Use Of Fluorescence Research Articles

George Gabriel Stokes as a biologist.

It is universally acknowledged that George Gabriel Stokes (1819-1903) was a polymath who made major contributions to the fields of mathematics, chemistry, physics, fluidics and optics. However, his contributions to biology have received far less attention and this brief communication examines two of Stokes' major biological contributions, namely his description of the phenomenon of fluorescence and his studies on the changes in the colour of blood following oxidation and reduction. The paper on fluorescence is discussed because in it, Stokes demonstrates his wide-ranging biological knowledge and because the use of fluorescence is an invaluable experimental tool in biology. It was by developing the experimental approaches and equipment used to investigate fluorescence that Stokes then applied these to other investigations, including that of blood. From what we now know, what Stokes was describing in his paper on blood were the changes in the configuration of the haemoglobin molecule upon the acquisition and release of oxygen. This article is part of the theme issue 'Stokes at 200 (part 2)'.

Intraoperative use of fluorescence with indocyanine green reduces anastomotic leak rates in rectal cancer surgery: an individual participant data analysis.

Fluorescence imaging by means of Indocyanine green (ICG) has been applied to intraoperatively determine the perfusion of the anastomosis. The purpose of this Individual Participant Database meta-analysis was to assess the effectiveness in decreasing the incidence of anastomotic leak (AL) after rectal cancer surgery. We searched PubMed, Embase, Cochrane Library and ClinicalTrial.gov, EU Clinical Trials and ISRCTN registries on September 1st, 2019. We considered eligible those studies comparing the assessment of anastomotic perfusion during rectal cancer surgery by intraoperative use of ICG fluorescence compared with standard practice. We defined as primary outcome the incidence of AL at 30days after surgery. The studies were assessed for quality by means of the ROBINS-I and the Cochrane risk tools. We calculated odds ratios (ORs) using the Individual patient data analysis, restricted to rectal lesions, according to original treatment allocation. The review of the literature and international registries produced 15 published studies and 5 ongoing trials, for 9 of which the authors accepted to share individual participant data. 314 patients from two randomized trials, 452 from three prospective series and 564 from 4 non-randomized studies were included. Fluorescence imaging significantly reduced the incidence of AL (OR 0.341; 95% CI 0.220-0.530; p < 0.001), independent of age, gender, BMI, tumour and anastomotic distance from the anal verge and neoadjuvant therapy. Also, overall morbidity and reintervention rate were positively influenced by the use of ICG. The incidence of AL may be reduced when ICG fluorescence imaging is used to assess the perfusion of a colorectal anastomosis. Limitations relate to the consistent number of non-randomized studies included and their heterogeneity in defining and assessing AL. Ongoing large randomized studies will help to determine the exact role of routine ICG fluorescence imaging may decrease the incidence of AL in surgery for rectal cancer.

Systematic use of fluorescence in-situ hybridisation and clinicopathological features in the screening of PDGFRB rearrangements of patients with myeloid/lymphoid neoplasms.

Rearrangement of the platelet-derived growth factor receptor B (PDGFRB) gene defines a unique group of myeloid/lymphoid neoplasms with frequent eosinophilia and high sensitivity to tyrosine kinase inhibitors. This genetic abnormality is also rarely reported in Philadelphia-like B-cell acute lymphoblastic leukaemia/lymphoma (B-ALL). PDGFRB rearrangement was initially thought to only occur in cases with 5q31-33 rearrangement as determined with conventional cytogenetics; however, there are reported cases with cryptic rearrangements. We aim to develop a broader strategy for screening of PDGFRB rearrangements of patients with myeloid/lymphoid neoplasms. We performed fluorescence in-situ hybridisation (FISH) for PDGFRB rearrangement in 197 patients, including 70 with B-ALL, 10 with myeloid neoplasms with 5q31-33 rearrangements, and 117 with eosinophilia (≥0.5×109 /l in peripheral blood or ≥5% in bone marrow), and identified PDGFRB rearrangement in four of 197 (2.0%) cases. In an attempt to identify clinicopathological and genetic features that may have a stronger association with PDGFRB rearrangement, we analysed 13 patients with confirmed PDGFRB rearrangements, including 10 with myeloid neoplasms and three with B-ALL. Among the 10 patients with myeloid neoplasms, eosinophilia was present in eight, monocytosis in two, 5q31-33 rearrangement in seven, and abnormal bone marrow morphology in all. All patients with myeloid neoplasms showed an excellent response to imatinib, including a patient in blast crisis. The three B-ALL patients presented denovo, showed no eosinophilia, had a complex karyotype including 5q31-33 rearrangement, and had clinically aggressive courses with ultimate patient demise. These findings suggest that a higher yield for the identification of PDGFRB rearrangement may result from an index of suspicion in patients with eosinophilia, monocytosis, bone marrow features of a myeloid neoplasm, and 5q31-33 rearrangement, and patients with Philadelphia-like B-ALL.

