Clinical role and importance of fluorescence in situ hybridization method in diagnosis of H pylori infection and determination of clarithromycin resistance in H pylori eradication therapy

H pylori is etiologically associated with gastritis, gas-tric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of related diseases with the use of more accurate diagnostic molecular tests. Indeed some of the tests cannot give the evidence of current infection; H pylori can be detected by noninvasive and invasive methods, the latter requiring an endoscopy. Eradication failure is a big problem in H pylori infection. Recently, clarithromycin resistance in H pylori strains is increasing and eradicati-on therapy of this bacterium is becoming more difficult. Molecular methods have frequently been applied besides phenotypic methods for susceptibility testing to detect clarithromycin resistance due to mutations in the 2143 and 2144 positions of 23S rRNA gene. Fluorescence in situ hybridization (FISH) method on paraffin embedded tissue is a rapid, accurate and cost-effective method for the detection of H pylori infection and to determine clarithromycin resistance within three hours according to the gold standards as a non-culture method. This method can also be applied to fresh biopsy samples and the isolated colonies from a culture of H pylori, detecting both the culturable bacillary forms and the coccoid forms of H pylori, besides the paraffin embedded tissue secti-ons. This technique is helpful for determining the bac-terial density and the results of treatment where clarith-romycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.

Study on State of Terbium Ion in Liposome by One order Derivative Fluorescence Quenching Method

The state of Tb3+ is investigated in liposome. When the concentration of PC is below CMC (critical micell concentration), most of Tb3+ is associated with PC, the binding constant is about 3.35×103 L/mol. When the concentration of PC is beyond CMC, most of Tb3+ is dimerized, the dimerization constant is about 3.92×104L/mol. In PC?CH?H2O system, the binding constant of Tb3+?CH complex 2.93×104L/mol is obtained.

A QUENCHING FLUORESCENCE IMMUNOASSAY METHOD FOR DETERMINATION OF TRACE ALBUMIN USING UNLABELED TERBIUM CHELATE

A fluoroimmunoassay method using unlabeled Terbium chelate is described.The principle is similar to that of fluoroimmunoassay method using lanthanide chelate as labels.The procedure is simpte because labeling process is unnecessary.The recovery of HSA and albumin in urine is 107% and 95% respectively.The standard deviation is tess than 10%.

A FLUORESCENCE QUENCHING IMNUNOASSAY METHOD FOR DETERMINATION OF TRACE ALBUMIN USING UNLABELED EUROPIUM CHELATE

A fluoroimmunoassay method using unlabeled europium chelate is described.The principle is similar to that of fluoroimmunoassay method using lanthanide chelate as labels.The procedure is simple because labeling process is omitted.The detection limit is about 10^(10) mol/L antigen.The relative standard deviation of immunoassay is less than 10%.The recoveries of human serum albumin and estradiol protein conjugate are 96-105% and 111% respectively.

Determination of Chlorobenzene by Hydroxypropyl-β- Cyclodextrin Sensitized Fluorescence Quenching Method with Neutral Red as a Fluorescence Probe

The fluorescence quenching of inclusion complex of neutral red （NR） and hydroxypropyl-β-cyclodextrin （HP-β-CD） carried by chlorobenzene was investigated. The fluorescence intensity of NR increased due to the formed inclusion complex of HP-β-CD and NR. But the fluorescence intensity of NR-HP-β-CD diminished when chlorobenzene was added, and there was a linear relationship between the fluorescence quenching value of the system （△IF = IF, NR-HP-β-CD - IF, CB-NB-NR-HP-β-CD） and the concentration of chlorobenzene. Based on this, a novel fluorescence quenching method for the determination of chlorobenzene with NR as a fluorescence probe has been developed. Under the optimal conditions, the linear range of calibration curve for the determination of chlorobenzene was 5.0 × 10^-8 - 8.0 × 10^-6 mol/L and the detection limit was 1.0 × 10^-8 mol/L. It has been applied to determination ofchlorobenzene in synthetic waste water samples with satisfactory results.

