Fluorogenic Method Research Articles

Tailoring Efficient Fluorogenic Tactic for Ultrasensitive Detection of Dopamine in Urine and Rat Brain through Real-Time and In Situ Formation of High-Performance Fluorophore.

Owing to the predominance of dopamine (DA) in controlling mental health, planning an innovative method for DA detection with simplicity and high efficacy is conducive to the assessment of neurological disorders. Herein, an efficient fluorogenic tactic has been elaborated for ultrasensitive detection of DA with remarkably enhanced turn-on response. Utilizing a twisted intramolecular charge-transfer (TICT)-suppressing strategy, a highly emissive azocine derivative 11-hydroxy-2,3,6,7,11,12,13,14-octahydro-1H,5H,10H-11,14a-methanoazocino[5',4':4,5]furo[2,3-f]pyrido[3,2,1-ij]quinolin-10-one (J-Aza) is generated via a one-step reaction between DA and 8-hydroxyjulolidine. It is marvelous that J-Aza not only possesses ideal fluorescence quantum yield (ΦF) as high as 0.956 but also exhibits bathochromic shifted fluorescence (green emissive) and stronger anti-photobleaching capacity superior to traditional azocine-derived 1,2,3,4-tetrahydro-5H-4,11a-methanobenzofuro[2,3-d]azocin-5-one (Aza) with moderate ΦF, blue fluorescence, and poor photostability. By confining the TICT process, the detection limit to DA can be reduced to 80 pM, which is competitive in contrast to previously reported fluorescence methods. Encouraged by the instant response (within 90 s), wide linear range (0.1-500 nM), great selectivity, and excellent sensitivity, this fluorogenic method has been used for the real-time measurement of DA contents in practical urine samples with satisfactory results. Furthermore, the cerebral DA level in the reserpine-induced depression rat model has also been evaluated by our designed method, demonstrating its potent analytical applicability in the biosensing field.

Zymograph profiling reveals a divergent evolution of sirtuin that may originate from class III enzymes

Sirtuins are a group of NAD+-dependent deacylases that conserved in three domains of life and comprehensively involved in the regulation of gene transcription, chromosome segregation, RNA splicing, apoptosis, and aging. Previous studies in mammalian cells have revealed that sirtuins not only exist as multiple copies, but also show distinct deacylase activities in addition to deacetylation. However, the understanding of sirtuin zymographs in other organisms with respect to molecular evolution remains at an early stage. Here, we systematically analyze the sirtuin activities in representative species from archaea, bacteria, and eukaryotes, using both the HPLC assay and a 7-amino-4-methylcoumarin-based fluorogenic method. Global profiling suggests that the deacylase activities of sirtuins could be divided into three categories and reveals undifferentiated zymographs of class III sirtuins, especially for those from bacteria and archaea. Nevertheless, initial differentiation of enzymatic activity was also observed for the class III sirtuins at both paralog and ortholog levels. Further phylogenetic analyses support a divergent evolution of sirtuin that may originate from class III sirtuins. Together, this work demonstrates a comprehensive panorama of sirtuin zymographs and provides new insights into the cellular specific regulation and molecular evolution of sirtuins.

A fluorogenic method to directly observe transfer and distribution of influenza viral contents to target vesicles.

There is a long-standing interest in studying viral infection processes using bottom-up biophysical models to dissect various underlying molecular determinants. Fusion of enveloped viruses with cellular membranes and subsequent release of viral contents into the cytoplasm is a key event in the infection cycle. In case of influenza virus, fusion of the viral membrane is mediated by conformational changes of its surface protein hemagglutinin triggered by acidic pH within endosomes. Previously, content transfer assays have relied on fluorescence enhancement of an encapsulated self-quenched water-soluble dye upon fusion of the viral compartment with a target membrane. However these assays do not track the transfer of actual viral macromolecular contents, and suffer from drawbacks such as the need to use high concentration of the content dye which may have perturbative effects on the membranes. To overcome these limitations, we developed a method utilizing nucleic acid-binding dyes to directly observe the transfer of viral ribonucleoprotein complexes to target vesicles by total internal reflection fluorescence (TIRF) microscopy. The dyes were encapsulated within surface-tethered vesicles and the unlabeled virus particles were bound via sialic acid receptors. The fluorogenic nature of the dyes allowed background-free detection of fusion events when pH was dropped. We determined the kinetics of viral content transfer and found that it is slower than that of lipid mixing. We further employed a super-localization strategy to investigate to what extent the viral contents are transferred to the target vesicle through the fusion pore. Overall, our method allows observation of viral fusion events on a single-particle level and will provide the groundwork for development of biosensors for rapid quantification of fusogenic virus particles.