Future Directions.

Transanal total mesorectal excision (taTME) is a novel technique that has evolved over the years to address the challenges of low rectal cancer surgery by applying the principles and benefits of laparoscopic surgery to more historic transanal techniques. It has been popularized through its use in rectal cancer, but the transanal approach is slowly being expanded to tackle different clinical scenarios including benign conditions such as inflammatory bowel disease and endometriosis. For all of these new indications, it is the desire to access and begin the dissection in native tissue beyond the pathology which makes this approach applicable to other diseases where anatomy can be challenging. Training pathways to safely introduce taTME in a standardized manner are being developed and implemented in a bid to ensure adequate training to all the surgeons using this technique and thus minimize complications and patient morbidity. The future directions of this promising technique include the use of image and optical technological enhancement to aid navigation, the use of pneumorectum stabilization, and perhaps the use of fluorescence as a safety improvement. Developments have come also from the field of robotics. After a demonstration of feasibility in cadaver models, a growing experience has been gathered in the robotic approach to taTME, covered in the last part of this chapter.

Use of fluorescence to guide surgical resection in vulvo-vaginal neoplasia: Two case reports

We report hereby two cases of fluorescence-guided surgical resection with Indocyanine green in vulvo-vaginal neoplasia. The first case was a 86-year old patient who had a high-grade squamous intraepithelial lesion of the left small lip and on the vulvar fork. After a first incomplete surgery, a second fluorescence-guided vulvectomy was performed with ICG injected intravenously to determine intraoperatively surgical margins. At final pathologic examination, surgical margins were free of disease and postoperative course was uneventful. The second case was a 44-year old patient who had a clear cell carcinoma of the upper vagina. She underwent a fluorescence-guided colpo-hysterectomy with pelvic lymphadenectomy. ICG was injected one centimeter around the tumor and highlighted intraoperatively the limits of the vaginal resection. On the specimens, surgical margins were also negative. Peritumoral or intravenous injection of ICG are promising techniques for the intraoperative identification of surgical margins in gynecologic malignancies.

Use of Fluorescence In Situ Hybridization (FISH) in Diagnosis and Tailored Therapies in Solid Tumors.

Fluorescence in situ hybridization (FISH) is a standard technique used in routine diagnostics of genetic aberrations. Thanks to simple FISH procedure is possible to recognize tumor-specific abnormality. Its applications are limited to designed probe type. Gene rearrangements e.g., ALK, ROS1 reflecting numerous translocational partners, deletions of critical regions e.g., 1p and 19q, gene fusions e.g., COL1A1-PDGFB, genomic imbalances e.g., 6p, 6q, 11q and amplifications e.g., HER2 are targets in personalized oncology. Confirmation of genetic marker is frequently a direct indication to start specific, targeted treatment. In other cases, detected aberration helps pathologists to better distinguish soft tissue sarcomas, or to state a final diagnosis. Our main goal is to show that applying FISH to formalin-fixed paraffin-embedded tissue sample (FFPE) enables assessing genomic status in the population of cells deriving from a primary tumor or metastasis. Although many more sophisticated techniques are available, like Real-Time PCR or new generation sequencing, FISH remains a commonly used method in many genetic laboratories.