Determination of heavy metal ion in landfill leachate with solvent extracting ＆ fluorescence quenching method

Add the masking agent and biscyclohexanoneoxalyldihydraone into the diluted clarificd liquid of the landfill leachate which was treatmented by digestion and centrifugal filtration to complexate all heavy metal ion in the trcatmented liquid, and extracted using CHCl3 many time, then it were demasking and decomposing respectively, and adjusted different pH and formed Me^n＋-PAN coordination compound when these metal ion reaction with PAN. The Fluoresence quenching of Rh6G （λex/λem=543mn/558nm） when the metal ion coordination compound was add into the Rh6G solution step by step, the quenching intensity was directly proportional to the concentration of the metal ion in the certain range. So a new method of fast and simple for determination of trace metal ion in landfill lcachate was established to determine metal ion in sample of different landfill leachate in Three Gorge Water Reservoir, and comparison the classic assay method with satisfactory results.

Determination of Anionic Surfactants in Environmental Water Samples Using Fluorescence Enhancement Method Based on Fe_3O_4@PAA-RB Fluorescent Nanoparticles

[ Objective] The study aims at developing a novel fluorescence enhancement method to determine anionic surfactants. [ Method] Based on Fe3O4 @ PAA-RB fluorescent nanoparticles as fluorescent probes, we have developed a novel fluorescence enhancement method for the determi- nation of an anionic surfactant sodium dodecyl sulfate （SDS） through the gradual optimization of experiment conditions. [ Result] Under the opti- mum conditions, the extent of fluorescence enhancement is directly proportional to SDS concentration varying from 0.5 to 16.0 μmol/L, and the de- tection limit reaches 0.051 μmol/L. The relative standard deviation （RSD） for 4.0 μmol/L SDS is 3.3% （ n =6）. The proposed method has been successfully applied to the determination of SDS in environmental water samples, with recovery of 96.3% -105.5%. E Conclusion] The novel fluo- rescence enhancement method is not only simple and rapid, but also has avoided using tedious solvent-extraction and toxic organic solvents.

Tumor cell death visualization of renal cell carcinoma under the combined effect of the Gratiola officinalis extract and cyclophosphamide using fluorescent staining methods

Objective of the study:We used fluorescence imaging methods of apoptosis and necrosis in human renal carcinoma A498 tumor cells in vitro to reveal the indicated forms of cell death under the combined effect of flavonoid-containing extract of Gratiola officinalis and cytostatic(cyclo-phosphamide).Materials and methods:The dyes were propidium iodide and acridine orange,which were used in the"alive and dead"test.This test helped us to identify the total number of dead cells in the forms of necrosis and apoptosis and the number of cells in which apoptosis had started,it was characterized by the appearance of apoptotic bodies or nucleus pyknosis.Results:We found the most pronounced cytotoxic activity at the ratio of extract of Gratiola officinalis and cyclophosphamide concentrations of 1∶1.The number of living cells decreased when exposed to the ratio of extract and cytostatic concentrations of 2∶1.When the ratio of concentration of the extract relative to the cytostatic increased to 3∶1,the cytostatic activity of the extract began to appear,the total number of tumor cells decreased.The number of cells with nucleus pyknosis and the number of cells with apoptosis signs significantly increased at a 3∶1 ratio of extract and cytostatic concentrations,which confirms the presence of pro-apoptotic activity of the studied combination.This trend indicates the dependence of a certain form of cell death(apoptosis,necrosis)on the ratio of extract and cytostatic doses,and it also demon-strates the cytostatic and cytotoxic effects of this combination.Conclusion:Fluorescence methods of investigation in the"alive and dead"test allowed us to visualize the forms of cell death of human kidney carcinoma A498 by combined exposure to the fiavonoid-containing extract of Gratiola officinalis and cytostatic(cyclophosphamide)24 h after exposure.We found that the combination with a concentration ratio of the extract and cyclophosphamide of 3:1 has the greatest effectiveness due to stimulation of the cytostatic effect and cytotoxic effect.

ACTIVATION OF NUCLEOLAR rRNA GENE TRANSCRIPTION IN LYMPHOCYTES BY TUMOR PROMOTERS: APPLIES STUDIES WITH IN SITU HYBRIDIZATION DETECTED BY FLUORESCENT METHOD

The distribution of rDNA was visualized in interphase nuclei of tumor promoter treated human lymphocytes in comparison with the mltogen Phytoheamagglutinin (PHA) effects by using an in situ hybridization fluorescent method. The procedure involves biotinylated rDNA as the probe and FITC-avidin as detection system. Silver (Ag) staining was used to visualize nucleoli. In the interphase nuclei of most of the nonstimulated control lymphocytes, only one small Ag-stained nucleolus could be seen. The in situ hybridization, however, revealed one to several agglomerations of rDNA fluorescent spots. With tumor promoting herb extract WCE (40 μg/ml) or TPA (60 ng/ ml) treatment, the Interphase nucleoli increased slightly in number with the morphology alteration into larger, reticular or compact granular types. Ag-stained particles also increased in number. The number of the in situ hybridization rDNA fluorescent spots and dots increased markedly and largereticulate formations of numerous rDNA spots were seen. This phenomenum resembles to the changes in PHA stimulated lymphocytes. Statistic analysized data showed signifcant difference between control and drug- treated cells. These results indicate that transcriptionally activated rDNA and amplification of total rDNA was induced by the used tumor promoters.

