Increase In Fluorescence Signal Research Articles

An Edaravone-Guided Design of a Rhodamine-Based Turn-on Fluorescent Probe for Detecting Hydroxyl Radicals in Living Systems.

The hydroxyl radical (·OH), one of the reactive oxygen species (ROS) in biosystems, is found to be involved in many physiological and pathological processes. However, specifically detecting endogenous ·OH remains an outstanding challenge owing to the high reactivity and short lifetime of this radical. Herein, inspired by the scavenging mechanism of a neuroprotective drug edaravone toward ·OH, we developed a new ·OH-specific fluorescent probe RH-EDA. RH-EDA is a hybrid of rhodamine and edaravone and exploits a ·OH-specific 3-methyl-pyrazolone moiety to control its fluorescence behavior. RH-EDA itself is almost nonfluorescent in physiological conditions, which was attributed to the formation of a twisted intramolecular charge transfer (TICT) state upon photoexcitation and the acylation of its rhodamine nitrogen at the 3' position. However, upon a treatment with ·OH, its edaravone subunit was converted to the corresponding 2-oxo-3-(phenylhydrazono)-butanoic acid (OPB) derivative (to afford RH-OPB), thus leading to a significant fluorescence increase (ca. 195-fold). RH-EDA shows a high sensitivity and selectivity to ·OH without interference from other ROS. RH-EDA has been utilized for imaging endogenous ·OH production in living cells and zebrafishes under different stimuli. Moreover, RH-EDA allows a high-contrast discrimination of cancer cells from normal ones by monitoring their different ·OH levels upon stimulation with β-Lapachone (β-Lap), an effective ROS-generating anticancer therapeutic agent. The present study provides a promising methodology for the construction of probes through a drug-guided approach.

Low Temperature Stress Mediates the Antioxidants Pool and Chlorophyll Fluorescence in Vitis vinifera L. Cultivars.

Grapes are sensitive to early autumn and spring low temperature damage. The current study aimed to assay the effects of cold stress (+1 °C for 4, 8, and 16 h) on three grapevine cultivars (Ghiziluzum, Khalili, and Perllete). The results showed that cold stress caused significant changes in the antioxidant and biochemicals content in the studied cultivars. Furthermore, examining the chlorophyll fluorescence indices, cold stress caused a significant increase in minimal fluorescence (F0), a decrease in maximal fluorescence (Fm), and the maximum photochemical quantum yield of photosystem II (Fv/Fm) in all cultivars. Among the studied cultivars, ‘Perllete’ had the highest increase in proline content and activity of antioxidant enzymes and also had the lowest accumulation of malondialdehyde, hydrogen peroxide, electrolyte leakage, and F0, as well as less of a decrease in Fm and Fv/Fm, and had a higher tolerance to cold stress than ‘Ghiziluzum’ and ‘Khalili’. ‘Perllete’ and ‘Ghiziluzum’ showed reasonable tolerance to the low temperature stress. ‘Khalili’ was sensitive to the stress. The rapid screening of grapevine cultivars in early spring low temperatures is applicable with the assaying of some biomolecules and chlorophyll fluorescence.

DNAzyme Sensor Uses Chemiluminescence Resonance Energy Transfer for Rapid, Portable, and Ratiometric Detection of Metal Ions.

DNAzymes have emerged as an important class of sensors for a wide variety of metal ions, with florescence DNAzyme sensors as the most widely used in different sensing and imaging applications because of their fast response time, high signal intensity, and high sensitivity. However, the requirements of an external excitation light source and its associated power increase the cost and size of the fluorometer, making it difficult to be used for portable detections. To overcome these limitations, we report herein a DNAzyme sensor that relies on chemiluminescence resonance energy transfer (CRET) without the need for external light. The sensor is constructed by combining the functional motifs from both Pb2+-dependent 8-17 DNAzyme conjugated to fluorescein (FAM) and hemin/G-quadruplex that mimics horseradish peroxidase to catalyze the oxidation of luminol by H2O2 to yield chemiluminescence. In the absence of Pb2+, the hybridization between the enzyme and substrate strands bring the FAM and hemin/G-quadruplex in close proximity, resulting in CRET. The presence of Pb2+ ions can drive the cleavage on the substrate strand, resulting in a sharp decrease in the melting temperature of hybridization and thus separation of the FAM from hemin/G-quadruplex. The liberated CRET pair causes a ratiometric increase in the donor's fluorescent signal and a decrease in the acceptor signal. Using this method, Pb2+ ions have been measured rapidly (<15 min) with a low limit of detection at 5 nM. By removing the requirement of exogenous light excitation, we have demonstrated a simple and portable detection using a smartphone, making the DNAzyme-CRET system suitable for field tests of lake water. Since DNAzymes selective for other metal ions or targets, such as bacteria, can be obtained using in vitro selection, the method reported here opens a new avenue for rapid, portable, and ratiometric detection of many targets in environmental monitoring, food safety, and medical diagnostics.

Lipid droplet polarity decreases during the pathology of muscle injury as revealed by a polarity sensitive sensor

Revealing the relationship between lipid droplets (LDs)polarity and disease is indispensable in clinicopathological diagnosis. So far, muscle injury is often ignored as it is not life-threatening as cardiovascular and cerebrovascular diseases, making the exploration of the internal relationship between muscle injury and LDs polarity a gray area. Herein, a fluorescent probe (CCB) with powerful polar-sensitive as well as precise LDs targeting was designed for visualizing the LDs polarity in the pathology of muscle injury. By means of the probe CCB, the identification of cancer cells and the monitoring of LDs polarity changes in dysfunctional cells were successfully realized. Furthermore, the penetration ability of CCB in tissues of mice was tested to verify the applicability of the probe in organisms. Importantly, by CCB, the relationship between muscle damage and LDs polarity was explored, revealing that muscle damage caused a significant decrease in LDs polarity accompanied by a significant increase in fluorescence. Most importantly, it is the first time to reveal the relationship between muscle damage and LDs polarity. Therefore, the probe CCB will be a powerful monitoring platform for diagnosing related diseases caused by abnormal LDs polarity.

