Incubation Of Serum Research Articles

Synthesis, gallium labelling and characterization of P04087, a functionalized phosphatidylserine-binding peptide

BackgroundRadiolabeled phosphatidylserine (PS)-binding peptides represent an innovative strategy for molecular imaging of apoptosis and thrombus. The hexapeptide PGDLSR was described as a selective and high affinity ligand for PS. In this work, we synthesized and evaluated a gallium labelled-PGDLSR peptide as a potential and selective radiopharmaceutical for nuclear imaging. PGDLSR-β-alanine-NODAGA (P04087) was prepared using Fmoc-based synthesis and then chelated with cold gallium, 68Ga and 67Ga. The affinity of Ga-P04087 for PS was evaluated by a competitive binding assay using biotinylated AnnexinV. The in vitro stability of the radiotracer was checked at room temperature and after incubation in human serum at 37 °C with and without a metalloprotease inhibitor. The in vivo binding of 67Ga-P04087 to phosphatidylserine was evaluated in a rat model of infective endocarditis.ResultsPGDLSR was successfully prepared with a yield of 31%. P04087 was obtained with a yield of 28% and in high chemical purity (>95%). The radiochemical purities of 67Ga-P04087 and 68Ga-P04087 exceeded 98% in all cases. IC50 of P04087 and Ga-P04087 were in the same order of magnitude (10−7M). The radiolabelled product was stable for 24 h at room temperature, but was very rapidly degraded in human serum in the absence of a protease inhibitor, which had a stabilizing effect. No focal uptake could be detected visually in the cardiac area on SPECT images. On autoradiography however, a focal uptake of 67Ga-P04087 in the valve area was present and histological slices demonstrated localization of peptide binding at the peripheral layer of vegetations.ConclusionIn spite of the preservation of the peptide affinity to the PS after its conjugation to the NODAGA chelator, and of the presence of 67Ga-P04087 uptake on autoradiography, the absence of detectable foci in vivo in the valve area may be attributed to both the low intensity of the signal and the presence of background activity originating from blood pool and surrounding tissues in the living animals. Further modifications are necessary to design a radiolabeled peptide with higher binding potencies to PS while possessing enhanced metabolic stability in vivo.

Peptide-Based Stealth Nanoparticles for Targeted and pH-Triggered Delivery.

Stealth agents are extensively investigated as a means by which to prolong nanostructure residence time in the bloodstream by avoiding uptake by the reticuloendothelial system. Unfortunately, commonly used agents such as poly(ethylene glycol) can adversely impact targeting efficiency and promote immune reaction by the host organism. Therefore, there is an increasing interest in developing biocompatible, non-PEGylated organic nanostructures able to perform targeted delivery to increase the efficacy of liposomal technology. Here, a lipopeptide is presented that can be mixed with lipids commonly used in liposomal formulations in percentages ranging from 20% to 60% w/w. The resulting vesicles are thermally and chemically stable. The peptide coating limits serum-protein adsorption even upon prolonged incubation in pure serum in physiological conditions, outperforming PEGylated liposomes. This architecture can be easily modified to allow straightforward derivatization by standard bio-orthogonal conjugation. Upon derivatization with an anti-transferrin receptor aptamer, these vesicles show highly selective cellular internalization with minimal nonspecific uptake and pH-triggered doxorubicin release.

In Vitro Mouse and Human Serum Stability of a Heterobivalent Dual-Target Probe That Has Strong Affinity to Gastrin-Releasing Peptide and Neuropeptide Y1 Receptors on Tumor Cells.