Facile Conversion of the Quinone-Semicarbazone Chromophore of Naftazone into a Fluorescent Quinol-Semicarbazide: Kinetic Study and Analysis of Naftazone in Pharmaceuticals and Human Serum.

Naftazone is a quinone-semi carbazone drug that possesses a strong orange color, and hence it was usually analyzed colorimetrically or by HPLC-UV. However, these methods are not sensitive enough to determine naftazone in biological samples. Naftazone lacks intrinsic fluorescence and does not possess easily derivatizable functional groups. In this contribution, we introduced the first spectrofluorimetric method for naftazone assay through reduction-elicited fluorogenic derivatization through the reduction of its quinone-semicarbazone moiety to the corresponding quinol-semicarbazide derivative by potassium borohydride as a reduction probe. The solvent-dependent fluorescence of the reaction product was studied in various protic and aprotic solvents. Eventually, the fluorescence of the reduced naftazone was measured in 2-propanol at λemission of 350 nm after excitation at λecxitation of 295 nm. The relative fluorescence intensity was linearly correlated to the drug concentration (r = 0.9995) from 10.0 to 500 ng/mL with high sensitivity, where the lower detection limit was 2.9 ng/mL. Hence, the method was effectively applied for naftazone tablets quality control with a mean %recovery of 100.3 ± 1.5, and the results agreed with those of the comparison HPLC-UV method. Furthermore, a new salting-out assisted liquid-liquid extraction (SALLE) method was established for naftazone extraction from human serum, followed by its determination using the developed reduction-based fluorogenic method. The developed SALLE method showed excellent recovery for naftazone from human serum (92.3–106.5%) with good precision (RSD ≤ 6.8%). Additionally, the reaction of naftazone with potassium borohydride was kinetically monitored, and it was found to follow pseudo-first-order kinetics with an activation energy of 43.8 kcal/mol. The developed method’s greenness was approved using three green analytical chemistry metrics.

Visualizing the Multistep Process of Protein Aggregation in Live Cells.

Protein aggregation is a biological phenomenon in which aberrantly processed or mutant proteins misfold and assemble into a variety of insoluble aggregates. Decades of studies have delineated the structure, interaction, and activity of proteins in either their natively folded structures or insoluble aggregates such as amyloid fibrils. However, a variety of intermediate species exist between these two extreme states in the protein folding landscape. Herein, we collectively term these intermediate species as misfolded protein oligomers, including soluble oligomers and preamyloid oligomers that are formed by unfolded or misfolded proteins. While extensive tools have been developed to study folded proteins or amyloid fibrils, research to understand the properties and activities of misfolded protein oligomers has been limited by the lack of methods to detect and interrogate these species in live cells.In this Account, we describe our efforts in the development of chemical methods that allow for the characterization of the multistep protein aggregation process, in particular the misfolded protein oligomers, in living cells. As the start of this journey, we attempted to develop a fluorogenic method wherein the misfolded oligomers could turn on the fluorescence of chemical probes that are conjugated to the protein-of-interest (POI). To this end, we produced a series of destabilized HaloTag variants, formulating the primary component of the AgHalo sensor, which misfolds and aggregates when cells are subjected to stress. When AgHalo is covalently conjugated with a solvatochromic fluorophore, misfolding of the AgHalo conjugate would activate fluorescence, resulting in the observation of misfolded oligomers. Following this work, we extended the scope of detection from AgHalo to any protein-of-interest via the AggTag method, wherein the POIs are genetically fused to self-labeling protein tags (HaloTag or SNAP-tag). Focusing on the molecular rotor-based fluorophores, we applied the modulated fluorescent protein (FP) chromophore core as a prototype for the AggTag probes, to enable the fluorogenic detection of misfolded soluble oligomers of multiple proteins in live cells. Next, we further developed the AggTag method to distinguish insoluble aggregates from misfolded oligomers, using two classes of probes that activate different fluorescence emission toward these two conformations. To enable this goal, we applied physical organic chemistry and computational chemistry to discover a new category of triode-like fluorophores, wherein the π orbitals of either an electron density regulator or the donor-acceptor linkages are used to control the rotational barriers of fluorophores in the excited states. This mechanism allows us to rationally design molecular rotor-based fluorophores that have desired responses to viscosity, thus extending the application of the AggTag method.In summary, our work allows the direct monitoring of the misfolded protein oligomers and differentiation of insoluble aggregates from other conformations in live cells, thus enabling studies of many currently unanswered questions in protein aggregation. Future directions are to develop methods that enable quantitative analyses of the protein aggregation process. Further, new methods are needed to detect and to quantify the formation and maturation of protein or RNA condensates that form membraneless organelles.

