Advancing ethanol content determination in hydrogels: non-destructive and operational methods for health and criminal inspections.

The significance of accurate determination of ethanol content in hydrogel formulations was accentuated during COVID-19 pandemic coinciding with the heightened demand for sanitizing agents. The present article proposes three robust methodologies for this purpose: Fourier Transform Infrared Spectroscopy (FTIR), Raman spectroscopy, and Densitometry with matrix effect correction by Near-Infrared Spectroscopy (NIR). All three methods demonstrated outstanding linearity (R2 ≥ 0.99) and minimal errors (< 1.7%), offering simplicity and operational efficiency. FTIR and Raman, being non-destructive and requiring minimal preparation, enable practical on-site analysis capabilities, underscoring the potential of the spectroscopic methods to expedite health investigations and inspections, empowering on-site ethanol determination, and relieving the burden on official laboratories. Additionally, the densitometry with NIR-based approach showcased superior accuracy and precision compared to spectroscopic methods, meeting validation criteria while offering operational advantages over the costly official distillation-based method. Therefore, it stands as a reliable and reproducible technique for comprehensive health and criminal compliance assessments, making it a compelling alternative for both industry and official laboratories.

Production of Gibberellic Acid by Solid-State Fermentation Using Wastes from Rice Processing and Brewing Industry.

Gibberellic acid (GA3) is a natural hormone present in some plants used in agricultural formulations as a growth regulator. Currently, its production on an industrial scale is performed by submerged fermentation using the fungus Gibberella fujikuroi, which is associated with low yields, leaving the purification stages with high costs. An alternative is solid-state fermentation (SSF), which makes it possible to obtain higher concentrations of product using low-cost substrates, such as agroindustrial by-products. This research investigated the use of raw rice bran (RRB) and barley malt residue (BMR) as substrates for GA3 production by the fungus Gibberella fujikuroi. Through two statistical designs, the effect of moisture (50 to 70 wt.%) and medium composition (RRB content between 30 and 70 wt.% to a mass ratio between RRB and BMR) was first evaluated. Using the best conditions previously obtained, the effect of adding glucose (carbon source, between 0 and 80 g·L-1) and ammonium nitrate-NH4NO3-(nitrogen source, between 0 and 5 g·L-1) on GA3 productivity was analyzed. The best yield was obtained using 30 wt.% RRB and 70 wt.% BMR for a medium with 70 wt.% of moisture after 7 days of process. It was also found that higher concentrations of NH4NO3 favor the GA3 formation for intermediate values of glucose content (40 g·L-1). Finally, a kinetic investigation showed an increasing behavior in the GA3 production (10.1 g·kg of substrate-1 was obtained), with a peak on the seventh day and subsequent tendency to stabilization.

Linking microbiome and stress hormone responses in wild tropical treefrogs across continuous and fragmented forests.

The amphibian skin microbiome is an important component of anti-pathogen defense, but the impact of environmental change on the link between microbiome composition and host stress remains unclear. In this study, we used radiotelemetry and host translocation to track microbiome composition and function, pathogen infection, and host stress over time across natural movement paths for the forest-associated treefrog, Boana faber. We found a negative correlation between cortisol levels and putative microbiome function for frogs translocated to forest fragments, indicating strong integration of host stress response and anti-pathogen potential of the microbiome. Additionally, we observed a capacity for resilience (resistance to structural change and functional loss) in the amphibian skin microbiome, with maintenance of putative pathogen-inhibitory function despite major temporal shifts in microbiome composition. Although microbiome community composition did not return to baseline during the study period, the rate of microbiome change indicated that forest fragmentation had more pronounced effects on microbiome composition than translocation alone. Our findings reveal associations between stress hormones and host microbiome defenses, with implications for resilience of amphibians and their associated microbes facing accelerated tropical deforestation.

Enoxaparin improves COVID-19 by reducing Neutrophils Extracellular Traps (NETs) production.