Receptor-targeting radiolabeled molecular probes with high affinity and specificity are useful in studying and monitoring biological processes and responses. Dual- or multiple-targeting probes, using radiolabeled metal chelates conjugated to peptides, have potential advantages over single-targeting probes as they can recognize multiple targets leading to better sensitivity for imaging and radiotherapy when target heterogeneity is present. Two natural hormone peptide receptors, gastrin-releasing peptide (GRP) and Y1, are specifically interesting as their expression is upregulated in most breast and prostate cancers. One of our goals has been to develop a dual-target probe that can bind both GRP and Y1 receptors. Consequently, a heterobivalent dual-target probe, t-BBN/BVD15-DO3A (where a GRP targeting ligand J-G-Abz4-QWAVGHLM-NH2 and Y1 targeting ligand INP-K [ɛ-J-(α-DO3A-ɛ-DGa)-K] YRLRY-NH2 were coupled), that recognizes both GRP and Y1 receptors was synthesized, purified, and characterized in the past. Competitive displacement cell binding assay studies with the probe demonstrated strong affinity (IC50 values given in parentheses) for GRP receptors in T-47D cells (18 ± 0.7 nM) and for Y1 receptors in MCF7 cells (80 ± 11 nM). As a further evaluation of the heterobivalent dual-target probe t-BBN/BVD15-DO3A, the objective of this study was to determine its mouse and human serum stability at 37°C. The in vitro metabolic degradation of the dual-target probe in mouse and human serum was studied by using a 153Gd-labeled t-BBN/BVD15-DO3A and a high-performance liquid chromatography/radioisotope detector analytical method. The half-life (t1/2) of degradation of the dual-target probe in mouse serum was calculated as 7 hours and only ∼20% degradation was seen after 6 hours incubation in human serum. The slow in vitro metabolic degradation of the dual-target probe can be compared with the degradation t1/2 of the corresponding monomeric probes, BVD15-DO3A and AMBA: 15, and ∼40 minutes for BVD15-DO3A and 3.1 and 38.8 hours for AMBA in mouse and human serum, respectively. A possible pathway for in vitro metabolic degradation of the t-BBN/BVD15-DO3A in mouse serum is proposed based on the chromatographic retention times of the intact probe and its degradants.

Comparability of the effect of storage time and temperature on serum anti-Müllerian hormone measurement between original and modified enzyme-linked immunosorbent assay

ObjectiveTo explore the effect of modified enzyme-linked immunosorbent assay on the AMH results is increased or decreased, and to investigate the effect of storage time and temperature on AMH measurements with and without sample premixing assay buffer using the Kangrun ELISA method. MethodSerum AMH concentration were measured by ELISA, consistency between two kits, and comparability between original and the modified assay under different stored conditions were analyzed by Passing-Bablok regression analysis and Bland-Altman bias evaluation. ResultThere was a strong consistency between AMH concentrations measured in Kangrun ELISA and Ansh Labs ultra-sensitive AMH ELISA. Pre-mixing serum specimens with assay buffer gave consistent results compared with original assay. Modified protocol can reduce the amplitude of increase affected by sample aged and give the most consistent results regardless of storage conditions. ConclusionPre-mixing protocol did not influence the results of fresh serum or frozen serum incubation <3days at 4°C and −80°C, but when specimens detected after collection and stored in other storage conditions, should be pre-mixed with assay buffer to insure its accuracy.

Fluselenamyl: A Novel Benzoselenazole Derivative for PET Detection of Amyloid Plaques (Aβ) in Alzheimer's Disease.

Fluselenamyl (5), a novel planar benzoselenazole shows traits desirable of enabling noninvasive imaging of Aβ pathophysiology in vivo; labeling of both diffuse (an earlier manifestation of neuritic plaques) and fibrillar plaques in Alzheimer’s disease (AD) brain sections, and remarkable specificity for mapping Aβ compared with biomarker proteins of other neurodegenerative diseases. Employing AD homogenates, [18F]-9, a PET tracer demonstrates superior (2–10 fold higher) binding affinity than approved FDA tracers, while also indicating binding to high affinity site on Aβ plaques. Pharmacokinetic studies indicate high initial influx of [18F]-9 in normal mice brains accompanied by rapid clearance in the absence of targeted plaques. Following incubation in human serum, [18F]-9 indicates presence of parental compound up to 3h thus indicating its stability. Furthermore, in vitro autoradiography studies of [18F]-9 with AD brain tissue sections and ex vivo autoradiography studies in transgenic mouse brain sections show cortical Aβ binding, and a fair correlation with Aβ immunostaining. Finally, multiphoton- and microPET/CT imaging indicate its ability to penetrate brain and label parenchymal plaques in transgenic mice. Following further validation of its performance in other AD rodent models and nonhuman primates, Fluselenamyl could offer a platform technology for monitoring earliest stages of Aβ pathophysiology in vivo.