Research progress on fluorescence analysis of antitumor Chinese medicine components

This paper classifies the anti-tumor traditional Chinese medicine's fluorescence analysis and finds that its research content mainly includes two aspects:the application of fluorescence analysis in the determination of the effective component content of anti-tumor traditional Chinese medicine;the study of the combined use of fluorescence imaging technology and the drug.According to literature analysis,it is found that although the research on the fluorescence analysis of anti-tumor Chinese medicine components is in a good state of development,it still needs to explore further in the identification of unknown components and deeper research on known components.Therefore,it can provide a certain basis for clinical guidance of tumor patients.

Current Problems of the Diagnostics and Treatment of Sepsis and Burn Injuries: The Modified Pathogenetic Concept

Background: The deep understanding of pathogenesis is a key moment in the formation of the modern strategy of modern medicine. We conducted the thorough analysis of the microscopic processes occurring in the bodies of patients with purulent-septic complications. The modified pathogenetic concept of the diagnostic and treatment model of diseases with septic complications is presented. The obtained information about the mechanisms of origin and development of these diseases is fundamentally important for finding the modern effective methods of treating patients. The aim of the research is to modify treatment tactics for patients with sepsis and burn injuries based on the modified pathogenetic concept using modern diagnostics, i.e. the method of fluorescence spectroscopy (MFS) and biomarkers. Materials and Methods: The proposed modified pathogenetic concept of the diagnostic and treatment model of diseases with purulent-septic complications along with standard methods was used successfully for effective treatment of 15 patients with sepsis and 25 with burn injuries. Results: 3 main scenarios of behaviour of spectral-fluorescence characteristics of patients with sepsis are illustrated. Spectral-fluorescence markers of sepsis were studied, which are informative 24 to 48 hours before the appearance of obvious clinical and laboratory signs of significant changes in the general somatic status of patients. Conclusions: The proposed diagnostic and therapeutic approach is new and fundamentally important for diagnostics and monitoring of the process of treatment of patients with purulent-septic diseases and burn injuries. An in-depth understanding of the dynamics of septic complications and the corresponding changes of the main markers of these diseases during treatment is especially relevant. The use of infusion therapy with solutions of donor albumin as an effective pathogenetic treatment is scientifically justified.

The synthesis of B,N-carbon dots by a combustion method and the application of fluorescence detection for Cu^2+

Heteroatom doping is an efficient approach to regulate the fluorescence properties of carbon dots.Using aminophenylboronic acid as the raw material,a combustion method was developed for the synthesis of boron,nitrogen-doped carbon dots（B,N-carbon dots）.The B,N-carbon dots emitted green fluorescence and displayed high resistance to both photo bleaching and ionic strength.A facile fluorescence sensing approach for Cu^2＋ was fabricated via static fluorescence quenching.Under optimal conditions,a rapid detection of Cu^2＋ could be completed in 2 min with a linearity ranging from 1 μmol/L to 25 μmol/L and a detection limit of 0.3 μmol/L Furthermore,the proposed method showed potential applications for the detection of Cu^2＋ in natural water samples.

Aggregating Properties of Compounds Cz-C-n in DMSO-H_2O Binary Solvent

The aggregating properties of Cz-C-n (n = 3, 6, 10) have been investigated by means of fluorescence method in DMSO-H2O binary solvent. The measured CAC and C F values indicate that the aggregating tendency of the amphiphilic compounds Cz-C-n containing crown ether increases with the length of alkyl chains, similar to that of carbazole compounds with long alkyl chains.