A Fluorogenic Assay: Analysis of Chemical Modification of Lysine and Arginine to Control Proteolytic Activity of Trypsin

The chemical modification of amino acids plays an important role in the modulation of proteins or peptides and has useful applications in the activation and stabilization of enzymes, chemical biology, shotgun proteomics, and the production of peptide-based drugs. Although chemoselective modification of amino acids such as lysine and arginine via the insertion of respective chemical moieties as citraconic anhydride and phenyl glyoxal is important for achieving desired application objectives and has been extensively reported, the extent and chemoselectivity of the chemical modification of specific amino acids using specific chemical agents (blocking or modifying agents) has yet to be sufficiently clarified owing to a lack of suitable assay methodologies. In this study, we examined the utility of a fluorogenic assay method, based on a fluorogenic tripeptide substrate (FP-AA1-AA2-AA3) and the proteolytic enzyme trypsin, in determinations of the extent and chemoselectivity of the chemical modification of lysine or arginine. As substrates, we used two fluorogenic tripeptide probes, MeRho-Lys-Gly-Leu(Ac) (lysine-specific substrate) and MeRho-Arg-Gly-Leu(Ac) (arginine-specific substrate), which were designed, synthesized, and evaluated for chemoselective modification of specific amino acids (lysine and arginine) using the fluorogenic assay. The results are summarized in terms of half-maximal inhibitory concentrations (IC50) for the extent of modification and ratios of IC50 values (IC50arginine/IC50lysine and IC50lysine/IC50arginine) as a measure of the chemoselectivity of chemical modification for amino acids lysine and arginine. This novel fluorogenic assay was found to be rapid, precise, and reproducible for determinations of the extent and chemoselectivity of chemical modification.

Methods to Study Myc-Regulated Cellular Senescence: An Update.

Cellular senescence plays a role in several physiological processes including aging, embryonic development, tissue remodeling, and wound healing and is considered one of the main barriers against tumor development. Studies of normal and tumor cells both in culture and in vivo suggest that MYC plays an important role in regulating senescence, thereby contributing to tumor development. We have previously described different common methods to measure senescence in cell cultures and in tissues. Unfortunately, there is no unique marker that unambiguously defines a senescent state, and it is therefore necessary to combine measurements of several different markers in order to assure the correct identification of senescent cells. Here we describe protocols for simultaneous detection of multiple senescence markers in situ, a quantitative fluorogenic method to measure senescence-associated β-galactosidase activity (SA-β-gal), and a new method to detect senescent cells based on the Sudan Black B (SBB) analogue GL13, which is applicable to formalin-fixed paraffin-embedded tissues. The application of these methods in various systems will hopefully shed further light on the role of MYC in regulation of senescence, and how that impacts normal physiological processes as well as diseases and in particular cancer development.

A lysosome targeting probe based on fluorescent protein chromophore for selectively detecting GSH and Cys in living cells

The low-molecular weight biothiols like glutathione (GSH) and cysteine (Cys) play many important roles in various biological processes, the imbalance of biothiols level will lead to many diseases. However, methods that can selectively detect the GSH and Cys was rarely reported because of the similar reactivity and structure. Here, a fluorogenic method was presented to selectively detect the GSH and Cys in vitro using probe ML-FP based on fluorescent protein mimics. Probe ML-FP is a fluorescence turn on probe with lysosome targeting capacity. 2,4-dinitrobenzenesulfonyl serves as fluorescence quench and detection group in probe ML-FP. Differentiating the GSH and Cys was realized benefit from the different reaction time as well as fluorescence response between probe and target species. Low detection limit (4.98 nM for Cys and 4.39 nM for GSH) as well as fast response time was founded in this work. Probe ML-FP possess excellent biocompatibility due to fluorescent protein chromophore and was successfully used for bioimaging in living cells.

