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Introduction: Ectopic gastrointestinal varicosities are defined as dilated portosystemic collateral veins that may localize anywhere in the gastrointestinal tract outside the gastroesophageal region. Ectopic colonic varices can be considered idiopathic when other etiology that related to portal hypertension or portal vein thrombosis have been excluded., Case Presentation: A forty-five-year-old female patient has been under treatment for histopathologically confirmed ulcerative colitis since the age of 17. In her forties, the patient developed worsening hematochezia leading to severe anemia. Routine colonoscopy was performed which confirmed extensive rectosigmoid varices. A thorough investigation did not confirm any underlying causes, such as portal hypertension or cirrhosis., Discussion: The selective percutaneous transhepatic mesenteric angiography, which is recommended as a diagnostic and therapeutic option, was not performed because the interventional radiologists did not consider embolization feasible. Laparoscopic rectosigmoid resection with high ligation of the inferior mesenteric vein led to complete remission of hematochezia. The final histological examination confirmed ectopic rectum and sigmoid varices, and ulcerative colitis was ruled out., Conclusions: Lower gastrointestinal bleeding from the colonic varices is very rare, with only a few cases reported in the literature. In the idiopathic form, the prognosis is very good, given the absence of other underlying diseases causing portal hypertension. Ectopic varices present a clinical challenge as they are difficult to diagnose and localize. There are currently no clear guidelines for diagnosis and therapy, and recommendations are based on different case reports. Idiopathic cases can be treated effectively by resection of the affected bowel segment., Competing Interests: Declaration of competing interest The study sponsor (University of Debrecen, Hungary) has no involvement in data collection and analysis, and in the writing of the manuscript., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: We present a technique for covering large midline loss of abdominal wall using a novel method by autologous tissues., Methods: Twenty-two patients (body mass index = 35,6 ± 6,9 kg/m2) were involved in the prospective cohort study. Acute and elective cases were included. The gap area was 450.1 ± 54 cm2. The average width of the midline gap was 16,3 ± 3,2 cm. The rectus muscles were mobilized from its posterior sheath. Both muscles were turned by180º medially, so that the complete abdominal wall gap could be covered without considerable tension. Changes in intra-abdominal pressure, quality of life and hernia recurrency were determined., Results: There was no significant increase in the intra-abdominal pressure. Wound infection and seroma occurred in four cases. Bleeding occurred in one case. Pre- and post-operative quality of life index significantly improved (23 ± 13 vs. 47 ± 6; p = 0,0013). One recurrent hernia was registered. The procedure could be performed safely and yielded excellent results. The method was applied in acute cases. The intact anatomical structure of rectus muscles was essential., Conclusions: The midline reconstruction with bilateral turned-over rectus muscles provided low tension abdominal wall status, and it did not require synthetic mesh implantation.

Introduction: There have been significant changes in the treatment protocol for rectal tumors in recent decades, greatly reducing the rate of local recurrence and distant metastasis, thereby increasing overall survival. Method: We performed a retrospective processing and statistical analysis of the data of 362 patients with rectal cancer who underwent local neoadjuvant chemoradiotherapy and then underwent surgical treatment between 1 January 2010 and 31 December 2017 at the Institute of Surgery of the University of Debrecen. We compared the response rate and overall survival results of our patients with local neoadjuvant treatment to the outcomes of total neoadjuvant treatment reported by the recent large international studies. Results: We experienced complete pathological regression in 8.6% of our patients. After neoadjuvant therapy, 10.7% of our patients experienced distant metastasis at the time of the operation or within 3 months period thereafter. In our study, the rate of response to the neoadjuvant treatment was a prognostic factor independent of the stage at di-agnosis and recognition. The groups with better response produced significantly better survival results. Conclusion: The total neoadjuvant treatment doubled the number of patients with complete pathological response, and the incidence of distant metastasis was by 7% lower in both recent international studies compared to the local neoadjuvant group. 85% of our patients were T3-4N+ stage at the time of recognition. Given the 10.7% rate of dis- tant metastases detected at the time of surgery or within 3 months in our patient population, we can state that ap- proximately half of our patients would have benefited from the administration of total neoadjuvant therapy which produced better outcomes. Based on this conclusion, we decided to introduce the total neoadjuvant therapy protocol in our department for treatment of patients with advanced rectal tumors.