PD19-04 VALIDATION OF HYBRID ICG-99mTc-NANOCOLLOID FOR SENTINEL NODE BIOPSY IN A LARGE COHORT OF PENILE CANCER PATIENTS

INTRODUCTION AND OBJECTIVE: Dynamic sentinel node (SN) biopsy is an invasive lymph node (LN) staging method that is typically performed using 99mTc-nanocolloid combined with blue dye to detect micro-metastatic disease for ≥T1G2 penile cancer (PC) patients with no palpable inguinal LNs (cN0). Recent reports suggested that the hybrid tracer indocyanine green (ICG)-99mTc-nanocolloid may improve intraoperative optical SN identification relative to the blue dye. However, this evidence is supported by small sample size series. The aim of the current study was to confirm these findings relying on the largest single center cohort of PC patients who underwent SN biopsy using ICG-99mTc-nanocolloid and blue dye METHODS: 404 patients with ≥T1G2N0 penile squamous cell carcinoma were scheduled for SN biopsy and treatment of the primary tumour at a single European centre. Of those, 255 received SN biopsy using ICG-99mTc-nanocolloid and blue dye. After peritumoural injection of ICG-99mTc-nanocolloid, SNs were preoperatively identified based on lymphoscintigraphy and SPECT/CT. Shortly before surgery, blue dye was administered in all patients using the same injection sites as the hybrid tracer. Intraoperatively, SNs were pursued via gamma tracing, visual identification (blue dye) and near-infrared fluorescence imaging (ICG). Intraoperative SN identification rates of blue dye and ICG-99mTc-nanocolloid were compared using a two-sample t-test RESULTS: There were no tracer-related adverse events. Lymphoscintigraphy and SPECT/CT visualized at least one SN in all patients. All preoperatively defined SNs were intra-operatively identified using a combination of radio-, fluorescence- and blue dye guidance. A total of 841 SNs were excised (median 3 SNs per patient; range 1-9). Of these, 95% were visible with fluorescence imaging, while only 56% were stained by blue dye (p<0.001). Pathologic analyses of the 841 SNs removed, identified 58 positive nodes in 43 patients. Of those, 100% (n=58) and 69% (n=40) were fluorescent or stained blue, respectively. In 45 patients, blue dye did not identify any LN. Of these patients, 7% had positive nodes. Conversely, fluorescence imaging using the hybrid tracer enabled optical detection of at least one SN in all patients CONCLUSIONS: The use of fluorescence improved the optical detection rate of SNs of 39% relative to blue dye. Its use is safe and can replace blue dye for SN biopsy in patients with PC Source of Funding: None

An Efficient Method for Generating Murine Hypothalamic Neurospheres for the Study of Regional Neural Progenitor Biology.

The hypothalamus is a key homeostatic brain region and the primary effector of neuroendocrine signaling. Recent studies show that early embryonic developmental disruption of this region can lead to neuroendocrine conditions later in life, suggesting that hypothalamic progenitors might be sensitive to exogenous challenges. To study the behavior of hypothalamic neural progenitors, we developed a novel dissection methodology to isolate murine hypothalamic neural stem and progenitor cells at the early timepoint of embryonic day 12.5, which coincides with peak hypothalamic neurogenesis. Additionally, we established and optimized a culturing protocol to maintain multipotent hypothalamic neurospheres that are capable of sustained proliferation or differentiation into neurons, oligodendrocytes, and astrocytes. We characterized media requirements, appropriate cell seeding density, and the role of growth factors and sonic hedgehog (Shh) supplementation. Finally, we validated the use of fluorescence activated cell sorting of either Sox2GFPKI or Nkx2.1GFPKI transgenic mice as an alternate cellular isolation approach to enable enriched selection of hypothalamic progenitors for growth into neurospheres. Combined, we present a new technique that yields reliable culturing of hypothalamic neural stem and progenitor cells that can be used to study hypothalamic development in a controlled environment.

Correlation between percutaneous biopsy and final histopathology for retroperitoneal sarcoma: a single-centre study.

Retroperitoneal sarcomas are rare soft tissue tumours accounting for 10-15% of soft tissue sarcomas. Patient prognosis and treatment recommendations (including extent of surgery and neoadjuvant strategies) are determined by the pre-operative histopathological subtype and grade obtained from biopsy and thus it is important to understand the accuracy of biopsy in retroperitoneal masses. This study presents a case series of primary retroperitoneal sarcomas managed at Peter MacCallum Cancer Centre (PMCC) between 2008 and 2019. Statistical analyses were performed to determine correlation between histopathology from percutaneous biopsy and surgical excision. A total of 117 patients who underwent percutaneous core biopsy and surgical excision of retroperitoneal sarcoma were included. Diagnostic accuracy varied with histopathological diagnosis, but overall precise concordance between biopsy and final histopathology was seen in 61% (κ = 0.57). Biopsy was most sensitive for identifying well-differentiated liposarcoma (WDLPS) (sensitivity 85%, 95% CI 0.06-0.96) and leiomyosarcoma (sensitivity 81%, 95% CI 0.54-0.96) and was least sensitive for identifying de-differentiated liposarcoma (DDLPS) (sensitivity 40%, 95% CI 0.25-0.56). Overall agreement between biopsy and final histopathology increased with use of PET/CT scan pre-biopsy and with use of fluorescence in situ hybridisation testing on biopsy, however, neither test improved recognition of de-differentiated components within WD/DDLPS on core biopsy. Pre-operative biopsy is important for clinical decision making in the treatment of retroperitoneal sarcoma. A significant portion of patients with a WDLPS will have a de-differentiated component identified at the time of resection that was not identified on initial biopsy.