Comparison of inversion of Chlorophyll concentration in the Bohai Bay based on GOCI Data

Bohai bay has poor water circulation capacity and low self-purification capacity,and a large number of land-sourced pollutants are accepted every year.The estimation of chlorophyll concentration based on remote sensing plays an important role in the study of marine red tide and the monitoring of water quality in the Bohai Bay.So it is necessary to select the best algorithm by comparing the performance of each algorithm.Choosing the best algorithm is helpful to improve the precision of chlorophyll concentration.Based on the in-situ measurement data of the Bohai Bay and the GOCI remote sensing reflectance data,the retrieval models of chlorophyll concentration were established in this paper by using the methods of fluorescence line height(FLH),OC3,blue-green band ratio and NIR-red band ratio.The above algorithms were evaluated by comparing the simulation results and the verification results.The results showed that there are obvious differences in the accuracy of different retrieval methods.The fitting and retrieval effect of FLH is the best and NIR-red band ratio is the worst,and the OC3is similar to the blue-green band ratio method.

The New Approach to the Diagnostics and Treatment of Endogenous Intoxication in Patients with Burn Injury

Background: The main causes of mortality in patients with burn injury are the development of systemic inflammatory process, multiple organ failure and septic complications. The aim of the research: Improvement of diagnostic and therapeutic approaches to the detection and elimination of endogenous intoxication in patients with burn injury. Materials and methods: The main study group consisted of 19 patients and the comparison group—of 10 patients with burn injury. 92 blood serum (BS) samples of the patients of the main group were tested, using the method of fluorescence spectroscopy (MFS). The advanced therapeutic tactics were proposed for the patients of both groups. The control group consisted of 40 healthy individuals (donors). BS of these patients was also tested using MFS. Results: Patients with burn injury have endogenous intoxication in their blood. The effective concentration of albumin is reduced in patients with burn injury due to the blockage of albumin binding centers by bacterial metabolism products. Fluorescence spectra (FS) of BS in patients with burn trauma and donors were obtained and investigated. Based on MFS results, an improved treatment regimen using infusion of albumin solution was proposed. Conclusions: An improved technique for the management of patients with burn injury is based on the use of MFS for the diagnostic evaluation of endogenous intoxication in them. The idea of pathological changes in albumin molecules in patients with burn injury is pathogenetically substantiated by the successful use of infusion of albumin solution in these patients on the basis of the MFS.

Current Problems of Diagnostics and Treatment of Purulent-Inflammatory Diseases and Sepsis in Medical Practice

Background: Poor treatment of burns can lead to sepsis and even death. Especially promising is the use of the method of fluorescence spectroscopy for its diagnostics. The aim of the research is to create a pathogenetic concept as the basis of a diagnostic and treatment model of purulent-inflammatory diseases and sepsis. Material and methods: The study lasted from 2001 to 2019 and included 4 stages. The experimental base of luminescent research at all stages was the luminescence laboratory of the Department of Experimental Physics of the Ivan Franko National University of Lviv. The study was performed by using optical monochromators MDR-2 and MDR-12. The excitation of the blood serum was performed with light with a wavelength of 280 nm, which corresponds to the glow region of human serum albumin. Results: The main indicators, used for the analysis, were the fluorescence intensity (IF) and the position of the maximum fluorescence band (λmax). The object of the study was samples of the blood serum of patients of the main and control groups. In the case of the presence of endogenous intoxication in the body, albumin binding centers are blocked by the products of bacterial metabolism and therefore such “pathological” albumin is unable to perform its functions, including transport and detoxification. Conclusions: The pathogenetic concept as the basis of the diagnostic and treating model of purulent-inflammatory diseases and sepsis was devised. In particular, the important role of albumin solution infusion in the treatment of the above-mentioned diseases was established.

The Pathogenetic Concept of the Diagnostic-Treatment Approach for Patients with Purulent-Septic Complications

Background: The significant improvement of immediate and long-term functional results of treating patients is the fundamental problem of modern medical science. A deep understanding of the pathogenesis is the key point in creating the management strategy for patients with various diseases. Information about the mechanisms of origin and development of purulent-inflammatory diseases and sepsis is essential for finding effective ways to prevent and treat them. The aim of the research is to use the method of fluorescence spectroscopy in creating the pathogenetic diagnostic and treatment model for the prevention and treatment of purulent-inflammatory diseases and sepsis, modification of treatment tactics, search for new markers of purulent-septic diseases, as well as monitoring of patients during the treatment. Materials and methods: The proposed approach, along with standard diagnostic methods, was used to organize the treatment process of 100 patients with purulent-inflammatory diseases, including 15 patients with sepsis, 35 with acute inflammatory abdominal pathology, 20 patients with burn injury (main group) and 35 patients with burn injury (comparison group). Results: The behavior of spectral-fluorescent characteristics in their dynamics has been studied, and the new markers for assessing patients’ conditions have been proposed. Their effectiveness for the diagnosis of purulent-septic diseases has been proved, which advances the results of standard research methods by 24 - 48 hours. Conclusions: The proposed diagnostic and treatment approach is fundamentally important for diagnosis and monitoring during the treatment of patients with purulent-septic diseases. Particularly relevant is the proposal to modify the treatment process for these patients, associated with the use of infusion of donor albumin solutions.