Calcium Phosphate Crystals from Uremic Serum Promote Osteogenic Differentiation in Human Aortic Smooth Muscle Cells.

Recent study demonstrated that calcium phosphate (CaP) crystals isolated from high phosphate medium were a key contributor to arterial calcification. The present study further investigated the effects of CaP crystals induced by uremic serum on calcification of human aortic smooth muscle cells. This may provide a new insight for the development of uremic cardiovascular calcification. We tested the effects of uremic serum or normal serum on cell calcification. Calcification was visualized by staining and calcium deposition quantified. Expression of various bone-calcifying genes was detected by real-time PCR, and protein levels were quantified by western blotting or enzyme-linked immunosorbent assays. Pyrophosphate was used to investigate the effects of CaP crystals' inhibition. Finally, CaP crystals were separated from uremic serum to determine its specific pro-calcification effects. Uremic serum incubation resulted in progressively increased calcification staining and increased calcium deposition in HASMCs after 4, 8 and 12days (P vs 0day <0.001 for all). Compared to cells incubated in control serum, uremic serum significantly induced the mRNA expression of bone morphogenetic factor-2, osteopontin and RUNX2, and increased their protein levels as well (P<0.05 for all). Inhibition of CaP crystals with pyrophosphate incubation prevented calcium deposition and bone-calcifying gene over-expression increased by uremic serum. CaP crystals, rather than the rest of uremic serum, were responsible for these effects. Uremic serum accelerates arterial calcification by mediating osteogenic differentiation. This effect might be mainly attributed to the CaP crystal content.

Abstract 575: Human serine protease therapeutics- potent constructs targeting Fn14 in multiple tumor types

Abstract The cytokine TWEAK, a TNF superfamily member, and its cognate receptor, Fn14, have emerged as potentially valuable targets for cancer therapy since their expression has been frequently identified as independent negative prognostic indicators in a variety of tumor types. The serine protease Granzyme B (GrB) is a highly cytotoxic component of human immune effector cells and induces multiple, intense pro-apoptotic signals when delivered to the cytoplasm of target cells. This human protein has been well-studied, operating through multimodal pathways which are both caspase-dependent and caspase-independent. Our laboratory has designed series of new Fn14 targeted fusion constructs containing an engineered GrB payload. The GrB-Fc-IT4 containing either WTCYS or EACYS mutants of GrB, and a human scFv targeting Fn14 were expressed in HEK293E and suspension CHO cells, harvested under serum-free conditions and purified to homogeneity. Both constructs display high-level protein production (&amp;gt;50 mg/L), enhanced serum stability and impressive cytotoxic activity (IC50 &amp;lt; 20 nM) against Fn14 positive cells. The specific activity of the GrB moiety, as assessed by cleavage of a synthetic chromogenic GrB substrate, was comparable to that of human GrB for the WTCYS while the EACYS variant showed a 50% reduction in specific activity. On the other hand, in vitro stability studies showed that EACYS was more stable after 48h incubation in human serum, retaining over 60% of its initial specific activity. Against a panel of 20 human cell lines expressing Fn14, both constructs showed high affinity and selective cytotoxicity within the nanomolar range (IC50 ranged from 4 to 284 nM) and were two to over a hundred times more potent than free GrB. Mechanistic studies demonstrated that GrB-Fc-IT4 constructs activated caspase cascades and cytochrome C related pro-apoptotic mechanisms in keeping with the known intracellular functions of GrB in target cells. Pharmacokinetic studies in mice revealed that GrB-Fc-IT4 fusion proteins exhibited a bi-exponential clearance from plasma with a rapid initial clearance (t ½ α = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2 β = 35 hours). Toxicity studies in mice demonstrated that the MTD is above 100 mg/kg total dose. Murine orthotropic tumor model efficacy and bio-distribution studies are ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Yunli Zhao, Kathryn E. Ruisaard, Jeffrey A. Winkles, Michael G. Rosenblum. Human serine protease therapeutics- potent constructs targeting Fn14 in multiple tumor types. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 575.