Human Serum Albumin Aggregation/Fibrillation and its Abilities to Drugs Binding.

Human serum albumin (HSA) is a protein that transports neutral and acid ligands in the organism. Depending on the environment’s pH conditions, HSA can take one of the five isomeric forms that change its conformation. HSA can form aggregates resembling those in vitro formed from amyloid at physiological pH (neutral and acidic). Not surprisingly, the main goal of the research was aggregation/fibrillation of HSA, the study of the physicochemical properties of formed amyloid fibrils using thioflavin T (ThT) and the analysis of ligand binding to aggregated/fibrillated albumin in the presence of dansyl-l-glutamine (dGlu), dansyl-l-proline (dPro), phenylbutazone (Phb) and ketoprofen (Ket). Solutions of human serum albumin, both non-modified and modified, were examined with the use of fluorescence, absorption and circular dichroism (CD) spectroscopy. The experiments conducted allowed observation of changes in the structure of incubated HSA (HSAINC) in relation to nonmodified HSA (HSAFR). The formed aggregates/fibrillation differed in structure from HSA monomers and dimers. Based on CD spectroscopy, previously absent β-structural constructs have been registered. Whereas, using fluorescence spectroscopy, the association constants differing for fresh and incubated HSA solutions in the presence of dansyl-amino acids and markers for binding sites were calculated and allowed observation of the conformational changes in HSA molecule.

Simultaneous nitrogen removal and phosphorus recovery using an anammox expanded reactor operated at 25 °C

While anammox is a cost-effective nitrogen treatment process for wastewater with high nutrient strength, phosphorus remains untouched during this process and needs further treatment. In this study, the nitrogen removal and phosphorus recovery were achieved simultaneously at 25 °C using an anammox expanded bed. A stable high nitrogen removal efficiency of 83.7 ± 4.8% at a 1500 mgN/L influent total nitrogen concentration and a phosphorus removal efficiency of 94.2 ± 1.2% at 100 mg P/L influent total phosphorus were obtained during continuous operation. The effects of the nitrogen loading rate, hydraulic retention time (HRT), pH, Ca2+ and PO43- concentration on the phosphorus removal was verified in the long-term operation of the reactor. The sludge produced contained a high content of phosphorus mainly in the form of hydroxyapatite (HAP), and the sludge composition strongly reflected the nitrogen and phosphorus loading. The structure of the anammox-HAP composite granules was illustrated by the use of fluorescence in situ hybridization (FISH) and Raman spectroscopic mapping analysis. The results in this study indicate that by controlling the operation parameters, it is possible to integrate a high efficiency phosphorus recovery with the anammox process, and significantly reduce the nutrient loading for further wastewater treatment.

Karyological, Molecular and Cytogenetical Studies on Tree Plants in V.N. Sukachev Institute of Forest