BACKGROUND: COVID-19 causes consequences such as imbalance of the immune system and thrombotic events. During the infection process, NETs in excess induce a pro-inflammatory response and disseminated intravascular coagulation. We evaluated the role of enoxaparin as a potential inhibitor of NETs. METHODS: K18-hACE2 animals infected with the SARS-CoV-2 virus and a group of 23 individuals admitted to the hospital with COVID-19 treated with enoxaparin or without treatment and controls without the disease were included. RESULTS: Enoxaparin decreased the levels of NETs, reduced the signs of the disease and mitigated lung damage in the animals infected with SARS-CoV-2. These effects were partially associated with prevention of SARS-CoV-2 entry and NETs synthesis. Clinical data revealed that treatment with enoxaparin decreased the levels of inflammatory markers, the levels of NETs in isolated neutrophils and the organ dysfunction. CONCLUSION: This study provides evidence for the beneficial effects of enoxaparin in COVID-19 in addition to its anticoagulant role.

NLRP3 is essential for neutrophil polarization and chemotaxis in response to leukotriene B4 gradient.

Neutrophil recruitment to sites of infection and inflammation is an essential process in the early innate immune response. Upon activation, a subset of neutrophils rapidly assembles the multiprotein complex known as the NLRP3 inflammasome. The NLRP3 inflammasome forms at the microtubule organizing center, which promotes the formation of interleukin (IL)-1ß and IL-18, essential cytokines in the immune response. We recently showed that mice deficient in NLRP3 (NLRP3-/-) have reduced neutrophil recruitment to the peritoneum in a model of thioglycolate-induced peritonitis. Here, we tested the hypothesis that this diminished recruitment could be, in part, the result of defects in neutrophil chemotaxis. We find that NLRP3-/- neutrophils show loss of cell polarization, as well as reduced directionality and velocity of migration toward increasing concentrations of leukotriene B4 (LTB4) in a chemotaxis assay in vitro, which was confirmed through intravital microscopy of neutrophil migration toward a laser-induced burn injury of the liver. Furthermore, pharmacologically blocking NLRP3 inflammasome assembly with MCC950 in vitro reduced directionality but preserved nondirectional movement, indicating that inflammasome assembly is specifically required for polarization and directional chemotaxis, but not cell motility per se. In support of this, pharmacological breakdown of the microtubule cytoskeleton via nocodazole treatment induced cell polarization and restored nondirectional cell migration in NLRP3-deficient neutrophils in the LTB4 gradient. Therefore, NLRP3 inflammasome assembly is required for establishment of cell polarity to guide the directional chemotactic migration of neutrophils.

Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory functions and clearance of apoptotic cells.

COVID-19 is a disease of dysfunctional immune responses, but the mechanisms triggering immunopathogenesis are not established. The functional plasticity of macrophages allows this cell type to promote pathogen elimination and inflammation or suppress inflammation and promote tissue remodeling and injury repair. During an infection, the clearance of dead and dying cells, a process named efferocytosis, can modulate the interplay between these contrasting functions. Here, we show that engulfment of SARS-CoV-2-infected apoptotic cells exacerbates inflammatory cytokine production, inhibits the expression of efferocytic receptors, and impairs continual efferocytosis by macrophages. We also provide evidence supporting that lung monocytes and macrophages from severe COVID-19 patients have compromised efferocytic capacity. Our findings reveal that dysfunctional efferocytosis of SARS-CoV-2-infected cell corpses suppresses macrophage anti-inflammation and efficient tissue repair programs and provides mechanistic insights for the excessive production of pro-inflammatory cytokines and accumulation of tissue damage associated with COVID-19 immunopathogenesis.

