Obliterative Bronchiolitis

The local immunoreactivity of C-reactive protein (CRP) was studied in a heterotopic porcine model of posttranplant obliterative bronchiolitis (OB). Bronchial allografts and control autografts were examined serially 2–28 days after subcutaneous transplantation. The autografts stayed patent. In the allografts, proliferation of inflammatory cells (P < .0001) and fibroblasts (P = .02) resulted in occlusion of the bronchial lumens (P < .01). Influx of CD4+ (P < .001) and CD8+ (P < .0001) cells demonstrated allograft immune response. CRP positivity simultaneously increased in the bronchial walls (P < .01), in macrophages, myofibroblasts, and endothelial cells. Local CRP was predictive of features characteristic of OB (R = 0.456–0.879, P < .05−P < .0001). Early obliterative lesions also showed CRP positivity, but not mature, collagen-rich obliterative plugs (P < .05). During OB development, CRP is localized in inflammatory cells, myofibroblasts and endothelial cells probably as a part of the local inflammatory response

Tumor margins that lead to reoperation in breast cancer: A retrospective register study of 4,489 patients

Background and Objectives Optimal margins for ductal carcinoma in situ (DCIS) remain controversial in breast-conserving surgery (BCS) and mastectomy. We examine the association of positive margins, reoperations, DCIS and age. Methods A retrospective study of histopathological reports (4489 patients). Margin positivity was defined as ink on tumor for invasive carcinoma. For DCIS, we applied 2 mm anterior and side margin thresholds, and ink on tumor in the posterior margin. Results The incidence of positive side margins was 20% in BCS and 5% in mastectomies (p p p = 0.013). Of BCS patients with invasive carcinoma in the side margin, 73% were reoperated on. A reoperation was performed in 70% of patients with a close (p = 0.002). The reoperation rates were 55% in invasive carcinoma with close DCIS, 66% in close extensive intraductal component (EIC), and 83% in close pure DCIS (p Conclusions Individual assessment as opposed to rigid adherence to guidelines was used in the decision on reoperation.</p

Medico-legal autopsy in postoperative hemodynamic collapse following coronary artery bypass surgery

Sudden unexpected postoperative hemodynamic collapse with a high mortality develops in 1–3% of patients undergoing coronary artery bypass surgery (CABG). The contribution of surgical graft complications to this serious condition is poorly known and their demonstration at autopsy is a challenging task. Isolated CABG was performed in 8,807 patients during 1988–1999. Of the patients, 76 (0.9%) developed sudden postoperative hemodynamic collapse resulting in subsequent emergency reopening of the median sternotomy and open cardiac massage. Further emergency reoperation could be performed in 62 (82%) whereas 14 patients died prior to reoperation and a further 21 did not survive the reoperation or died a few days later. All 35 (46%) patients who did not survive were subjected to medico-legal autopsy combined with postmortem cast angiography. By combining clinical data with autopsy and angiography data, various types of graft complications were observed in 27 (36%, 1.3 per patient) of the 76 patients with hemodynamic collapse. There were no significant differences in the frequency (33 vs. 40%) or number of complicated grafts per patient (1.2 vs. 1.4) between those who survived reoperation and who did not. Autopsy detected 25 major and minor findings not diagnosed clinically. Postmortem cast angiography visualized 2 graft twists not possible to detect by autopsy dissection only. Surgical graft complications were the most frequent single cause for sudden postoperative hemodynamic collapse in CABG patients leading to a fatal outcome in almost half of the cases. Postmortem angiography improved the accuracy of autopsy diagnostics of graft complications

Oncoplastic breast consortium recommendations for mastectomy and whole breast reconstruction in the setting of post-mastectomy radiation therapy

Aim: Demand for nipple-and skin-sparing mastectomy (NSM/SSM) with immediate breast reconstruction (BR) has increased at the same time as indications for post-mastectomy radiation therapy (PMRT) have broadened. The aim of the Oncoplastic Breast Consortium initiative was to address relevant questions arising with this clinically challenging scenario. Methods: A large global panel of oncologic, oncoplastic and reconstructive breast surgeons, patient advocates and radiation oncologists developed recommendations for clinical practice in an iterative process based on the principles of Delphi methodology. Results: The panel agreed that surgical technique for NSM/SSM should not be formally modified when PMRT is planned with preference for autologous over implant-based BR due to lower risk of long-term complications and support for immediate and delayed-immediate reconstructive approaches. Nevertheless, it was strongly believed that PMRT is not an absolute contraindication for implant-based or other types of BR, but no specific recom-mendations regarding implant positioning, use of mesh or timing were made due to absence of high-quality evidence. The panel endorsed use of patient-reported outcomes in clinical practice. It was acknowledged that the shape and size of reconstructed breasts can hinder radiotherapy planning and attention to details of PMRT techniques is important in determining aesthetic outcomes after immediate BR. Conclusions: The panel endorsed the need for prospective, ideally randomised phase III studies and for surgical and radiation oncology teams to work together for determination of optimal sequencing and techniques for PMRT for each patient in the context of BRPeer reviewe