Investigation of a recently detected 11-nor-9-carboxy-Δ9-tetrahydrocannabinol isomer: Studies on the degradation of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol glucuronide

An isomer of the tetrahydrocannabinol (THC) metabolite 11-nor-9-carboxy-Δ9-THC (THCCOOH) had been detected in blood of cannabis users. The present study was initiated to elucidate whether the labile metabolite THCCOOH-glucuronide could be the precursor. THCCOOH-glucuronide was incubated in human serum and albumin (HSA) solution at various temperatures (−18, 4.5, 22 and 37°C) and pH values (pH 7.4 and 8.3) for seven days in the presence or absence of the esterase inhibitor sodium fluoride. Analysis of incubation samples was performed using LC–MS/MS. Marked degradation of THCCOOH-glucuronide was observed at 37°C. It was found that not only THCCOOH, but also the isomer is a degradation product of THCCOOH-glucuronide and its in-vivo production is assumed. Degradation to THCCOOH and the isomer occurred at alkaline pH, in the presence of fluoride-sensitive esterases and of HSA alone. To inhibit isomer formation during sample storage, refrigeration and controlling of the pH are recommended. However, THCCOOH and the isomer exhibit similar properties during incubations in serum, but differ in their interaction with HSA. The present study confirmed the nature of the isomer as degradation product of the abundant THC metabolite THCCOOH-glucuronide. Serum albumin and esterases are obviously involved. The isomer is formed not only during storage, but also under physiological conditions, suggesting that it can be considered an in-vivo metabolite. However, the chemical structure of the isomer remains unknown and further research is necessary.

Reversal of Acute Kidney Injury-Induced Neutrophil Dysfunction: A Critical Role for Resistin.

To assess the reversibility of acute kidney injury-induced neutrophil dysfunction and to identify involved mechanisms. Controlled laboratory experiment and prospective observational clinical study. University laboratory and hospital. C57BL/6 wild-type mice. Patients with septic shock with or without acute kidney injury. Murine acute kidney injury was induced by intraperitoneal injections of folic acid (nephrotoxic acute kidney injury) or by IM injections of glycerol (rhabdomyolysis-induced acute kidney injury). After 24 hours, we incubated isolated neutrophils for 3 hours in normal mouse serum or minimum essential medium buffer. We further studied the effects of plasma samples from 13 patients with septic shock (with or without severe acute kidney injury) on neutrophilic-differentiated NB4 cells. Experimental acute kidney injury significantly inhibited neutrophil migration and intracellular actin polymerization. Plasma levels of resistin, a proinflammatory cytokine and uremic toxin, were significantly elevated during both forms of acute kidney injury. Incubation in serum or minimum essential medium buffer restored normal neutrophil function. Resistin by itself was able to induce acute kidney injury-like neutrophil dysfunction in vitro. Plasma resistin was significantly higher in patients with septic shock with acute kidney injury compared with patients with septic shock alone. Compared with plasma from patients with septic shock, plasma from patients with septic shock and acute kidney injury inhibited neutrophilic-differentiated NB4 cell migration. Even after 4 days of renal replacement therapy, plasma from patients with septic shock plus acute kidney injury still showed elevated resistin levels and inhibited neutrophilic-differentiated NB4 cell migration. Resistin inhibited neutrophilic-differentiated NB4 cell migration and intracellular actin polymerization at concentrations seen during acute kidney injury, but not at normal physiologic concentrations. Acute kidney injury-induced neutrophil dysfunction is reversible in vitro. However, standard renal replacement therapy does not correct this defect in patients with septic shock and acute kidney injury. Resistin is greatly elevated during acute kidney injury, even with ongoing renal replacement therapy, and is sufficient to cause acute kidney injury-like neutrophil dysfunction by itself.

The cytoprotective capacity of processed human cardiac extracellular matrix.

