Dual-Laser Tetra-Polarization FRET (4polFRET) for Site-Selective Control of Homo-FRET in Hetero-FRET Systems on the Cell Surface: The Homo-FRET Gate.

The effects of donor homo-Förster resonance energy transfer (homo-FRET) taking place in hetero-FRET systems is described in the context of hetero-FRET detection via donor and acceptor fluorescence anisotropies in cell surface receptor clusters. Donor homo-FRET can influence both the efficiency of detection as well as the magnitude of the detectable hetero-FRET. A 4-fold polarized FRET detection scheme-tetrapolarization FRET (4polFRET)-is proposed not only for discriminating the effects of homo-FRET from those of hetero-FRET, but also for correlating homo-associations of the donors and acceptors at different donor-acceptor distances, even beyond the critical Förster distance for hetero-FRET ( R0). The method is based on suppressing homo-FRET at the donor side with red-edge excitation. After the anisotropy effects of physical rotation and homo-FRET were separated by site-selective spectroscopy, the magnitude of the effect of homo-FRET on hetero-FRET has been estimated. It has been found significant, offering a new sensitive technique for detecting conformational dynamics via the homo-FRET mediated component of hetero-FRET, the "homo-FRET enhanced hetero-FRET" or "homo-FRET gate". The method is realizable in flow, as well as in image cytometry equipped with polarization detecting facility.

Perrin and Förster unified: Dual-laser triple-polarization FRET (3polFRET) for interactions at the Förster-distance and beyond.

Dual laser flow cytometric energy transfer (FCET)--elaborated by Trón et al. in 1984--is an efficient and rapid way of measuring FRET on large cell populations. FRET efficiency and the donor and acceptor concentrations are determined from one donor and two acceptor signals. In this communication this method is extended towards the domain of receptor dynamics by the detection of polarized components of the three intensities. By enabling a complete description of the proximity and dynamics of FRET-systems, the new measuring scheme allows a more refined description of both the structure and dynamics of cell surface receptor clusters at the nano-scale and beyond. Associated donor fraction, limiting anisotropy and rotational correlation time of the donor, acceptor anisotropy and cell-by-cell estimation of the orientation factor for FRET (κ2) are available in the steady state on a single FRET sample in a very rapid and statistically efficient way offered by flow cytometry. For a more sensitive detection of conformational changes the "polarized FRET indices"--quantities composed from FRET efficiency and anisotropies--are proposed. The method is illustrated by measurements on a FRET system with changing FRET-fraction and on a two donor-one acceptor-system, when the existence of receptor trimers are proven by the detection of "hetero-FRET induced homo-FRET relief", i.e. the diminishing of homo-FRET between the two donors in the presence of a donor quencher. The method also offers higher sensitivity for assessing conformational changes at the nano-scale, due to its capability for the simultaneous detection of changes of proximity and relative orientations of the FRET donor and acceptor. Although the method has been introduced in the context of FRET, it is more general: It can be used for monitoring triple-anisotropy correlations also in those cases when FRET actually does not occur, e.g. for interactions occuring beyond the Förster-distance R0. Interpretation of κ2 has been extended.

Depolarized FRET (depolFRET) on the cell surface: FRET control by photoselection.

Sensitivity of FRET in hetero- and homo-FRET systems on the photoselected orientation distribution of donors has been proven by using polarized and depolarized light for excitation. FRET as well as donor and acceptor anisotropies have been simultaneously measured in a dual emission-polarization scheme realized in a conventional flow cytometer by using single laser excitation and applying fluorophore-conjugated mAbs against the MHCI and MHCII cell surface receptors. Depolarization of the originally polarized light have been achieved by using crystal depolarizers based on Cornu's principle, a quarter-wave plate for circular polarization, and a parallel beam splitter acting as a diagonal-polarizer for dual-polarization excitation. Simultaneous analysis of intensity-based FRET efficiency and acceptor depolarization equivocally report that depolarization of light may increase FRET in an amount depending on the acceptor-to-donor concentration ratio. Acceptor depolarization turned to be more sensitive to FRET than donor hyper-polarization and even than intensity-based FRET efficiency. It can be used as a sensitive tool for monitoring changes in the dynamics of the donor-acceptor pairs. The basic observations of FRET enhancement and increased acceptor depolarization obtained for hetero-FRET are paralleled by analog observations of homo-FRET enhancements under depolarized excitation. In terms of the orientation factor for FRET, the FRET enhancements on depolarization in the condition of the macroscopically isotropic orientation distributions such as those of the cell surface bound fluorophores report on the presence of local orientation mismatches of the donor and acceptor preventing the optimal FRET in the polarized case, which may be eliminated by the excitation depolarization. A theory of fluorescence anisotropy for depolarized excitation is also presented.