SARS-CoV-2 productively infects primary human immune system cells in vitro and in COVID-19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a hyperinflammatory state and lymphocytopenia, a hallmark that appears as both signature and prognosis of disease severity outcome. Although cytokine storm and a sustained inflammatory state are commonly associated with immune cell depletion, it is still unclear whether direct SARS-CoV-2 infection of immune cells could also play a role in this scenario by harboring viral replication. We found that monocytes, as well as both B and T lymphocytes, were susceptible to SARS-CoV-2 infection in vitro, accumulating double-stranded RNA consistent with viral RNA replication and ultimately leading to expressive T cell apoptosis. In addition, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from coronavirus disease 2019 (COVID-19) patients. The rates of SARS-CoV-2-infected monocytes in peripheral blood mononuclear cells from COVID-19 patients increased over time from symptom onset, with SARS-CoV-2-positive monocytes, B cells, and CD4+ T lymphocytes also detected in postmortem lung tissue. These results indicated that SARS-CoV-2 infection of blood-circulating leukocytes in COVID-19 patients might have important implications for disease pathogenesis and progression, immune dysfunction, and virus spread within the host.

Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients.

Severe cases of COVID-19 are characterized by a strong inflammatory process that may ultimately lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation via cleavage and activation of key inflammatory molecules including active caspase-1 (Casp1p20), IL-1ß, and IL-18. Although participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease are unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and is active in COVID-19 patients. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of postmortem patients upon autopsy. Inflammasome-derived products such as Casp1p20 and IL-18 in the sera correlated with the markers of COVID-19 severity, including IL-6 and LDH. Moreover, higher levels of IL-18 and Casp1p20 are associated with disease severity and poor clinical outcome. Our results suggest that inflammasomes participate in the pathophysiology of the disease, indicating that these platforms might be a marker of disease severity and a potential therapeutic target for COVID-19.

Eu3+ -tetrakis ß-diketonate complexes for solid-state lighting application.

Eu3+ -ß-diketonate complexes are used, for example, in solid-state lighting (SSL) or light-converting molecular devices. However, their low emission quantum efficiency due to water molecules coordinated to Eu3+ and low photostability are still problems to be addressed. To overcome such challenges, we synthesized Eu3+ tetrakis complexes based on [Q][Eu(tfaa)4 ] and [Q][Eu(dbm)4 ] (Q1 = C26 H56 N+ , Q2 = C19 H42 N+ , and Q3 = C17 H38 N+ ), replacing the water molecules in the tris stoichiometry. The tetrakis ß-diketonates showed desirable thermal stability for SSL and, under excitation at 390 nm, they displayed the characteristic Eu3+ emission in the red spectral region. The quantum efficiencies of the dbm complexes achieved values as high as 51%, while the tfaa complexes exhibited lower quantum efficiencies (28-33%), but which were superior to those reported for the tris complexes. The structures were evaluated using the Sparkle/PM7 model and comparing the theoretical and the experimental Judd-Ofelt parameters. [Q1][Eu(dbm)4 ] was used to coat a near-UV light-emitting diode (LED), producing a red-emitting LED prototype that featured the characteristic emission spectrum of [Q1][Eu(dbm)4 ]. The emission intensity of this prototype decreased only 7% after 30 h, confirming its high photostability, which is a notable result considering Eu3+ complexes, making it a potential candidate for SSL.

Neutrophil extracellular traps (NETs) exacerbate severity of infant sepsis.

BACKGROUND: Neutrophil extracellular traps (NETs) are innate defense mechanisms that are also implicated in the pathogenesis of organ dysfunction. However, the role of NETs in pediatric sepsis is unknown. METHODS: Infant (2 weeks old) and adult (6 weeks old) mice were submitted to sepsis by intraperitoneal (i.p.) injection of bacteria suspension or lipopolysaccharide (LPS). Neutrophil infiltration, bacteremia, organ injury, and concentrations of cytokine, NETs, and DNase in the plasma were measured. Production of reactive oxygen and nitrogen species and release of NETs by neutrophils were also evaluated. To investigate the functional role of NETs, mice undergoing sepsis were treated with antibiotic plus rhDNase and the survival, organ injury, and levels of inflammatory markers and NETs were determined. Blood samples from pediatric and adult sepsis patients were collected and the concentrations of NETs measured. RESULTS: Infant C57BL/6 mice subjected to sepsis or LPS-induced endotoxemia produced significantly higher levels of NETs than the adult mice. Moreover, compared to that of the adult mice, this outcome was accompanied by increased organ injury and production of inflammatory cytokines. The increased NETs were associated with elevated expression of Padi4 and histone H3 citrullination in the neutrophils. Furthermore, treatment of infant septic mice with rhDNase or a PAD-4 inhibitor markedly attenuated sepsis. Importantly, pediatric septic patients had high levels of NETs, and the severity of pediatric sepsis was positively correlated with the level of NETs. CONCLUSION: This study reveals a hitherto unrecognized mechanism of pediatric sepsis susceptibility and suggests that NETs represents a potential target to improve clinical outcomes of sepsis.