Specific and Quantitative Detection of Albumin in Biological Fluids by Tetrazolate-Functionalized Water-Soluble AIEgens.

The analysis of albumin has clinical significance in diagnostic tests and obvious value to research studies on the albumin-mediated drug delivery and therapeutics. The present immunoassay, instrumental techniques, and colorimetric methods for albumin detection are either expensive, troublesome, or insensitive. Herein, a class of water-soluble tetrazolate-functionalized derivatives with aggregation-induced emission (AIE) characteristics is introduced as novel fluorescent probes for albumin detection. They can be selectively lighted up by site-specific binding with albumin. The resulting albumin fluorescent assay exhibits a low detection limit (0.21 nM), high robustness in aqueous buffer (pH = 6-9), and a broad tunable linear dynamic range (0.02-3000 mg/L) for quantification. The tetrazolate functionality endows the probes with a superior water solubility (>0.01 M) and a high binding affinity to albumin (KD = 0.25 μM). To explore the detection mechanism, three unique polar binding sites on albumin are computationally identified, where the multivalent tetrazolate-lysine interactions contribute to the tight binding and restriction of the molecular motion of the AIE probes. The key role of lysine residues is verified by the detection of poly-l-lysine. Moreover, we applied the fluorogenic method to quantify urinary albumin in clinical samples and found it a feasible and practical strategy for albumin analysis in complex biological fluids.

Red emission cysteine probe with high selectivity based on fluorescent protein chromophores and turn-on fluorescence in cell cultures

Cysteine (Cys) plays a vital role in many physiological processes, and also is closely related to various diseases, thus the development of novel high selectivity and sensitivity fluorescent probes for in situ monitoring of biothiol (Cys) is crucial. Herein, we present a fluorogenic method to detect Cys in vitro and live cells based on fluorescent protein chromophores, which have been modulated by chemical reaction. Probe (RFP) is a fluorescence turn on probe with red emission wavelength and large Stokes shift (127 nm). This Michael-addition reaction-based probe exhibits low detection limit (18.7 μM), fast response time, and low cytotoxicity. RFP can differentiate the Cys and Hcy, using the difference of reaction time between probe and Cys or Hcy. Together, this probe exhibited excellent cell permeability, and can be successfully applied to detect the endogenous Cys in living cells.

A Fluorogenic AggTag Method Based on Halo- and SNAP-Tags to Simultaneously Detect Aggregation of Two Proteins in Live Cells.

Protein aggregation involves the assembly of partially misfolded proteins into oligomeric and higher-order structures that have been associated with several neurodegenerative diseases. However, numerous questions relating to protein aggregation remain unanswered due to the lack of available tools for visualization of these species in living cells. We recently developed a fluorogenic method named aggregation tag (AggTag), and presented the AggTag probe P1, based on a Halo-tag ligand, to report on the aggregation of a protein of interest (POI) in live cells. However, the Halo-tag-based AggTag method only detects the aggregation of one specific POI at a time. In this study, we have expanded the AggTag method by using SNAP-tag technology to enable fluorogenic and biorthogonal detection of the aggregation of two different POIs simultaneously in live cells. A new AggTag probe-P2, based on a SNAP-tag ligand bearing a green solvatochromic fluorophore-was synthesized for this purpose. Using confocal imaging and chemical crosslinking experiments, we confirmed that P2 can also report both on soluble oligomers and on insoluble aggregates of a POI fused with SNAP-tag in live cells. Ultimately, we showed that the orthogonal fluorescence of P1 and P2 allows for simultaneous visualization of two different pathogenic protein aggregates in the same cell.

Modulation of Fluorescent Protein Chromophores To Detect Protein Aggregation with Turn-On Fluorescence.

We present a fluorogenic method to visualize misfolding and aggregation of a specific protein-of-interest in live cells using structurally modulated fluorescent protein chromophores. Combining photophysical analysis, X-ray crystallography, and theoretical calculation, we show that fluorescence is triggered by inhibition of twisted-intramolecular charge transfer of these fluorophores in the rigid microenvironment of viscous solvent or protein aggregates. Bioorthogonal conjugation of the fluorophore to Halo-tag fused protein-of-interests allows for fluorogenic detection of both misfolded and aggregated species in live cells. Unlike other methods, our method is capable of detecting previously invisible misfolded soluble proteins. This work provides the first application of fluorescent protein chromophores to detect protein conformational collapse in live cells.