Dual-laser homo-FRET on the cell surface.

Inhomogeneous broadening and red-edge effects have been detected on a highly mobile system of fluorescently conjugated mAbs targeted to cell surface receptors. By exploiting site-selective spectroscopy and the characteristic loss of homo-FRET on increasing excitation and decreasing emission wavelengths, contributions of physical rotation and homo-FRET to the depolarization of fluorescence anisotropy have been separated. Absolute homo-FRET efficiency has been determined by ratioing two anisotropies: a homo-FRET-sensitive one, which is excited at the absorption main band and detected at the long wavelength region of emission, and a homo-FRET-insensitive one, which is excited at the long wavelength region of absorption and detected at the short wavelength region of emission. Because the anisotropies are simultaneously detected in a unified detection scheme of a dual T-format arrangement, the method is applicable for the real-time tracking of dynamical changes of physical rotations and proximities. The utility of the method is demonstrated in the context of the MHCII molecule and the heavy and light chains of the MHCI molecule, a system of three receptors with well-characterized close mutual proximities. Although the method is presented for a flow cytometer, it can also be realized in a fluorescence microscope capable for dual-laser excitation and dual-anisotropy detection.

Single-laser polarization FRET (polFRET) on the cell surface.

A new method for the simultaneous detection of rotational mobility and proximity of cell surface receptors is presented based on cell-by-cell basis measurement of polarized fluorescence intensity components of the donor and acceptor of a FRET system. In addition to the FRET efficiency and the donor and acceptor concentrations, the method makes also possible the determination of the rotational characteristics and the associated fraction of the donors (FRET-fraction). The method is illustrated with flow cytometric and rFLIM measurements on donor-acceptor systems comprising fluorescently labeled whole antibodies and their Fab fragments against epitopes of the MHCI and MHCII cell surface receptors on human lymphoblast cells. Fluorescence anisotropy of donor and acceptor and FRET efficiency were measured for samples of different acceptor-to-donor concentration ratios. Acceptor anisotropy proved to be more sensitive than the donor anisotropy for sensing FRET. After determining the rotational constants of the donor-conjugated antibodies by measurements of FRET in the steady state, and by rFLIM as a reference, the associated fractions of the MHCI and MHCII molecules in their clusters were determined. Besides the flow cytometer and the wide-field rFLIM used in this study, the method can be applied also in other devices capable of dual-anisotropy detection.

The effects of treatment with liraglutide on atherothrombotic risk in obese young women with polycystic ovary syndrome and controls.

BACKGROUND: Polycystic ovary syndrome (PCOS) is associated with obesity and increased cardiovascular (CV) risk markers. In this study our aim was to assess the effects of six months treatment with liraglutide 1.8 mg od on obesity, and CV risk markers, particularly platelet function, in young obese women with PCOS compared to controls of similar age and weight. METHODS: Carotid intima-media wall thickness (cIMT) was measured by B-mode ultrasonography, platelet function by flow cytometry, clot structure/lysis by turbidimetric assays and endothelial function by ELISA and post-ischaemic reactive hyperemia (RHI). Data presented as mean change (6-month - baseline) ± standard deviation. RESULTS: Nineteen obese women with PCOS and 17 controls, of similar age and weight, were recruited; baseline atherothrombotic risk markers did not differ between the two groups. Twenty five (69.4%) participants completed the study (13 PCOS, 12 controls). At six months, weight was significantly reduced by 3.0 ± 4.2 and 3.8 ± 3.4 kg in the PCOS and control groups, respectively; with no significant difference between the two groups, P = 0.56. Similarly, HOMA-IR, triglyceride, hsCRP, urinary isoprostanes, serum endothelial adhesion markers (sP-selectin, sICAM and sVCAM), and clot lysis area were equally significantly reduced in both groups compared to baseline. Basal platelet P-selectin expression was significantly reduced at six months in controls -0.17 ± 0.26 but not PCOS -0.12 ± 0.28; between groups difference, 95% confidence interval = -0.14 - 0.26, P = 0.41. No significant changes were noted in cIMT or RHI. CONCLUSIONS: Six months treatment with liraglutide (1.8 mg od) equally affected young obese women with PCOS and controls. In both groups, liraglutide treatment was associated with 3-4% weight loss and significant reduction in atherothrombosis markers including inflammation, endothelial function and clotting. Our data support the use of liraglutide as weight loss medication in simple obesity and suggest a potential beneficial effect on platelet function and atherothrombotic risk at 6 months of treatment. TRIAL REGISTRATION: Clinical trial reg. no. ISRCTN48560305. Date of registration 22/05/2012.