Stochastic resonance in clusters of major histocompatibility molecules is extended by a more detailed description of adaptive thresholding and by applying the notion of suprathreshold stochastic resonance as a stochastically quantizing encoder of transmembrane signaling downstream of major histocompatibility molecules and T-cell receptors on the side of presenting and recognizing cells, respectively. The adaptive nature of thresholding is partly explained by a mirroring of the noncognate-cognate dichotomy shown by the T-cell receptor structure and the kinetic-segregation model of the onset of T-cell receptor triggering. Membrane clusters of major histocompatibility molecules and T-cell receptors on their host cells are envisioned as places of the temporal encoding of downstream signals via the suprathreshold stochastic resonance process. The ways of optimization of molecular prostheses, such as chimeric antigen receptors against cancer in transmembrane signaling, are suggested in the framework of suprathreshold stochastic resonance. The analogy between Förster resonance energy transfer and suprathreshold stochastic resonance for information transfer is also discussed. The overlap integral for energy transfer parallels the mutual information transferred by suprathreshold stochastic resonance.

The original version of this article unfortunately contained a mistake. The variants listed in Table 3 of the original version of this article are not in line with the latest HGVS (Human Genome Variation Society) nomenclature (version 19.01).

Introduction: Lynch syndrome is an autosomal dominant disorder, most frequent leading to colon cancer. Identification of patients with Lynch syndrome and screening of their family members are available prevention approach that can significantly decrease mortality. Unfortunately, routine screening still does not belong to standard of care in Hungary. In this study, we performed a comprehensive screening in order to identify patients with mismatch repair (MMR) mutation between the years of 2011 and 2014. Identified mutations were compared with those already published in the international databases., Patients and Methods: Patients who underwent treatment for colorectal cancer at the Surgical Institute of the University of Debrecen were screened using the modified Amsterdam and Bethesda Criteria. Immunohistochemistry and microsatellite analyses were performed in order to identify possible mutation carrier cases. Suspicious cases underwent DNA sequencing to detect mutations in the mismatch repair genes (hMLH1, hMSH2)., Results: All together 760 colorectal cancer patients were screened. A total of 28 patients were identified as possible MMR mutation carrier and underwent further genetic evaluation. Pathogenic sequence variants of the MMR gene were found in 5 patients. Hypermethylation of the promoter region of the hMLH1 gene was identified in 2 patients. Two out of the 5 pathogenic sequence variants of the MMR gene were first identified by our group while other 2 mutations were previously published as possible founder mutations., Conclusion: Identification of families with Lynch syndrome, while challenging because of variable phenotypes at diagnosis, is feasible with available molecular biological technologies and crucial to reduce mortality caused by this syndrome.

Highly conserved 2D receptor clusters (membrane rafts) of immunological signaling molecules with MHCI and MHCII antigens as their cores have been observed in the past on the surface of T- and B-cell lines of lymphoid origin, as well as on cells from patients with colon tumor and Crohn's disease. Conservativity is related to the ever presence of MHCI molecules. Although they are suspected to play a role in maintaining these clusters and facilitating transmembrane signaling, their exact role has been left largely enigmatic. Here we are suggesting stochastic resonance (SR), or "noise-assisted signal detection", as a general organizing principle for transmembrane signaling events evoked by processes like immune recognition and cytokine binding taking place in these clusters. In the conceptual framework of SR, in immune recognition as a prototype of transmembrane signaling, the sea of self-peptide-MHC complexes around a nonself-peptide presenting MHC is conceived as a source of quickly fluctuating unspecific signal ("athermal noise") serving the extra energy for amplifying the weak sub-threshold specific signal of the nonself-peptide presenting MHC. This same noise is also utilized for a readjustment of the threshold - and also the sensitivity and specificity - of detection by a closed loop feedback control of the TcR-CD8 (CD4) proximity on the detecting T-cell. The weak sub threshold specific signal of nonself-peptide presenting MHC is amplified by the superposing unspecific signals of the neighboring self peptide-MHC complexes towards the T-cell receptor as the detector. Because in a successful detection event both self- and nonself-peptides are detected simultaneously, the principle of coincidence (or lock-in) detection is also realized. The ever presence of MHC islands gets a natural explanation as a source of extra power - in a form of "athermal noise" - needed for coincidence detection and frequency encoding the evoked downstream signals. The effect is quite general, because the actual type of molecules surrounding a chief signaling molecule - like nonself-peptide holding MHC, interleukin-2 and -15 cytokine receptors (IL-2R/15R) - as the fluctuating interaction energy sources is immaterial. The model applies also for other types of signaling, such as those evoked by cytokine binding. The phenomenon of SR can also be interpreted as sampling of a low frequency, specific signal with a high frequency unspecific signal, the "noise". Recipes for identifying other forms of SR in membrane clusters with biophysical tools are recommended., (Copyright © 2019 European Federation of Immunological Societies. All rights reserved.)