Effects of electromyographic biofeedback as an adjunctive therapy in the treatment of swallowing disorders: a systematic review of the literature.

PURPOSE: To describe the primary effects of electromyographic biofeedback therapy on swallowing via a systematic review. METHODS: A blind search was carried out by two researchers in the PubMed and Bireme platforms and in the Medline, Lilacs, SciELO, PsycINFO, CINAHL, and Web of Science databases, and the journal articles identified therein were evaluated for inclusion in the study. Original articles associated with the theme were selected with no population-, region-, or language-associated limits. A protocol was created for this study with the following points: author, year, place, number and characteristics of participants, activities evaluated, instruments used, and main results. The PEDro scale was used to analyze the quality of the studies. RESULTS: Among the 686 articles identified in the combined searches, 566 were duplicates. A total of 65 articles were discarded after the title and abstract were read, and a further 29 articles were discarded after the full text was read, yielding a total of six articles for inclusion. In summary, the results lead us to believe that positive effects on the laryngeal lifting capacity, improved swallowing functions, and increased excursion and maximal elevation of the hyoid bone, may be directly related to this method of therapy. CONCLUSIONS: Adjunctive therapeutic protocols with biofeedback electromyography exert positive effects on swallowing function.

Solid Lipid Nanoparticles for Dibucaine Sustained Release.

Dibucaine (DBC) is among the more potent long-acting local anesthetics (LA), and it is also one of the most toxic. Over the last decades, solid lipid nanoparticles (SLN) have been developed as promising carriers for drug delivery. In this study, SLN formulations were prepared with the aim of prolonging DBC release and reducing its toxicity. To this end, SLN composed of two different lipid matrices and prepared by two different hot-emulsion techniques (high-pressure procedure and sonication) were compared. The colloidal stability of the SLN formulations was tracked in terms of particle size (nm), polydispersity index (PDI), and zeta potential (mV) for 240 days at 4 °C; the DBC encapsulation efficiency was determined by the ultrafiltration/centrifugation method. The formulations were characterized by differential scanning calorimetry (DSC), electron paramagnetic resonance (EPR), and release kinetic experiments. Finally, the in vitro cytotoxicity against 3T3 fibroblast and HaCaT cells was determined, and the in vivo analgesic action was assessed using the tail flick test in rats. Both of the homogenization procedures were found suitable to produce particles in the 200 nm range, with good shelf stability (240 days) and high DBC encapsulation efficiency (~72⁻89%). DSC results disclosed structural information on the nanoparticles, such as the lower crystallinity of the lipid core vs. the bulk lipid. EPR measurements provided evidence of DBC partitioning in both SLNs. In vitro (cytotoxicity) and in vivo (tail flick) experiments revealed that the encapsulation of DBC into nanoparticles reduces its intrinsic cytotoxicity and prolongs the anesthetic effect, respectively. These results show that the SLNs produced are safe and have great potential to extend the applications of dibucaine by enhancing its bioavailability.

Paclitaxel Reduces Tumor Growth by Reprogramming Tumor-Associated Macrophages to an M1 Profile in a TLR4-Dependent Manner.