Time-dependent effects of castration on the bladder function and histological changes in the bladder and blood vessels

We examined the effect of androgens on bladder blood flow （BBF）, bladder function and histological changes in castrated male rats. Male Wistar rats were classified into unoperated group （control group）, groups castrated at the age of 8weeks （group 8wPC） and groups castrated at the age of 4weeks （group 4wPC）. Each rat was used at the age of 20weeks. BBF was measured using fluorescent microspheres. Bladder cystometry was performed without anesthesia or restraint; the bladder was first irrigated with saline and then with 0.25% acetic acid （AA） solution. Maximum voiding pressure and voiding interval were measured. The bladder and lilac artery were histologically examined for differences in smooth muscle and quantity of collagen fiber to analyze the effect of castration on the smooth muscle content. No differences were noted in BBF following castration. The voiding intervals for all groups were shortened （P 〈 0.001） following AA irrigation. No significant difference was noted in the maximum voiding pressure. Histological changes were observed in bladder and lilac artery. Smooth muscle/collagen ratio at the bladder was lower in groups 8wPC and 4wPC compared to the control group （P 〈 0.01）, while that at the lilac artery was decreased in group 4wPC compared to the control group （P〈 0.001）. In conclusion, our findings indicate that castration does not alter BBF, but leads to histological changes in the bladder as well as its associated blood vessels.

Studies on the Refolding of Egg White Lysozyme Denatured by Urea Using ＂Phase Diagram＂ Method of Fluorescence

The refolding of reduced and non-reducing egg white lysozymes in a urea solution was studied by a ＂phase diagram＂ method of fluorescence. The result showed that in the refolding of the reduced egg white lysozyme, an intermediate state of an egg white lysozyme exists at the urea concentrations in a final renaturation solution being about 4.5 mol/L, their refolding follows a three-state model; while in the refolding of the non-reducing egg white lysozyme, two intermediate states exist at the urea concentrations being separately 4.0 and 2.5 mol/L, and their refolding follows a four-state model. Through the comparison between the unfolding and refolding of an egg white lysozyme in the urea solution, it was found that both of the refolding of reduced and non-reducing egg white lysozyme molecules was irreversible to their unfolding in the urea solution. Finally, a suggested refolding was separately presented for the reduced and non-reducing egg white lysozymes in the urea solution.

In vitro fluorescence displacement investigation of thyroxine transport disruption by bisphenol A

Bisphenol A （BPA） is a chemical with high production volume and wide applications in many industries. Although BPA is known as an endocrine disruptor, its toxic mechanisms have not been fully characterized. Due to its structural similarity to thyroid hormones thyroxine （T4） and triiodothyronine （T3）, one possible mechanism of BPA toxicity is disruption of hormone transport by competitive binding with the transport proteins. In this study, the binding interactions of BPA, T4, and T3 with three thyroid hormone transport proteins, human serum albumin （HSA）, transthyretin （TTR）, and thyroxine-binding globulin （TBG） were investigated by fluorescence measurement. Using two site-specific fluorescence probes dansylamide and dansyl-L-proline, the binding constants of BPA with HSA at drug site I and site II were determined as 2.90 × 10^4 and 3.14 × 10^4 L/mol, respectively. By monitoring the intrinsic fluorescence of tryptophan, a binding constant of 4.70 ×10^3 L/mol was obtained. Similarly, by employing 8-anilino-l-naphthalenesulfonic acid as fluorescence probe, the binding affinity of BPA with TTR and TBG was measured to be 3.10 × 10^5 and 5.90× 10^5 L/mol, respectively. In general, BPA showed lower binding affinity with the proteins than T3 did, and even lower affinity than T4. Using these binding constants, the amount of BPA which would bind to the transport proteins in human plasma was estimated. These results suggest that the concentrations of BPA commonly found in human plasma are probably not high enough to interfere with T4 transport.