Intensity correlation-based calibration of FRET.

Dual-laser flow cytometric resonance energy transfer (FCET) is a statistically efficient and accurate way of determining proximity relationships for molecules of cells even under living conditions. In the framework of this algorithm, absolute fluorescence resonance energy transfer (FRET) efficiency is determined by the simultaneous measurement of donor-quenching and sensitized emission. A crucial point is the determination of the scaling factor α responsible for balancing the different sensitivities of the donor and acceptor signal channels. The determination of α is not simple, requiring preparation of special samples that are generally different from a double-labeled FRET sample, or by the use of sophisticated statistical estimation (least-squares) procedures. We present an alternative, free-from-spectral-constants approach for the determination of α and the absolute FRET efficiency, by an extension of the presented framework of the FCET algorithm with an analysis of the second moments (variances and covariances) of the detected intensity distributions. A quadratic equation for α is formulated with the intensity fluctuations, which is proved sufficiently robust to give accurate α-values on a cell-by-cell basis in a wide system of conditions using the same double-labeled sample from which the FRET efficiency itself is determined. This seemingly new approach is illustrated by FRET measurements between epitopes of the MHCI receptor on the cell surface of two cell lines, FT and LS174T. The figures show that whereas the common way of α determination fails at large dye-per-protein labeling ratios of mAbs, this presented-as-new approach has sufficient ability to give accurate results. Although introduced in a flow cytometer, the new approach can also be straightforwardly used with fluorescence microscopes.

Impella-Driven High-Risk Percutaneous Coronary Intervention: A Novel, Single Non-Surgical-Centre Case Report.

Complex percutaneous coronary intervention (PCI) procedures have been routinely performed in non-surgical centres in the UK for more than two decades. These procedures follow strict guidelines and recommendations by the British Cardiovascular Intervention Society to ensure a more effective running of PCI programs. Even more so, expected guiding principles necessary for the safe optimisation of complex PCI procedures have also been created.  An 81-year-old male was admitted with non-ST-elevation myocardial infarction (NSTEMI) and severely impaired left ventricle ejection fraction (LVEF; 26% according to the cardiac MRI report). Angiogram findings revealed severe multiple-vessel coronary artery disease affecting the following arteries: right coronary artery (RCA), left anterior descending artery (LAD), left circumflex artery (LCx), and intermediate artery (IM). There was also severe disease in the distal left main stem (LMS) bifurcation extending to the ostia of the LAD, LCx, and IM branches. Following a multidisciplinary meeting, the patient underwent Impella-supported high-risk PCI (complex PCI) using the DK crush technique with no peri- and post-procedure complication and a significant LV function improvement (45-49%). This is the first known case of this procedure performed at the Royal Cornwall Hospital in Treliske (RCHT), Truro, Cornwall. This case report highlights that when the decision to choose between coronary artery bypass graft (CABG) and PCI is not straightforward following an individualised risk-stratification scoring system analysis and in the setting of patient comorbidities, a high-risk PCI supported with the Impella device is a suitable alternative with promising short-term and long-term outcomes.

[Reducing the risk of radiation-induced cardiotoxicity in patients with left breast tumor]. / A sugárkezelés okozta cardiotoxicitas kockázatának csökkentése bal oldali emlotumoros betegek kezelése során.