Paclitaxel is an antineoplastic agent widely used to treat several solid tumor types. The primary mechanism of action of paclitaxel is based on microtubule stabilization inducing cell-cycle arrest. Here, we use several tumor models to show that paclitaxel not only induces tumor cell-cycle arrest, but also promotes antitumor immunity. In vitro, paclitaxel reprogrammed M2-polarized macrophages to the M1-like phenotype in a TLR4-dependent manner, similarly to LPS. Paclitaxel also modulated the tumor-associated macrophage (TAM) profile in mouse models of breast and melanoma tumors; gene expression analysis showed that paclitaxel altered the M2-like signature of TAMs toward an M1-like profile. In mice selectively lacking TLR4 on myeloid cells, for example, macrophages (LysM-Cre+/-/TLR4fl/fl), the antitumor effect of paclitaxel was attenuated. Gene expression analysis of tumor samples from patients with ovarian cancer before and after treatment with paclitaxel detected an enrichment of genes linked to the M1 macrophage activation profile (IFNγ-stimulated macrophages). These findings indicate that paclitaxel skews TAMs toward an immunocompetent profile via TLR4, which might contribute to the antitumor effect of paclitaxel and provide a rationale for new combination regimens comprising paclitaxel and immunotherapies as an anticancer treatment.Significance: This study provides new evidence that the antitumor effect of paclitaxel occurs in part via reactivation of the immune response against cancer, guiding tumor-associated macrophages toward the M1-like antitumor phenotype.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5891/F1.large.jpg Cancer Res; 78(20); 5891-900. ©2018 AACR See related commentary by Garassino et al., p. 5729.

Dibucaine in Ionic-Gradient Liposomes: Biophysical, Toxicological, and Activity Characterization.

Administration of local anesthetics is one of the most effective pain control techniques for postoperative analgesia. However, anesthetic agents easily diffuse into the injection site, limiting the time of anesthesia. One approach to prolong analgesia is to entrap local anesthetic agents in nanostructured carriers (e.g., liposomes). Here, we report that using an ammonium sulphate gradient was the best strategy to improve the encapsulation (62.6%) of dibucaine (DBC) into liposomes. Light scattering and nanotracking analyses were used to characterize vesicle properties, such as, size, polydispersity, zeta potentials, and number. In vitro kinetic experiments revealed the sustained release of DBC (50% in 7 h) from the liposomes. In addition, in vitro (3T3 cells in culture) and in vivo (zebrafish) toxicity assays revealed that ionic-gradient liposomes were able to reduce DBC cyto/cardiotoxicity and morphological changes in zebrafish larvae. Moreover, the anesthesia time attained after infiltrative administration in mice was longer with encapsulated DBC (27 h) than that with free DBC (11 h), at 320 µM (0.012%), confirming it as a promising long-acting liposome formulation for parenteral drug administration of DBC.

Nanocapsules Containing Neem (Azadirachta Indica) Oil: Development, Characterization, And Toxicity Evaluation.

In this study, we prepared, characterized, and performed toxicity analyses of poly(ε-caprolactone) nanocapsules loaded with neem oil. Three formulations were prepared by the emulsion/solvent evaporation method. The nanocapsules showed a mean size distribution around 400 nm, with polydispersity below 0.2 and were stable for 120 days. Cytotoxicity and genotoxicity results showed an increase in toxicity of the oleic acid + neem formulations according to the amount of oleic acid used. The minimum inhibitory concentrations demonstrated that all the formulations containing neem oil were active. The nanocapsules containing neem oil did not affect the soil microbiota during 300 days of exposure compared to the control. Phytotoxicity studies indicated that NC_20 (200 mg of neem oil) did not affect the net photosynthesis and stomatal conductance of maize plants, whereas use of NC_10 (100:100 of neem:oleic acid) and NC_15 (150:50 of neem:oleic acid) led to negative effects on these physiological parameters. Hence, the use of oleic acid as a complement in the nanocapsules was not a good strategy, since the nanocapsules that only contained neem oil showed lower toxicity. These results demonstrate that evaluation of the toxicity of nanopesticides is essential for the development of environmentally friendly formulations intended for applications in agriculture.

Highly fluorescent and superparamagnetic nanosystem for biomedical applications.