GWAS on longitudinal growth traits reveals different genetic factors influencing infant, child, and adult BMI

Early childhood growth patterns are associated with adult health, yet the genetic factors and the developmental stages involved are not fully understood. Here, we combine genome-wide association studies with modeling of longitudinal growth traits to study the genetics of infant and child growth, followed by functional, pathway, genetic correlation, risk score, and colocalization analyses to determine how developmental timings, molecular pathways, and genetic determinants of these traits overlap with those of adult health. We found a robust overlap between the genetics of child and adult body mass index (BMI), with variants associated with adult BMI acting as early as 4 to 6 years old. However, we demonstrated a completely distinct genetic makeup for peak BMI during infancy, influenced by variation at the LEPR/LEPROT locus. These findings suggest that different genetic factors control infant and child BMI. In light of the obesity epidemic, these findings are important to inform the timing and targets of prevention strategies

GWAS on longitudinal growth traits reveals different genetic factors influencing infant, child, and adult BMI

This is the final version. Available on open access from AAAS via the DOI in this recordData and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.Early childhood growth patterns are associated with adult health, yet the genetic factors and the developmental stages involved are not fully understood. Here, we combine genome-wide association studies with modeling of longitudinal growth traits to study the genetics of infant and child growth, followed by functional, pathway, genetic correlation, risk score, and colocalization analyses to determine how developmental timings, molecular pathways, and genetic determinants of these traits overlap with those of adult health. We found a robust overlap between the genetics of child and adult body mass index (BMI), with variants associated with adult BMI acting as early as 4 to 6 years old. However, we demonstrated a completely distinct genetic makeup for peak BMI during infancy, influenced by variation at the LEPR/LEPROT locus. These findings suggest that different genetic factors control infant and child BMI. In light of the obesity epidemic, these findings are important to inform the timing and targets of prevention strategies.Medical Research Council (MRC)Wellcome TrustNational Institutes of Health (NIH)Danish National Research FoundationLundbeck FoundationDanish Medical Research Counci

Effect of doping and crystallite size on the electrochemical performance of Li4Ti5O12

Defect spinel phase lithium titanate (Li4Ti5O12) has been suggested as a promising negative electrode material for next generation lithium ion batteries. Flame spray pyrolysis has been shown to be a viable fast, one-step process for synthesis of nanoparticulate Li4Ti5O12. However, due to the rapid quenching that is integral to the process the crystallite size remain very small and non-uniform. To overcome this shortcoming a vertical flow tube furnace was used to increase the high-temperature residence time. This resulted in an increase in the crystallite size and crystallinity of the product. As a result of this increase the electrochemical performance of the Li4Ti5O12 was markedly improved. Furthermore, silver doping of the Li4Ti5O12 material can be carried out simultaneously with its synthesis in the FSP process. The resulting nanosized silver particles on the surface of the Li4Ti5O12 particles further improve the electrochemical performance during high current operations. The specific capacities of these high-temperature synthesised pure and silver-doped Li4Ti5O12 nanoparticles were found to increase by up to 6% and 19%, respectively, compared to a commercial reference. Thus the technique provides a simple method for synthesising superior quality Li4Ti5O12 for battery applications.peerReviewe

Homogeneous Detection of Avidin Based on Switchable Lanthanide Luminescence

We have developed switchable lanthanide luminescence-based binary probe technology for homogeneous detection of avidin, which is a tetrameric protein. Two different nonluminescent label moietiesa light-absorbing antenna ligand and a lanthanide ion carrier chelatewere conjugated to separate biotins, which is known as avidin’s natural ligand. The assay was based on binding of the two differently labeled biotins on separate binding sites on the target protein and consequent self-assembly of a luminescent complex from the two label moieties. Specific luminescence signal was observed only at the presence of the target protein. The characteristics of the switchable lanthanide luminescence assay were compared to the reference assay, based on lanthanide resonance energy transfer. Both assays had a limit of detection in the low-picomolar concentration range; however, the lanthanide chelate complementation-based assay had wider dynamic range and its optimization was more straightforward. The switchable lanthanide luminescence technology could be further applied to generic protein detection, using reagents that are analogous to the proximity ligation assay principle