Aims: In severe acute pancreatitis the timing of necrosectomy is ideally should be postponed 4-6 weeks after the onset of the disease when the walled-of pancreatic necrosis (WOPN) has developed. The authors present their experience with open transgastric necrosectomy for extended WOPN., Patients and Methods: The authors performed 17 (12 male, 5 female with a mean age of 61.6 ± 15.1 years) open transgastric necrosectomies for extended WOPN in a period of 1, January 2012 and 31, December 2017. Before the operations conservative and semiconservative therapy was used for an average of 74.6 ± 83.1 days. The mean size of the WOPNs was 13.8 ± 5.2 cm with localisation of the retrocolic and retroduodenal regions. All necroses were septic., Results: Complications related to the operation were not observed. The mean time of hospitalization after the surgery was 11.6 ± 12.8 days. The mortality rate was 5.9%. Late operation or other interventions for pseudocyst or pancreas fistula formation was not performed. Two patients needed endoscopic dilatation with lavage in the early postoperative period because of fever. New diabetes mellitus was not observed but worsening of previously existed diabetes developed in 6.3% of the cases., Conclusions: The open transgastric necrosectomy is safe and effective for extended WOPN. The advantage of this type of necrosectomy is the prevention of pancreatic pseudocyst and fistula formation.

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 ( R 0 ). 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.

Orientation factor (κ 2 ) for FRET from a rotating donor dipole, transferred helicity of the donor field and torque exerted on the acceptor by the donor have been investigated in the framework of classical electrodynamics. It is shown that for rotating dipole, κ 2 is significantly higher as compared to linear dipole independently of the orientation distribution of the donor and acceptor and whether the static or the dynamic rotational regimes are used for averaging κ 2 . By this property of κ 2 , FRET serves as an example for a phenomenon where local field interference may take place in a "natural" way for emitters possessing rotating dipoles in their excited states by nature. The overlapping spatial distributions for the helicity of donor local field, torque exerted on the acceptor by the donor and for the FRET orientational factor suggest that transfer of both energy and helicity take place predominantly in the plane of rotation by keeping the original direction of helicity, i.e. in accordance with the conservation law for helicity. Orienting FRET has been proposed by engineering local field structure by using elliptically polarized light for donor excitation or by using linearly polarized light coupled with electromagnetic modification of the donor environment. The phenomenon of increased κ 2 can be exploited for checking helicity conservation for different FRET donors without the need for polarized detection optics. Modulation of FRET with changing ellipticity of the excited donor state might supply structural and dynamical information on the orientational distribution of dye-holding matrices even on the surface of living cells, e.g. on the level of cell surface receptor clusters. Furthermore, it might also be exploited in sensing local electromagnetic fields. Rotating excited donor states might also facilitate turning on photoswitchable acceptors., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