This work reports on highly fluorescent and superparamagnetic bimodal nanoparticles (BNPs) obtained by a simple and efficient method as probes for fluorescence analysis and/or contrast agents for MRI. These promising BNPs with small dimensions (ca. 17 nm) consist of superparamagnetic iron oxide nanoparticles (SPIONs) covalently bound with CdTe quantum dots (ca. 3 nm). The chemical structure of the magnetic part of BNPs is predominantly magnetite, with minor goethite and maghemite contributions, as shown by Mössbauer spectroscopy, which is compatible with the x-ray diffraction data. Their size evaluation by different techniques showed that the SPION derivatization process, in order to produce the BNPs, does not lead to a large size increase. The BNPs saturation magnetization, when corrected for the organic content of the sample, is ca. 68 emu g-1, which is only slightly reduced relative to the bare nanoparticles. This indicates that the SPION surface functionalization does not change considerably the magnetic properties. The BNP aqueous suspensions presented stability, high fluorescence, high relaxivity ratio (r 2/r 1 equal to 25) and labeled efficiently HeLa cells as can be seen by fluorescence analysis. These BNP properties point to their applications as fluorescent probes as well as negative T 2-weighted MRI contrast agents. Moreover, their potential magnetic response could also be used for fast bioseparation applications.

Removal of glyphosate herbicide from water using biopolymer membranes.

Enormous amounts of pesticides are manufactured and used worldwide, some of which reach soils and aquatic systems. Glyphosate is a non-selective herbicide that is effective against all types of weeds and has been used for many years. It can therefore be found as a contaminant in water, and procedures are required for its removal. This work investigates the use of biopolymeric membranes prepared with chitosan (CS), alginate (AG), and a chitosan/alginate combination (CS/AG) for the adsorption of glyphosate present in water samples. The adsorption of glyphosate by the different membranes was investigated using the pseudo-first order and pseudo-second order kinetic models, as well as the Langmuir and Freundlich isotherm models. The membranes were characterized regarding membrane solubility, swelling, mechanical, chemical and morphological properties. The results of kinetics experiments showed that adsorption equilibrium was reached within 4 h and that the CS membrane presented the best adsorption (10.88 mg of glyphosate/g of membrane), followed by the CS/AG bilayer (8.70 mg of glyphosate/g of membrane). The AG membrane did not show any adsorption capacity for this herbicide. The pseudo-second order model provided good fits to the glyphosate adsorption data on CS and CS/AG membranes, with high correlation coefficient values. Glyphosate adsorption by the membranes could be fitted by the Freundlich isotherm model. There was a high affinity between glyphosate and the CS membrane and moderate affinity in the case of the CS/AG membrane. Physico-chemical characterization of the membranes showed low values of solubility in water, indicating that the membranes are stable and not soluble in water. The SEM and AFM analysis showed evidence of the presence of glyphosate on CS membranes and on chitosan face on CS/AG membranes. The results showed that the glyphosate herbicide can be adsorbed by chitosan membranes and the proposed membrane-based methodology was successfully used to treat a water sample contaminated with glyphosate. Biopolymer membranes therefore potentially offer a versatile method to eliminate agricultural chemicals from water supplies.

Antispasmodic and myorelaxant effects of the flavoring agent methyl cinnamate in gut: potential inhibition of tyrosine kinase.

Methyl cinnamate (MC) is a safe flavoring agent useful to food industry. Although chemically analog to tyrosine kinase inhibitors, there is little information regarding its biological actions. Here, we aimed at assessing the MC effects on gastrointestinal contractility and the putative involvement of tyrosine kinase in the mediation of these effects. Isometric contractions were recorded in rat isolated strips from stomach, duodenum and colon segments. In gastric strips, MC (3-3000 µM) showed antispasmodic effects against carbachol-induced contractions, which remained unchanged by either l-NAME or tetraethylammonium pretreatment and occurred with potency similar to that obtained against contractions evoked by potassium or U-46619. In colon strips, MC was four times more potent than in gastric ones. MC and the positive control genistein inhibited phasic contractions induced by acetylcholine in Ca2+-free medium, an effect fully prevented by sodium orthovanadate. Both MC and genistein decreased the spontaneous contractions of duodenal strips and shortened the time necessary for gastric fundic tissues to reach 50% of maximal relaxation. In freshly isolated colon myocytes, MC decreased the basal levels of cytoplasmic Ca2+, but not the potassium-elicited cytoplasmic Ca2+ elevation. Colon strips obtained from rats subjected to intracolonic acetic acid instillation showed reduced contractility to potassium, which was partially recovered in MC-treated rats. Inhibitory effect of nifedipine against cholinergic contractions, blunted in acetic acid-induced colitis, was also recovered in MC-treated rats. In conclusion, MC inhibited the gastrointestinal contractility with a probable involvement of tyrosine kinase pathways. In vivo, it was effective to prevent the deleterious effects of colitis resulting from acetic acid injury.