INTRODUCTION: Breast cancer is one of the most common malignancies affecting women. Treatment with drugs and radiotherapy increases the incidence of late cardiovascular disease. It is therefore particularly important to protect the heart from radiation exposure. METHOD: We prepared an irradiation plan for 45 patients with left breast cancer using deep breathing and normal breathing techniques. The plans were compared and analyzed. The irradiation plans were created in the Philips Pinnacle v. 16 planning system. RESULTS: At the same target volume coverage, the use of the deep breathing technique leads to a reduction of the dose burden to the heart and to the left descending coronary branch, thus reducing the incidence of late cardiovascular complications. DISCUSSION: The results obtained show that the use of the deep breathing technique during adjuvant radiotherapy of left-sided breast cancer patients has a beneficial effect on the radiation exposure of the heart. Our results are in good agreement with similar data from national centres. We were not only able to maintain planning target volume coverage, but also to achieve an improvement of 1%. There is a significant difference in dose to the heart and coronary artery. By using the deep breathing technique, we were able to reduce the average cardiac dose by almost half (deep breathing: 2.87 Gy, normal breathing: 5.4 Gy). The coronary exposure was reduced from 19.5 Gy to 10.98 Gy. CONCLUSION: The accuracy of treatment can be further improved by using a respiratory gating system with a surface-guided radiotherapy system. The successful use of deep breathing technique requires professionalism of the treatment staff and good patient cooperation. It is less equipment intensive than a respiration-guided system. The deep breathing technique is no longer considered state-of-the-art in the era of breath-holding, but the experience gained in our department is worth describing because of its relevance to oncocardiology. Orv Hetil. 2023; 164(11): 420-425.

[Introduction of high-precision stereotactic body radiotherapy of lung tumors at Markusovszky Hospital]. / Tüdodaganatok nagy pontosságú stereotaxiás kezelésének bevezetése a Markusovszky Kórházban.

INTRODUCTION: A new, modern computed tomograph simulator was installed in the Oncology Department of the Markusovsky University Teaching Hospital from September 2021. The computed tomography simulator not only makes the work of specialists easier with its automatic contouring tool, but is also able to produce four-dimensional computed tomography scans. This facility is essential for radiotherapy of lung and breast cancer patients. OBJECTIVE: In this paper, we briefly review lung tumors and their treatment options, focusing on radiotherapy requiring high precision. We summarize patient selection criteria, the quality assurance processes for planning and treatment, and the experience gained in treating patients. METHOD: 5 patients were selected for our study. Their disease met the following criteria: 1 nodule, tumor diameter not exceeding 5 cm, patient was inoperable or negated surgery. The planning computed tomography scan was performed with Siemens Somatom go.Sim. At each respiratory phase, the tumor conturs were drawn and then aggregated as an integrated volume and an irradiation plan was prepared on this image. The treatments were performed on a Varian TrueBeam accelerator. RESULTS: Before each treatment, an adjusting CT scan was taken. The higher dose (4 × 12 Gy) treatment caused a reduction in tumor size on the last adjustment scan. DISCUSSION: Stereotaxic treatment, which is already available in Szombathely, may be a good alternative in the treatment of patients with inoperable lung cancer. The method is not burdensome for patients: fewer sessions, short treatment time. CONCLUSION: In the future, we would like to improve the accelerator with a breath capture system, which will allow even more precise treatment. Orv Hetil. 2022; 163(52): 2079-2087.

Various clinical scenarios leading to development of the string sign of the internal thoracic artery after coronary bypass surgery: the role of competitive flow, a case series.

BACKGROUND: The left internal mammary artery (LIMA) is the choice for grafting of the left anterior descending coronary artery (LAD). One possible mechanism of the rare graft failure involve the presence of competitive flow. METHOD: 105 patients who had undergone coronary bypass grafting between 1998 and 2000 were included in this observational study. The recatheterizations were performed 28 months after the operations. The rate of patency the LIMA grafts was determined, and the cases with graft failure were analyzed. RESULTS: The LIMA graft was patent in 99 patients (94%). Six patients (6%) exhibited diffuse involution of the graft (string sign). The string sign was always associated with competitive flow as the basis of the LIMA graft involution. In one case quantitative re-evaluation of the preoperative coronary angiography revealed merely less than 50% diameter stenosis on the LAD with a nonligated side-branch of the LIMA. At recatheterization in two patients the pressure wire measurements demonstrated only a non-significant decrease of the fractional flow reserve (0.83 and 0.89), despite the 53% and 57% diameter stenosis in the angiogram. Another patient displayeda significant regression of the LAD lesion between the pre- and postoperative coronary angiography (from 76% to 44%) as the cause of the development of the competitive flow. In one instance, a radial artery graft on the LAD during a redo bypass operation resulted in competitive flow in the radial graft due to the greater diameter than that of the LIMA. In a further patient, competitive flow developed from a short sequential part of the LIMA graft between the nonsignificantly stenosed diagonal branch and the LAD, with involution of the main part of the graft to the diagonal branch. CONCLUSIONS: The most common cause of the development of the string sign of a LIMA graft due to competitive flow is overassessment of the lesion of the LAD. Regression of a previous lesion or some other neighboring graft can also cause the phenomenon.