Aim. The aim of the article is to find out the karyological features and conduct molecular and cytogenetic analysis on tree plants for biological diversity studies, solve problems of taxonomics, evolutionary and population genetics. Methods. Classic methods with acetohematoxylin staining of slides and new method of fluorescent in situ hybridization (FISH) had been used. Results . More than 250 populations and provenances of representatives of different genera of gymnosperms from the families Pinaceae, Cupressaceae, Ephedraceae and tree angiosperms from the families Rosaceae, Aceraceae, Polygonaceae, Nitrariaceae, Rhamnaceae, Fabaceae, Ericaceae were studied. Investigations had been conducted in natural populations and during the introduction, in optimal and extreme conditions, in disturbed ecosystems, botanical gardens and parks; in addition, various intraspecific forms and unique trees had been studied. The variability of chromosome numbers and a wide range of chromosomal mutations had been revealed. Use of fluorescence in situ hybridization (FISH) with the 45S and 5S ribosomal RNA gene probes and DAPI staining allowed to identify of homologous chromosome pairs in the karyotypes of conifers and to facilitate the comparative karyotype analysis of those species. Analysis of the chromosome numbers of woody angiosperms showed a significant role of polyploidy in their evolution. Conclusions. The studies on karyotypes in tree species showed their diversity, variability of chromosome numbers, higher level of chromosomal anomalies and their wide spectrum in extreme conditions, under introduction and also in cultivars and abnormal form of trees. The use of molecular cytogenetic markers made it possible to obtain new information on the structure of conifer chromosomes. The role of polyploidy in the evolution of woody angiosperms was shown.

Contemporary Approach to Locally Advanced Oral Cavity Squamous Cell Carcinoma.

Surgical management of locally advanced oral cavity squamous cell carcinomas (OCSCC) has long been recognized as a primary treatment modality. Technological advances have led to significant improvements in our surgical approach, from improvement in the visualization of tumors to more efficient and precise reconstruction. Here, we review the latest technological advances in surgical extirpation and reconstruction of locally advanced OCSCCs. The focus of technological innovation in surgical extirpation has been on improving visualization, with the use of intraoperative ultrasound for margin delineation, intraoperative navigation, narrow-band imaging, and the use of fluorescence. Though early, these are promising steps to ensuring complete resection of the cancer. Advances in reconstruction have been centered on the incorporation of computer assisted design, manufacturing, and virtual surgical planning, allowing for more complex three-dimensional defects to be expeditiously reconstructed. As these technologies are still under development, their impact on oncologic outcomes are not yet robustly defined; however, as technology continues to advance and become more widely available, new technologies will undoubtedly become integrated into enhancing surgical precision and planning.

Accuracy of the neurosurgeons estimation of extent of resection in glioblastoma

BackgroundThe surgeons’ estimate of the extent of resection (EOR) shows little accuracy in previous literature. Considering the developments in surgical techniques of glioblastoma (GBM) treatment, we hypothesize an improvement in this estimation. This study aims to compare the EOR estimated by the neurosurgeon with the EOR determined using volumetric analysis on the post-operative MR scan.MethodsPre- and post-operative tumor volumes were calculated through semi-automatic volumetric assessment by three observers. Interobserver agreement was measured using intraclass correlation coefficient (ICC). A univariate general linear model was used to study the factors influencing the accuracy of estimation of resection percentage.ResultsICC was high for all three measurements: pre-operative tumor volume was 0.980 (0.969–0.987), post-operative tumor volume 0.974 (0.961–0.984), and EOR 0.947 (0.917–0.967). Estimation of EOR by the surgeon showed moderate accuracy and agreement. Multivariable analysis showed a statistically significant effect of operating neurosurgeon (p = 0.01), use of fluorescence (p < 0.001), and resection percentage (p < 0.001) on the accuracy of the EOR estimation.ConclusionAll measurements through semi-automatic volumetric analysis show a high interobserver agreement, suggesting this to be a reliable assessment of EOR. We found a moderate reliability of the surgeons’ estimate of EOR. Therefore, (early) post-operative MRI scanning for evaluation of EOR remains paramount.

RUNX2 (6p21.1) amplification in osteosarcoma

Prior cytogenetic profiling of osteosarcomas has suggested that amplifications at the 6p12-21 locus are relatively common alterations in these tumors. However, these studies have been limited by variable testing methodologies used as well as by the relatively small numbers of cases that have been analyzed. To better define the frequency of this alteration, 111 osteosarcomas were profiled using hybridization capture-based next-generation sequencing (NGS) platform (Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets) as part of an institutional clinical cancer genomics initiative. Using this platform, amplification at the 6p12-21 locus was determined by copy number assessment of the VEGFA and CCND3 genes. In addition, fluorescence in situ hybridization was used to assess copy number status for RUNX2, a known transcriptional regulator of osteoblastic differentiation which has previously been reported to be dysregulated in osteosarcomas. 6p12-21 amplification using NGS-based copy number assessment was confirmed in more than a fifth of all cases tested (24 of 111, 21.6%). Most of these cases, when tested using fluorescence in situ hybridization, were found to include RUNX2 within the amplified locus (17 of 18, 94.4%). Whereas many laboratories lack access to large-panel NGS assays, the use of fluorescence in situ hybridization to identify 6p12-21 amplification events by targeting RUNX2 represents a widely available diagnostic modality for the identification of such cases. This could help better define the role of RUNX2 in osteoblastic differentiation and serve as a surrogate for the identification of potentially targetable alterations such as VEGFA amplification at this locus.