Tissue factor expressed by adherent cells contributes to hemodialysis-membrane thrombogenicity

End-stage renal patients present a high risk of thrombosis and bleeding. Consequently, it is challenging to prevent clotting during hemodialysis. If a contact system induces thrombin generation in the extra corporeal circuit, recent data suggest a role of tissue factor (TF) in hemodialysis-associated thrombosis. Using a method of elution, we collected adhering cells to an acrylonitrile membrane layered by polythyleneimine (AN69-ST). Using optic microscopy and flow cytometry, we observed that adherent cells were mainly constituted by activated polymorphonuclear neutrophils (PMNs). Using a sensitive fluorogenic method of thrombin generation, we found that adhering cells triggered thrombin generation in a TF-dependent manner. We next identified the presence of TF mRNA (Q-PCR) in adhering cells. Using immunofluorescence, we observed the presence of TF in PMNs and of TF-decorated neutrophil extracellular traps (NETs). As TF triggers thrombin generation after binding to serine protease FVIIa, we evaluated the effect of an inactivated human recombinant factor VIIa (hrFVIIai) in a sheep model of hemodialysis (HD). One single bolus of hrFVIIai maintained the full patency of the hemodialysis circuit without any measurable systemic anticoagulant effect. TF is a promising target for preventing thrombosis during HD.

Thymopolypeptides combined with matrine type alkaloids suppress HBV replication

To investigate the antiviral effect of thymopolypeptides combined with 4 kinds of matrine type alkaloids on HepG2.2.15 cells, oxymatrine, sophocarpidine, sophocarpine, and sophoridine (at concentration of 0.2 mmol•L⁻¹ respectively) were respectively combined with thymopolypeptides (0.025, 0.1 g•L⁻¹), and after 48 h and 72 h treatment on HepG2.2.15 cells, the cells and supernatants were collected. The cells activity in various groups was determined by CCK-8 method to evaluate the toxic effects of the drugs on HepG2.2.15 cells. Enzyme linked immunosorbent assay (ELISA) was used to determine HBeAg and HBsAg levels in cellular supernatants. HBV DNA levels in cellular supernatants andcells were quantified with fluorogenic quantitative PCR method; and the expression level of IFN-α in supernatants was detected with CBA method. The results indicated that single thymopolypeptides at 0.025-0.4 g•L⁻¹ had no toxicity to cells. Thymopolypeptides in this concentration range combined with 0.2 mmol•L⁻¹ matrine type alkaloids also had no toxicity to cells. Anti-HBV activity of drug combination was better than that of alkali or thymopolypeptides alone. Thymopolypeptides at 0.025 g•L⁻¹ had better inhibitory effect than thymopolypeptides at 0.1 g•L⁻¹ on intracellular HBV DNA expression, but the inhibitory effect on supernatant HBeAg level was on the contrary. Anti-HBV activity was similar between alkaloids combined with 0.1 g•L⁻¹ and alkaloids combined with 0.025 g•L⁻¹. There was no statistical difference in anti-HBV effect between various combined groups (P<0.05). In general, 72 h anti-HBV effect was better than 48 h anti-HBV effect (P<0.05). The expression of IFN-α was increased after drug combination, with positive correlation to the changes of other four indicators (P<0.05). In conclusion, oxymatrine, sophocarpidine, sophocarpine and sophoridine combined with thymopolypeptides could inhibit HBsAg and HBeAg secretion in HepG2.2.15 cells and HBV DNA replication, and further promote the antiviral effect by promoting the expression of IFN-α.

A new ratiometric fluorescent chemodosimeter based on an ICT modulation for the detection of Hg2+

We design and synthesize a new ratiometric fluorescent chemodosimeter (S1) for the selective and sensitive detection of Hg2+. Upon addition of Hg2+, the emission of the S1 exhibits a large bathochromic shift from 393 to 515nm (up to 122nm) which is ascribed to an intramolecular charge transfer process in the resultant. The Hg2+-induced dethioacetalization for sensing mechanism has been demonstrated by using high-performance liquid chromatography analysis of the sensing process. The interference experiments further demonstrate that S1 exhibits very high selectivity towards Hg2+ over other coexisting cations/anions. Subsequently, a good linearity of the concentrations of Hg2+ (0–15μM) vs the ratiometric signals (I515/I393) allows a fluorogenic method for the quantitative detection of Hg2+, with the limitation of detection determined to be 5.22×10−7M.