Freshly isolated human cardiac extracellular matrix sheets (cECM) have been shown to support stem cell proliferation and tissue-specific lineage commitment. We now developed a protocol for standardized production of durable, bio-functional hcECM microparticles and corresponding hydrogel, and tested its cytoprotective effects on contractile cells subjected to ischemia-like conditions. Human ventricular myocardium was decellularized by a 3-step protocol, including Tris/EDTA, SDS and serum incubation (cECM). Following snap-freezing and lyophilization, microparticles were created and characterized by laser diffraction, dynamic image analysis (DIA), and mass spectrometry. Moreover, cECM hydrogel was produced by pepsin digestion. Baseline cell-support characteristics were determined using murine HL-1 cardiomyocytes, and the cytoprotective effects of ECM products were tested under hypoxia and glucose/serum deprivation. In cECM, glycoproteins (thrombospondin 1, fibronectin, collagens and nidogen-1) and proteoglycans (dermatopontin, lumican and mimecan) were preserved, but residual intracellular and blood-borne proteins were also detected. The median particle feret diameter was 66μm (15-157μm) by laser diffraction, and 57μm (20-182μm) by DIA with crystal violet staining. HL-1 cells displayed enhanced metabolic activity (39±12%, P<0.05) and proliferation (16±3%, P<0.05) when grown on cECM microparticles in normoxia. During simulated ischemia, cECM microparticles exerted distinct cytoprotective effects (MTS conversion, 240±32%; BrdU uptake, 45±14%; LDH release, -72±7%; P<0.01, each). When cECM microparticles were solubilized to form a hydrogel, the cytoprotective effect was initially abolished. However, modifying the preparation process (pepsin digestion at pH 2 and 25°C, 1mg/ml final cECM concentration) restored the cytoprotective cECM activity. Extracellular matrix from human myocardium can be processed to yield standardized durable microparticles that exert specific cytoprotective effects on cardiomyocyte-like cells. The use of processed cECM may help to optimize future clinical-grade myocardial tissue engineering approaches.

From the Cover: Disease-Induced Disparities in Formation of the Nanoparticle-Biocorona and the Toxicological Consequences.

Nanoparticle (NP) association with macromolecules in a physiological environment forms a biocorona (BC), which alters NP distribution, activity, and toxicity. While BC formation is dependent on NP physicochemical properties, little information exists on the influence of the physiological environment. Obese individuals and those with cardiovascular disease exist with altered serum chemistry, which is expected to influence BC formation and NP toxicity. We hypothesize that a BC formed on NPs following incubation in hyperlipidemic serum will result in altered NP-BC protein content, cellular association, and toxicity compared to normal serum conditions. We utilized Fe3O4 NPs, which are being developed as MRI contrast and tumor targeting agents to test our hypothesis. We used rat aortic endothelial cells (RAECs) within a dynamic flow in vitro exposure system to more accurately depict the in vivo environment. A BC was formed on 20nm PVP-suspended Fe3O4 NPs following incubation in water, 10% normal or hyperlipidemic rat serum. Addition of BCs resulted in increased hydrodynamic size and decreased surface charge. More cholesterol associated with Fe3O4 NPs after incubation in hyperlipidemic as compared with normal serum. Using quantitative proteomics, we identified unique differences in BC protein components between the 2 serum types. Under flow conditions, formation of a BC from both serum types reduced RAECs association of Fe3O4 NPs. Addition of BCs was found to exacerbate RAECs inflammatory gene responses to Fe3O4 NPs (Fe3O4-hyperlipidemic > Fe3O4-normal > Fe3O4) including increased expression of IL-6, TNF-α, Cxcl-2, VCAM-1, and ICAM-1. Overall, these findings demonstrate that disease-induced variations in physiological environments have a significant impact NP-BC formation, cellular association, and cell response.

Interaction of non-human primate complement and antibodies with hypermucoviscous Klebsiella pneumoniae.

Emergent hypermucoviscosity (HMV) phenotypes of Klebsiella pneumoniae have been associated with increased invasiveness and pathogenicity in primates. In this study, we investigated the interaction of African green monkeys (AGM) (Chlorocebus aethiops sabaeus) complement and antibody with HMV and non-HMV isolates as in vitro models of primate infection. Significantly greater survival of HMV isolates was evident after incubation in normal serum or whole blood (p < 0.05) of AGM donors when compared to non-HMV strains. Greater survival of HMV strains (p < 0.05) was found after incubation in whole blood and serum from seropositive donors when compared to seronegative donor samples. Additionally, significantly greater amounts of K. pneumoniae were phagocytozed by AGM leukocytes when complement was active (p < 0.05), but no difference in uptake was observed when serum from seropositive or seronegative animals was used in challenged cells utilizing flow cytometry. Results demonstrate that interaction of cellular and humoral immune elements play a role in the in vitro killing of K. pneumoniae, particularly HMV isolates. Neither AGM serum, nor washed whole blood effectively killed HMV isolates; however, assays using heparinized whole blood of seronegative donors significantly reduced viability of HMV and non-HMV strains. The lack of bacterial killing observed in seropositive donors treatments could be at least partially associated with low IgG2 present in these animals. A better understanding of the pathogenesis of klebsiellosis in primates and host immune response is necessary to identify surface molecules that can induce both opsonizing and bactericidal antibody facilitating killing of Klebsiella, and the development of vaccines in human and animals.