Relationship of donor and acceptor fluorescence anisotropies as well as efficiency of fluorescence resonance energy transfer (FRET) has been investigated in a confocal microscope in the context of FRET systems comprised of donor and acceptor-labeled MHCI and MHCII receptors on the surface of Kit-225 K6 human T-cells. The measurements have been carried out in a 2-laser, 5-signal platform where the total donor fluorescence intensity and 2 acceptor fluorescence intensities with their anisotropies - one at the donor's excitation wavelength, the other at the acceptor's excitation wavelength - have been detected. This configuration enabled the determination of FRET efficiency and correlating it with the two acceptor fluorescence anisotropies as a kind of calibration. Estimations for the FRET-enhanced donor fluorescence anisotropy, the directly excited acceptor fluorescence anisotropy, and the fluorescence anisotropy of sensitized emission have been obtained. Procedures for determining FRET by measuring only the total donor intensity and the acceptor intensity and its anisotropy, or two acceptor intensities and their anisotropies have been elaborated, the errors of which have been estimated based on the fluorescence anisotropy values obtained in the calibration with the method of flow cytometric energy transfer (FCET). The combined detection of the donor and acceptor fluorescence anisotropies enabled also the determination of the lower and upper limits of the orientation factor for FRET (κ 2 ). An increase in range for κ 2 with increasing FRET efficiency has been observed, with average κ 2 values different from the dynamic random average of 2/3. These observations call for the need of κ 2 determination in proximity measurements, where the donor and acceptor orientations are not predictable. An increasing range of κ 2 with increasing intermolecular proximity of the MHCI and MHCII receptors has been observed. This indicates that molecular flexibility in the clusters of the MHCI and MHCII receptors reduces with increasing cluster density, i.e. a "fluidity gradient" exists in the clusters. More specifically, the local density dependent flexibility can also be taken as a direct proof for that the association of these receptors is non-random, but mediated by some type of physical interaction, a finding as a benefit of FRET detection by polarization spectroscopy. Two new quantities - the quenched donor fluorescence anisotropy and a fluorescence anisotropy analogue, the "dissymmetry index" of the polarized FRET efficiency components - have also been introduced for the characterization of the orientational dynamics of the excited state during FRET., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Hereditary nonpolyposis colorectal carcinoma (HNPCC) is an autosomal dominant disease, which shows familial clustering., Aim: We would like to emphasize the importance of monitoring the HNPCC syndrome patients by presenting a case of a proven MMR gene mutation carrier and her family tree encompassing 10 years., Materials and Method: To screen a suspected HNPCC Hungarian family member we are taking thorough family histories. If the diagnosis of HNPCC was further supported by immunohistology and the microsatellite status, sequencing of the MMR genes was carried out., Results: A novel mutation in exon 6 of the hMSH2 gene leading to the deletion of two nucleotide pairs [c.969-970delTC] was detected in our patient. During the 10-year follow-up period of our patient new HNPCC-associated tumors have developed in several family members. Conslusion: Close surveillance of the patient and its family members at risk was effective, although it requires compliance from the subjects. Orv Hetil. 2017; 158(30): 1182-1187.

Introduction: The eventrated, giant abdominal wall hernias represent a considerable challenge in our practice. Presently, preoperative evaluation of the musculo-aponeurotic elements of the abdominal wall by CT imaging is not part of routine planning of surgery., Aim: Evaluation of the abdominal wall hernia progression in time. Moreover, follow up the changes of the abdominal wall structures following series of intraabdominal surgeries., Method: Abdominal CT imaging were performed on the 1st, 3rd, 6th, 12th, 18th, and 24th postoperative months after the primary series of surgeries in the cases of 12 patients, whose reconstructive surgeries were not possible. A prospective data collection was applied. Changing of the bilateral rectus muscle morphology, the evolution in time of the midline gap, and the progressive dynamism of the midline wall defects were determined., Results: A characteristic and progressive midline defect enlargement could be settled. Data analysis yielded that the combined width of the bilateral rectus muscles is sufficient to cover the midline abdominal wall defect, although there is an "optimal" timeframe for performing the intervention., Conclusion: CT evaluation of abdominal wall prior to reconstructive surgeries of loss of abdominal wall domain has a strong significance on determining and designing the adequate surgical procedure. Orv. Hetil., 2017, 158(7), 257-263.

Introduction: Double-layer dermal grafts are used for the management of complicated abdominal wall hernias in obese, high risk patients. The method has not yet been used in case of emergency in septic/dirty environment., Case Report: A 76-year old female patient (BMI 36.7kg/m 2 ) was admitted with mechanical bowel obstruction and sepsis caused by a third time recurred, incarcerated and eventrated abdominal wall hernia. During the emergency surgery perforation of the terminal ileum and the ascending colon was detected, along with a feculent peritonitis and extended abdominal wall necrosis. Extended right hemicolectomy and necrectomy of the abdominal wall were performed. The surgery resulted in an abdominal wall defect measuring 223cm 2 , for the management of which direct closure was not possible. Using a specific method, an autologous dermal graft was prepared from the redundant skin. The first dermal graft was placed under the abdominal wall with 5cm overlap, and the second layer was placed onto the first layer with 3cm overlap in a perforated fashion. The operating time was 250min. No significant intra-abdominal pressure elevation was measured. No reoperation was performed. On the fifth postoperative day, the patient was mobilised. She was discharged in satisfactory general condition on the 18th postoperative day. There is no recurrent hernia 8 months after the surgery., Discussion: Abdominal wall reconstruction was possible in a necrotic, purulent environment by using a de-epithelised autologous double layer dermal graft, without synthetic or biological graft implantation. The advantage of the procedure was cost-effectivity, and the disadvantage was that only in an obese patient is the sufficient quantity of dermal graft available., Conclusion: A homogeneous internal and perforated outer dermal graft was suitable for bridging the abdominal gap in the case of an obese, high risk patient. Autologous dermal grafts can be a safe and feasible alternative to biological meshes in emergency abdominal wall surgeries. Evaluation of a case series can be the next cornerstone of the method described above., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: Indication and timing of allograft nephrectomy is still uncertain in some cases., Aim: The aim of the authors was to summarize their experience with graftectomies., Method: Data from patients who underwent kidney transplantation between January 1, 2004 and December 31, 2015 were retrospectively analyzed. Frequency, indications, timing, complications as well as early and late allograft nephrectomies were reviewed., Results: From 480 renal transplants, 55 graftectomies were performed (11%). Frequent indications included chronic allograft nephropathy (47%), arterial blood supply complications (13%), ureter complications (9%). 22 cases (40%) of allograft nephrectomies were urgent while 33 cases (60%) were elective. 24% of graftectomies were performed within 30 days after transplantation and 76% thereafter., Conclusions: The main indications for early graftectomies were arterial complications (31%) and chronic allograft nephropathy (62%) in cases of late graftectomies. The majority of the graftectomies were elective. Leading indication was chronic allograft nephropathy. Early and late graftectomies have different characteristics.