Introducing Fluorescence Guided Surgery into Neuro-Oncology Practice and Getting It Right First Time

Abstract Introduction Fluorescence Guided Surgery (FGS) using 5-ALA is recommended for patients undergoing resection of suspected high grade glioma and this type of surgery is increasingly being used in more units throughout the UK. We report on our experience of introducing 5-ALA guided surgery for suspected HGG in a regional neurosciences unit. Methods Consecutive series of the first 24 patients in our unit to receive 5-ALA guided surgery for suspected HGG from June 2017 to October 2018. Data was prospectively collected. Results 5-ALA fluorescence guided surgery was used for a range of histologically confirmed WHO grade III-IV gliomas. All but 1 tumour (GBM) fluoresced with 5-ALA, with strong fluorescence in recurrent GBM and anaplastic foci of grade 3 tumours. EOR varied according to the eloquence of the lesion with 70% of cases achieving gross total resection. 40% of cases used awake mapping or intraoperative neurophysiology. 5 patients (20.8%) had temporary neurological deficits which resolved in all cases. Individual surgeon learning curves resulted in 2 styles of practice- complete FGS with fluorescence used throughout the case, and intermittent use of fluorescence as an adjunct often at the end of debulking under white light. Conclusions Adopting 5-ALA use comes with specific challenges in terms of service organisation, learning curves and complication avoidance. Our experience is that it is useful both to improve EOR and as an adjunct to identify tumour tissue in eloquent region tumours with monitoring of function. This is possible through a day admission pathway in line with GIRFT.

NIR fluorescence-guided tumor surgery: new strategies for the use of indocyanine green.

Surgery is the frontline treatment for a large number of cancers. The objective of these excisional surgeries is the complete removal of the primary tumor with sufficient safety margins. Removal of the entire tumor is essential to improve the chances of a full recovery. To help surgeons achieve this objective, near-infrared fluorescence-guided surgical techniques are of great interest. The concomitant use of fluorescence and indocyanine green (ICG) has proved effective in the identification and characterization of tumors. Moreover, ICG is authorized by the Food and Drug Administration and the European Medicines Agency and is therefore the subject of a large number of studies. ICG is one of the most commonly used fluorophores in near-infrared fluorescence-guided techniques. However, it also has some disadvantages, such as limited photostability, a moderate fluorescence quantum yield, a high plasma protein binding rate, and undesired aggregation in aqueous solution. In addition, ICG does not specifically target tumor cells. One way to exploit the capabilities of ICG while offsetting these drawbacks is to develop high-performance near-infrared nanocomplexes formulated with ICG (with high selectivity for tumors, high tumor-to-background ratios, and minimal toxicity). In this review article, we focus on recent developments in ICG complexation strategies to improve near-infrared fluorescence-guided tumor surgery. We describe targeted and nontargeted ICG nanoparticle models and ICG complexation with targeting agents.

Induction of chlorophyll fluorescence use for assessment of functioning of Gentiana lutea L. plants photosynthetic apparatus in different conditions of culturing in vitro