Simultaneous bioimaging recognition of cation Al3+ and anion F− by a fluorogenic method

A new highly selective and sensitive chemosensor 1 for both Al3+ and F− was designed and synthesized. 1 could detect both Al3+ and F− with turn-on fluorescence behavior in a nearly perfect aqueous solution. The binding stoichiometry of receptor 1 with Al3+ and F− was proposed to be 1:1, respectively, based on Job plot, 1H NMR titration and ESI-mass spectrometry analysis. The detection limits (1.12 μM for Al3+ and 7.01 μM for F−) of 1 for Al3+ and F− were lower than the World Health Organization guidelines (7.41 μM for Al3+ and 79 μM for F−) for drinking water. Chemosensor 1 was also successfully applied to living cells for simultaneous detection and quantification of both Al3+ and F−. Moreover, the sensing mechanisms of 1 toward Al3+ and F− were explained by theoretical calculations.

A simple rhodamine hydrazide-based turn-on fluorescent probe for HOCl detection.

Hypochlorous acid (HOCl) plays a crucial role in daily life and mediates a variety of physiological processes, however, abnormal levels of HOCl have been associated with numerous human diseases. It is therefore of significant interest to establish a simple, selective, rapid and sensitive fluorogenic method for the detection of HOCl in environmental and biological samples. A hydrazide-containing fluorescent probe based on a rhodamine scaffold was facilely developed that could selectively detect HOCl over other biologically relevant reactive oxygen species, reactive nitrogen species and most common metal ions in vitro. Via an irreversible oxidation-hydrolysis mechanism, and upon HOCl-triggered opening of the intramolecular spirocyclic ring during detection, the rhodamine hydrazide-based probe exhibited large fluorescence enhancement in the emission spectra with a fast response, low detection limit and comparatively wide pH detection range in aqueous media. The probe was further successfully applied to monitoring trace HOCl in tap water and imaging both exogenous and endogenous HOCl within living cells. It is anticipated that this simple and useful probe might be an efficient tool with which to facilitate more HOCl-related chemical and biological research. Copyright © 2015 John Wiley & Sons, Ltd.

Colorimetric and Fluorometric Detection of Neomycin Based on Conjugated Polydiacetylene Supramolecules

Utilizing the colorimetric and fluorogenic changes, a system based on polydiacetylenes (PDAs) is developed for the detection of neomycin. The PDA supramolecules polymerized from the mixed liposome composed of N-(3-hydroxyphenyl)pentacosa-10,12-diynamide (PCDA-AP) and pentacosa-10,12-diynoic acid (PCDA) at an optimized ratio of 1:9 display a unique colorimetric change (blue to red) and fluorescent enhancement in the presence of neomycin. The detection limit for neomycin is estimated to be 2.55 × 10(-7) M by the fluorogenic method. The optical changes induced by neomycin can be attributed to the disruption of the hydrogen bonding between phenol and carboxylic acid from PCDA-AP and PCDA.

Expanding the Use of a Fluorogenic Method to Determine Activity and Mode of Action ofBacillus thuringiensisBacteriocins Against Gram-Positive and Gram-Negative Bacteria

Previously we described a rapid fluorogenic method to measure the activity of five bacteriocins produced by Mexican strains of Bacillus thuringiensis against B. cereus 183. Here we standardize this method to efficiently determine the activity of bacteriocins against both Gram-positive and Gram-negative bacteria. It was determined that the crucial parameter required to obtain reproducible results was the number of cells used in the assay, that is, ~4 × 108 cell/mL and ~7 × 108 cell/mL, respectively, for target Gram-positive and Gram-negative bacteria. Comparative analyses of the fluorogenic and traditional well-diffusion assays showed correlation coefficients of 0.88 to 0.99 and 0.83 to 0.99, respectively, for Gram-positive and Gram-negative bacteria. The fluorogenic method demonstrated that the five bacteriocins of B. thuringiensis have bacteriolytic and bacteriostatic activities against all microorganisms tested, including clinically significant bacteria such as Listeria monocytogenes, Proteus vulgaris, and Shigella flexneri reported previously to be resistant to the antimicrobials as determined using the well-diffusion protocol. These results demonstrate that the fluorogenic assay is a more sensitive, reliable, and rapid method when compared with the well-diffusion method and can easily be adapted in screening protocols for bacteriocin production by other microorganisms.