Loxoprofen sodium induces the production of complement C5a in human serum

Basophil activation test (BAT) is an in vitro allergy test that is useful to identify allergens that cause IgE-dependent allergies. The test has been used to detect not only food allergies and allergies caused by environmental factors but also to detect drug hypersensitivity, which has been known to include IgE-independent reactions. In our preliminary studies in which BAT was applied to detect hypersensitivity of loxoprofen, a non-steroidal anti-inflammatory drug (NSAID), conventional BAT with incubation for 30min did not show basophil activation by means of increased CD203c expression. In this study, we extended the incubation time to 24h on the basis of the hypothesis that loxoprofen indirectly activates basophils. Basophils from healthy control donors as well as allergic patients showed up-regulation of CD203c after incubation with loxoprofen for 24h. Activation was induced using loxoprofen-treated serum. Proteomic and pharmacologic analyses revealed that serum incubation with loxoprofen generated an active complement component C5a, which induced CD203c expression via binding to the C5a receptor on basophils. Because C3a production was also detected after incubation for 24h, loxoprofen is likely to stimulate the complement classical pathway. Our findings suggest that the complement activation is involved in drug hypersensitivity and the suppression of this activation may contribute to the elimination of false positive of BAT for drug allergies.

Ultra-Fast Glyco-Coating of Non-Biological Surfaces.

The ability to glycosylate surfaces has medical and diagnostic applications, but there is no technology currently recognized as being able to coat any surface without the need for prior chemical modification of the surface. Recently, a family of constructs called function-spacer-lipids (FSL) has been used to glycosylate cells. Because it is known that lipid-based material can adsorb onto surfaces, we explored the potential and performance of cell-labelling FSL constructs to “glycosylate” non-biological surfaces. Using blood group A antigen as an indicator, the performance of a several variations of FSL constructs to modify a large variety of non-biological surfaces was evaluated. It was found the FSL constructs when optimised could in a few seconds glycosylate almost any non-biological surface including metals, glass, plastics, rubbers and other polymers. Although the FSL glycan coating was non-covalent, and therefore temporary, it was sufficiently robust with appropriate selection of spacer and surface that it could capture anti-glycan antibodies, immobilize cells (via antibody), and withstand incubation in serum and extensive buffer washing, making it suitable for diagnostic and research applications.

Development of a targeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T

Cardiac troponin T (cTnT) is a highly cardiospecific protein commonly used in the diagnosis of acute myocardial infarction (AMI), but is subject to proteolytic degradation upon its release in the circulation. In this study, a targeted mass spectrometry assay was developed to detect peptides which are differentially present within the different degradation products. cTnT was spiked in human serum and incubated at 37°C to induce proteolytic degradation. Isolation and fractionation of cTnT and its fragments from serum were performed using immunoprecipitation and SDS-PAGE. Bands migrating to 37kDa (intact cTnT), 29kDa (primary fragment), and 19, 18, and 16kDa (secondary fragments) were excised, digested, and subsequently analysed using targeted selected ion monitoring on a UHPLC-coupled quadrupole-Orbitrap mass spectrometer. Sixteen precursor ions from a total of 11 peptides unique to cTnT were targeted. Precursor ions were detectable up until 1200ng/L cTnT, which is a typical cTnT concentration after AMI. With tandem-MS and relative quantification, we proved the formation of cTnT fragments upon incubation in human serum and identified differentially present peptides in the fragment bands, indicative of N- and C-terminal proteolytic cleavage. These findings are of importance for the development of future cTnT assays, calibrators, and quality control samples. Biological significanceIn this study we have developed a gel-based targeted mass spectrometry assay which is able to differentiate between different molecular forms of cTnT. The unravelling of the molecular presentation of cTnT in human serum is of importance in the field of clinical chemistry, where this highly specific and sensitive biomarker is being measured on a routinely basis in patient samples. Knowledge of the amino acid sequence of the different cTnT fragments may aid in the development of improved calibrators and quality control samples. In addition, different fragmentation patterns may be indicative of different underlying pathologies. New antibodies for future assays targeting specific areas of cTnT can thus be created based on this information. This assay will be used in future experiments to assess the fragmentation pattern of cTnT in serum of multiple patient groups in our laboratory.