Aim. To study the peculiarities of functioning of photosynthetic apparatus of Gentiana lutea L. plants in vitro under different light qualities and source of carbon in the composition of nutrient medium by using the induction of chlorophyll fluorescence (ICF) method. Methods. Chlorophyll fluorescence was determined in light-adapted leaves of cultivated in vitro G. lutea plants by use of PAM fluorometry MultispeQ. The parameters change of photosynthetic apparatus functioning of cultivated in vitro plants was assessed with regard to light qualities (Variant 1: light intensity – 85 W/m2, when different spectra combinations of blue-wave band (Еb) and green-wave band (Еg) and red-wave band (Еr) was 33% : 42% : 25%; Variant 2: light intensity – 100 W/m2, with wave bands of spectra – Еb : Еg : Еr = 25% : 27% : 48%) and the source of carbon (10 g/l of sucrose or 3 g/l of mannite) in the composition of nutrient medium МS/2 (MS medium with half amount of macro- and microsalts), supplemented with 0.1mg/l of kinetin. Results. It was established that G. lutea plants cultivated in vitro for 90 days in light conditions of Variant 2 had a quantum yield photochemical of PS ІІ 8.3 % higher in comparison to G. lutea specimens cultured in light conditions of Variant 1. The vitality value of Variant 2 plants was 23 % higher. Change of carbon source in the composition of nutrient medium from sucrose (10 g/l) to mannite (3 g/l) was both increasing the efficiency of functioning of photosynthetic apparatus of Gentiana lutea L. plants in vitro and activating their resistance mechanisms to water deficit. Conclusions. The ICF method use shows that functioning of photosynthetic apparatus of G. lutea plants in vitro depends different qualities light and source of carbon in the composition of nutrient medium.&#x0D; Key words: induction of chlorophyll a fluorescence, plants in vitro, Gentiana lutea L.

Effect of daptomycin and vancomycin on Staphylococcus epidermidis biofilms: An in vitro assessment using fluorescence in situ hybridization.

Colonization of in-dwelling catheters by microbial biofilms is a major concern in patient health eventually leading to catheter-related blood stream infections. Biofilms are less susceptible to standard antibiotic therapies that are effective against planktonic bacteria. Standard procedure for the detection of microorganisms on the catheter tip is culture. However, viable but non-culturable cells (VBNCs) may be missed. The aim of this study was to evaluate the use of fluorescence in situ hybridization (FISH) as an indicator to visualize and quantify the effect of the antibiotics daptomycin and vancomycin on biofilms in situ. We established an in vitro catheter biofilm model of Staphylococcus epidermidis biofilms on polyurethane catheters. Biofilm activity was measured by FISH and correlated to colony forming units (CFU) data. Digital image analysis was used for quantification of total biofilm mass and the area of the FISH positive biofilm cells. FISH showed a pronounced effect of both antibiotics on the biofilms, with daptomycin having a significantly stronger effect in terms of both reduction of biofilm mass and number of FISH-positive cells. This supports the anti-biofilm capacity of daptomycin. Interestingly, neither antibiotic was able to eradicate all of the FISH-positive cells. In summary, FISH succeeded in visualization, quantification, and localization of antibiotic activity on biofilms. This technique adds a new tool to the arsenal of test systems for anti-biofilm compounds. FISH is a valuable complementary technique to CFU since it can be highly standardized and provides information on biofilm architecture and quantity and localization of survivor cells.

The use of fluorescence in situ hybridization analysis on sperm: indications to perform and assisted reproduction technology outcomes.

To determine the consequences of an altered sperm fluorescence in situ hybridization (FISH) result for ART outcomes and the indications for a sperm FISH analysis. Data from 439 infertile men were collected. Bivariate analyses were performed to determine the association of men's age, seminal alterations, and sperm FISH indication, with the incidence of X, Y, 13, 18, and 21 sperm chromosomal abnormalities. A multivariate logistic regression analysis was performed to establish the most predictive variables for altered sperm FISH. Results from the IVF/ICSI cycles were collected for 248 out of 439 patients. Two distinct groups were established: 151 couples that used their own oocytes and 97 couples involved in egg donation programs. In both groups, ART outcomes were compared between normal and altered sperm FISH. Teratozoospermia and oligozoospermia were associated with sperm chromosome anomalies (p < 0.05). Indications for sperm FISH analysis with the highest predictability were teratozoospermia, male age, oligozoospermia, and implantation failure (AUC = 0.702). Embryo quality (p = 0.096), pregnancy rate (p = 0.054), and implantation rate (p = 0.089) were higher in own-oocytes couples with normal sperm FISH than in altered sperm FISH couples, although differences were not statistically significant. In donor-oocytes couples, in which high-quality embryos were transferred later than in own-oocytes couples (3.8 vs. 3.0 days), we did not identify differences in the ART outcome between normal and altered sperm FISH couples. In both groups, the possible interference of woman age was negligible. Sperm FISH is indicated in middle-aged oligoteratozoospermic patients with implantation failures in previous IVF/ICSI cycles. Sperm chromosome anomalies have a moderate detrimental impact on embryo quality, implantation, and pregnancy rates.