A fluorescent spectroscopy and modelling analysis of anti-heparanase aptamers-serum protein interactions.

Aptamers are short, single stranded oligonucleotide or peptide molecules that bind a specific target molecule and can be used for the delivery of therapeutic agents and/or for imaging and clinical diagnosis. Several works have been developed aiming at the production of aptamers and the study of their applications, but few results have been reported on plasmatic dynamics of such products. Aptamers against the heparanase enzyme have been previously described. In this work, the interactions of two constructs of the most promising anti-heparanase aptamer (molecular weights about 9200Da and 22000Da) to human and bovine serum albumins were studied by fluorescence quenching technique. Stern-Volmer graphs were plotted and quenching constants were estimated. Stern-Volmer plots obtained from experiments carried out at 25°C and 37°C showed that the quenching of fluorescence of HSA and BSA by the low molecular weight aptamer was a collisional phenomenon (estimated Stern-Volmer constant: 3.22 (±0.01)×10(5)M(-1) for HSA at 37°C and 2.47 (±0.01)×10(5)M(-1) for HSA at 25°C), while the high molecular weight aptamer quenched albumins by static process (estimated Stern-Volmer constant: 4.05 (±0.01)×10(5)M(-1) for HSA at 37°C and 6.20 (±0.01)×10(5)M(-1) for HSA at 25°C), interacting with those proteins constituting complexes. Linear Stern-Volmer plot from HSA titrated with the low MW aptamer suggested the existence of a single binding site for the quencher in this albumin. Differently, for aptamer 2, the slightly downward curvature of the Stern-Volmer plot of the titration for that albumin suggested a possible conformational change that led to the exposition of lower affinity binding sites in HSA at 25°C. Similarly, although short aptamerdoes not appear to form a stable complex (collisional interaction), the longer aptamer is found to form a stable complex with HSA. In addition, the behaviour of quenching curves for HSA and BSA and values estimated for ratio R1/R2 from model developed by Silva et al. suggest that the primary binding site in both aptamers is located closer to the tryptophan residue in sub domain IIA. It is likely that both aptamers are competing for the same primary site in albumin.

Interactions of aptamers with sera albumins.

The interactions of two short aptamers to human and bovine serum albumins were studied by fluorescence spectroscopic techniques. Intrinsic fluorescence of BSA and HSA were measured by selectively exciting their tryptophan residues. Gradual quenching was observed by titration of both proteins with aptamers. Aptamers are oligonucleic acid or peptide molecules that bind a specific target and can be used for both biotechnological and clinical purposes, since they present molecular recognition properties like that commonly found in antibodies. Two aptamers previously selected against the MUC1 tumour marker were used in this study, one selected for the protein core and one for the glycosylated MUC1. Stern-Volmer graphs were plotted and quenching constants were estimated. Plots obtained from experiments carried out at 25 °C and 37 °C showed the quenching of fluorescence of by aptamers to be a collisional phenomenon. Stern-Volmer constants estimated for HSA quenched by aptamer A were 1.68 × 10(5) (± 5 × 10(3))M(-1) at 37 °C, and 1.37 × 10(5) (± 10(3))M(-1) at 25 °C; and quenched by aptamer B were 1.67 × 10(5) (± 5 × 10(3))M(-1) at 37 °C, and 1.32 × 10(5) (± 10(3))M(-1) at 25 °C. Results suggest that the primary binding site for aptamers on albumin is close to tryptophan residues in sub domain IIA.