Evaluation of Thrombin Generation in the Early Stages of Sepsis in Patients with Hematological Malignancies and Febrile Neutropenia,

Abstract 3347 Introduction:Coagulation activation is a consequences of the host response to sepsis. Up-regulation of tissue factor expression and down-regulation of natural anticoagulant and fibrinolytic systems observed in non-clinical and clinical studies of sepsis lead to disseminated intravascular coagulation (DIC), regarded as a contributor to the pathogenesis of sepsis. Nonetheless, clinical strategies targeting coagulation activation have not yet shown benefits for sepsis patients. DIC is a dynamic process, and hypercoagulability is regarded as a hallmark of its early stages. However, assessment of this hypercoagulable state rely on tests that measure specific compartments of hemostasis, such as levels of anticoagulant proteins, fibrinogen and markers of fibrin degradation. Because hemostasis is a complex system, whether these assays actually depict the net result of interactions of all elements in the host is a fair concern. In fact, disconnection between classical assays and global assays of hemostasis is being demonstrated in patients with cirrhosis. Methods:Here we performed laboratory evaluation of hemostasis in 24 patients with hematological malignancies and febrile neutropenia, in the early stages of sepsis. In addition to classical assays used in DIC such as platelet count (PC), fibrinogen (Fib), prothrombin time (PT) and D dimer (DD), thrombin generation (TG) was evaluated using a fluorogenic method (Technoclone, Vienna, Austria) in platelet poor plasma samples. Selection of patients with hematological malignancies and chemotherapy-induced neutropenia allowed us to obtain a baseline sample before the inititiation of chemotherapy, against which all comparisons were made. In addition, this strategy allowed us to obtain the 1st sample within 12h from fever onset, thus standardizing the time of evaluation, a key variable in studies of coagulation in sepsis. Laboratory evaluation was repeated 48h after fever onset. A sepsis severity score (SOFA) was recorder daily. Results:Median age was 42 years-old (16–62). Baseline diagnosis included acute leukemias in 14 and other hematological malignancies in 10. At fever onset, mean neutrophil count was 125/ml (range 20–500). Sepsis was defined according do ACCP/SSCM criteria. SOFA score at fever onset and 48 hours thereafter were 3 (range 0–13) and 4 (range 1–12) respectively. The proportion of patients with overt DIC based on classical ISTH score was overestimated by the presence of severe thrombocytopenia. Using an arbitrary modification to correct for this factor, only 1 patient presented overt DIC. Septic shock developed in 8 patients, in a median of 4 days from fever onset. Classical parameters of coagulation behaved as expected for the early phases of sepsis: PT remained stable, Fib and DD increased progressively. PC analysis was hampered by severe myelotoxicity. Total TG, expressed by the area under the curve of the thrombin generation curve (AUC), peak thrombin and lag time were not changed at fever onset and 48 hours thereafter compared to baseline values, and to values obtained in healthy individuals. An increase in the time to peak thrombin generation and a decrease in the velocity index were the only significant changes observed in our study. A positive correlation was observed between the lag time to TG and the SOFA score, and a negative correlation was observed between the SOFA score and AUC at the time of fever onset (all r>0.5 with P<0.05). In an exploratory analysis of the association of TG parameters with septic shock development, no significant association was observed. Discussion:In a population of patients with hematological malignancies and febrile neutropenia, TG was not increased in the early stages of sepsis. Our analysis also suggests that the kinetics of TG might be changed in the early stages of sepsis, but towards a slower activation of coagulation. These results are in accordance with recently published papers that evaluated septic patients with whole blood thromboelastography. The prognostic value of TG parameters in sepsis deserves further studies since sample size our study was not powered to draw definite conclusions about this. No evidence of hypercoagulability in patients with early sepsis could be demonstrated in our study using a global hemostasis test. Disclosures:No relevant conflicts of interest to declare.