Electrochemical Immunosensor for Qualitative Diagnosis of the American Trypanosomiasis Based on Gold Modified with 3-Mercaptopropionic

This paper describes the development of a label-free electrochemical immunosensor for the qualitative detection of American trypanosomiasis in serum samples. The immunosensor was constructed using self assembled monolayer technique with gold substrate modified with monolayer of 3-mercaptopropionic acid. The T. cruzi antigens were immobilized after activation carboxylic groups in monolayer with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. The parameters that can interfere the impedimetric response of the immunosensor response such as, concentration and incubation time in the solution of T. cruzi antigens, concentration of bovine serum albumin and incubation time in the solution of T. cruzi antibodies were optimized in the presence of the probe molecule hexacyanoferrate(II)/(III), using cyclic voltammetry and electrochemical impedance spectroscopy. The interaction between antigen–antibody was monitored using electrochemical impedance spectroscopy and the results were expressed in terms of the increase of the charge transfer resistance across the interface. The immunosensor showed selective and sensitive in the qualitative detection of T. cruzi antibodies in serum samples infected with toxoplasma antibodies. Therefore, the proposed methodology is a promising alternative for the development of a immunosensor for serological qualitative diagnosis of Chagas disease.

In vivo evaluation of PEGylated ⁶⁴Cu-liposomes with theranostic and radiotherapeutic potential using micro PET/CT.

The objective of this study was to evaluate the potential of PEGylated (64)Cu-liposomes in clinical diagnostic positron emission tomography (PET) imaging and PEGylated (177)Lu-liposomes in internal tumor radiotherapy through in vivo characterization and dosimetric analysis in a human xenograft mouse model. Liposomes with 5 and 10 mol% PEG were characterized with respect to size, charge, and (64)Cu- and (177)Lu-loading efficiency. The tumor imaging potential of (64)Cu-loaded liposomes was evaluated in terms of in vivo biodistribution, tumor accumulation and tumor-to-muscle (T/M) ratios, using PET imaging. The potential of PEGylated liposomes for diagnostic and therapeutic applications was further evaluated through dosimetry analysis using OLINDA/EXM software. The (64)Cu-liposomes were used as biological surrogates to estimate the organ and tumor kinetics of (177)Lu-liposomes. High remote loading efficiency (>95 %) was obtained for both (64)Cu and (177)Lu radionuclides with PEGylated liposomes, and essentially no leakage of the encapsulated radionuclide was observed upon storage and after serum incubation for 24 h at 37 °C. The 10 mol% PEG liposomes showed higher tumor accumulation (6.2 ± 0.2 %ID/g) than the 5 mol% PEG liposomes, as evaluated by PET imaging. The dosimetry analysis of the (64)Cu-liposomes estimated an acceptable total effective dose of 3.3·10(-2) mSv/MBq for diagnostic imaging in patients. A high absorbed tumor dose (114 mGy/MBq) was estimated for the potential radiotherapeutic (177)Lu-liposomes. The overall preclinical profile of PEGylated (64)Cu-liposomes showed high potential as a new PET theranostic tracer for imaging in humans. Dosimetry results predicted that initial administered activity of 200 MBq of (64)Cu-liposomes should be acceptable in patients. Work is in progress to validate the utility of PEGylated (64)Cu-liposomes in a clinical research programme. The high absorbed tumor dose (114 mGy/MBq) estimated for (177)Lu-liposomes and the preliminary dosimetric studies justify further therapeutic and dosimetry investigation of (177)Lu-liposomes in animals before potential testing in man.