Introduction: The first renal transplantation was completed in 1991 at the University of Debrecen. In 2013 Hungary joined Eurotransplant., Aim: The authors retrospectively compared the trends., Method: Comparison between Period A (from January 1, 2008 to August 31, 2013) and Period B (from September 1, 2013 to October 22, 2015)., Results: The proportion of living transplants rose from 3.5% to 9.1 %. During period B over 25% of utilized donors were over 60 years of age. Recipients with body mass index above 30 kg/m(2) increased from 12% to 31%. Prevalence of diabetes among recipients rose twofold. Uretero-neocystostomy was used during period A (99%) while in period B end to side uretero-ureteral anastomosis has also gained popularity (68%). In 2013 the authors introduced routine use of induction treatment. Acute rejection rate decreased from 34% to 8%. The rate of surgical complications did not change. Acute bacterial infections decreased from 41% to 33%. Cumulative renal allograft 1, 3 and 5 year survival rates were 86.6%, 85% and 82.7% in group A vs. projected rates 88%, 84% and 84% in group B, respectively., Conclusions: Despite the growing proportion of expanded criteria donors, the authors were able to maintain a low incidence of delayed graft function and a favorable graft survival. Since 2013 the authors introduced treatments for acute humoral rejection according to international standards.

Introduction: Complicated incisional hernias (at least one time recurrent and/or multilocular and/or infected synthetic mesh) still represent a significant problem., Aim: Presentation of operating techniques desribed so far, as well as publication of a novel procedure and results invented by the authors., Method: Between 01/2011 and 09/2015, 41 patients with recurrent and/or infected incisional hernias with or without entero- and subcutaneous fistulas were operated using the method of the s.c. double-layer autologous tension free dermal flap technique. An accurate follow-up method and a continuous registration of the results was conducted in case of every patient. The body mass index (BMI) and presence of diabetes mellitus (DM) were distinguished factors out of the patients' clinical data. Surgical complications, bulking or laxity, recurrence and the patients' satisfactory index - among other things - were recorded considering the procedure. Patients' clinical data and results: Average age was 59.2 years (13 male / 28 female) in the cohort. 1, 2, 3 times recurrent incisional hernias had 12, 23, 6 patients, respectively. Average BMI was 32,1 kg/m2. 12 patients were treated with type II diabetes. 13 patients had entero- or subcutaneous fistulas and/or infected synthetic meshes at the time of operations. Average follow-up time was 32 months (2-58 months). Seroma formation was registered in 13 cases (31.7%). Fistula formation was registered in one case (2.4%). Bulking formation or laxity was observed in 3 patients (7.3%) and recurrence was noticed in 3 patients (7.3%), 13, 17 and 19 months later in the postoperative period. All the entero- and subcutaneous fistulas developed prior to the last procedure were completely healed. There was no lethal outcome., Conclusion: The method developed by the authors is recommended and suitable for the solution to complicated and/or infected incisional abdominal wall hernias especially in cases of obese (BMI ≥ 25 kg/m(2)) and diabetic, high risk patients. After acquiring the precise operative technique, the method is safe, feasible and it comes along with lower complication and recurrence rate, compared to other applied and well established ones. Further clinical trials need to be conducted in the future in order to be evaluated definetely this procedure.

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)