Functionalized Gold Nanoparticles for Inhibition of Vascular Endothelial Growth Factor in Arthritic Patients

The idea behind this study is the use of Nanotechnological approaches in treatment of arthritic patients through targeting of Vascular Endothelial Growth factor as angiogenic factor. Anti-Vascular Endothelial Growth Factor A/Gold nanoparticles (anti-VEGFA /GNPs) conjugate have been prepared and applied to serum of Osteoarthritis (OA) and Rheumatoid Arthritis (RA) patients for scavenger of VEGFA antigen present in serum. The level of vascular endothelial growth factor antigen (VEGFA) in RA and OA patients has been estimated before and after incubation of serum of arthritic patients ‘with (anti-VEGFA /GNPs) anti-VEGFA/GNPs conjugate. It assumed that the anti-VEGFA/GNPs conjugate bind with VEGFA antigen so it will inhibit proliferation and angiogenesis in serum of arthritic patients as primary treatment of arthritis. The characterization of both anti-VEGFA/GNPs anti-VEGFA/GNPs conjugate and gold nanoparticles (GNPs) such as particle size, zeta potential (ZP), transmission electron microscopy (TEM) and FTIR were investigated. This anti-VEGFA/GNPs conjugate has been applied to “thirty three” serum samples of human patients with OA and RA in vitro. The Optimization of anti-VEGFA/GNPs gives that 150 ng/mL is an optimum concentration of anti-VEGFA/GNPs conjugate. It can decrease the level of VEGFA in serum of human patients upon incubation for 2 hr at room temperature. The anti-VEGFA/GNPs conjugate gives cure percent 59% and 62% in OA and RA patients ‘respectively through a single addition of anti-VEGFA/GNPs conjugate to serum that was validated by ELISA method. This technique open new modality and hope for OA and RA patients using novel Nanoechnological approach.

Lactobacillus rhamnosus may change the virulence of Candida albicans

To investigate the influence of Lactobacillus rhamnosus in the expression of virulence factors of Candida albicans in vitro. A suspension of L. rhamnosus was initially grown in MRS agar. The other day, Sabouraud dextrose agar was added on the growth of lactobacilli and C. albicans was seeded for 24, 48 and 72 hours. Candida strains were then isolated for investigation of the ability of biofilm formation, by means of cultivation into 96 wells plaque, and reading the optical densities and counting colony forming units per mL. Also the ability of germ tube formation was investigated, after incubation in horse serum and counting of 200 cells. The results were compared to Candida strains grown in the absence of L. rhamnosus, using Student's t test for statistical analysis. there was a significant reduction in the growth of C. albicans in the presence of lactobacilli after 24, 48 or 72 hours. Significant reduction was also observed in germ tube formation after interaction for 48 or 72 hours. For biofilm formation, no statistically significant difference was observed between the Candida strains grown in the presence or absence of lactobacilli. The results suggest that L. rhamnosus is able to influence significantly the growth and expression of virulence factors of C. albicans in vitro, and may interfere with pathogenicity of these micro-organisms.

Viable but Nonculturable and Persister Cells Coexist Stochastically and Are Induced by Human Serum.

Dormancy holds a vital role in the ecological dynamics of microorganisms. Specifically, entry into dormancy allows cells to withstand times of stress while maintaining the potential for reentry into an active existence. The viable but nonculturable (VBNC) state and antibiotic persistence are two well-recognized conditions of dormancy demonstrated to contribute to bacterial stress tolerance and, as a consequence, yield populations that are tolerant to high-dose antibiotics. Aside from this commonality, more evidence is being presented that indicates the relatedness of these two states. Here, we demonstrate that VBNC cells are present during persister isolation experiments, further indicating that these cells coexist and are induced by the same conditions. Interestingly, we reveal that VBNC cells can exist stochastically in unstressed growing cultures, a finding that is characteristic of persisters. Furthermore, human serum induces the formation of both VBNC cells and persisters, a finding not previously described for either dormancy state. Lastly, we describe the role of toxin-antitoxin systems (TAS) in the induction of the VBNC state and report that these TAS, which are classically implicated in persister cell formation, are also induced during incubation in human serum. This study provides evidence for the recently proposed "dormancy continuum hypothesis" and substantiates the physical and molecular relatedness of VBNC and persister cells in a standardized model organism. Notably, these results provide new evidence for the clinical significance of VBNC and persister cells.