An mTORC1/2 Kinase Inhibitor Remarkably Enhances the Cytotoxicity of Gemtuzumab Ozogamicin By Activating Lysosomal Function and Cell Cycle Promotion in AML Cells

Objective: Gemtuzumab ozogamicin (GO), the first antibody-drug conjugate (ADC), has attracted the interest of hematologists because over 90% of AML express its target, CD33. Paradoxically, to exert its cytotoxicity, GO is dependent on the cellular function of leukemic cells, including CD33 expression, internalization of CD33-GO complex, and digestion of the acid-labile linker between CD33 antibody and calicheamicin in acidic lysosomes. GO was voluntarily withdrawn from the US market following poor results in the Phase III SWOG S0601 trial. However, we speculate that this result was due to inhibition of the function of these leukemic cells by the chemotherapeutic agents given in combination with GO, hindering its efficacy. Accordingly, to better demonstrate the essential efficacy of GO, we considered that it would be better to enhance the cellular function of leukemic cells than to combine GO with cytotoxic agents. We previously reported that a low, non-toxic dosage of decitabine enhanced GO's cytotoxicity (Kurimoto et al, Leukemia 2013). Based on this result, another group conducted a Phase II trial and revealed significant benefit in a subset of patients with AML (Naval Daver et al, Leukemia 2016). Although GO and subsequent current ADCs depend on lysosomes for activation, lysosome number and activity in tumor cells has not been well elucidated, especially in AML blasts.Here, for the first time, we report that modulation of the lysosomal activity of leukemic cells enhanced the cytotoxicity of GO. We utilized autophagy to enhance lysosomal activity because autophagy consists of autophagosome formation followed by digestion in the acidic lysosome environment. mTOR kinase is known to inhibit autophagy. An mTORC1/2 kinase inhibitor, PP242, was reported to activate lysosomal function (Jing Zhou, et al. Cell Research, 2013) rather than induce severe cell death at low concentrations and was therefore speculated to increase free calicheamicin by improving the efficiency of linker hydrolysis. Thus, in this study, we examined whether PP242-induced inhibition of mTORC1/2 kinase potentiates the cytotoxicity of GO in AML cells and sought to investigate the mechanism by which such enhancement occurs.Methods: Human AML cell lines U937, THP-1, HL-60, SKM-1, SKNO-1, MARIMO and KO52 were used. Apoptosis assay and cell cycle analysis was performed by flow cytometry. Lysosomal compartments were labeled with Lysotracker Red DND-99 and fluorescence images were captured by confocal microscopy. The apoptosis mechanism was analyzed by Western blotting.Results: Cytotoxic effect of GO was enhanced by concurrent treatment with a non-toxic concentration (500 nM) of PP242 in U937 (specific apoptosis 15.3% (GO) vs 34.6% (GO+PP242), respectively, P < 0.001), THP-1 (14.5% vs 21.5%, P < 0.001), SKM-1 (11.7% vs 18.9%, P < 0.001) and SKNO-1 (33.3% vs 56.1%, P < 0.001) cells. In contrast, no combinatory effect was detected in HL-60, MARIMO or KO52 cells (Figure, left panel). Next, we used Lysotracker to label and quantify acidic compartments (lysosomes) in U937, THP-1, MARIMO and KO52 cells. The basal Lysotracker fluorescence was weak in all tested AML cell lines. Fluorescence was dramatically increased by the administration of PP242 and the intensified fluorescence was retained in PP242+GO in both U937 and THP-1 cells, as expected (Figure, right panel). However, we observed no significant increase in Lysotracker fluorescence by PP242 in MARIMO or KO52 cells, consistent with the lack of combinatory cytotoxicity. Moreover, PP242 suppressed GO-induced Chk1 activation and G2/M cell cycle arrest, which was considered to be another mechanism of the enhancement of cytotoxicity.Conclusions: Inhibition of mTORC1/2 kinase by PP242 enhanced the cytotoxicity of GO by increasing lysosomal compartments and promoting the cell cycle by suppressing GO-induced Chk1 activation. Such combination may represent an attractive new anti-leukemia therapeutic strategy. Further, it may help pave the way for the new application of mTORC1/2 kinase inhibitors in effective targeted drug delivery. This use may also benefit the use of other ADCs via utilization of its intrinsic mechanism of action, rather than by the simple combination of strongly cytotoxic drugs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Fluorescent Dye 1,6-Diphenyl-1,3,5-hexatriene Binds to Amyloid Fibrils Formed by Human Amylin and Provides a New Probe of Amylin Amyloid Kinetics.

The fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is widely used as a probe of membrane order. We show that DPH also interacts with amyloid fibrils formed by human amylin (h-amylin, also known as islet amyloid polypeptide) in solution, and this results in a 100-fold increase in DPH fluorescence for a sample of 20 μM h-amylin and 0.25 μM DPH. No increase in DPH fluorescence is observed with the non-amyloidogenic rat amylin or with freshly dissolved, nonfibrillar h-amylin. The time course of amyloid formation by amylin was followed by monitoring the fluorescence of added DPH as a function of time and was similar to that monitored by the standard fluorescent probe thioflavin-T. The inclusion of DPH in the buffer did not perturb the time course of amyloid formation under the conditions examined, and the time course was independent of the range of DPH concentrations tested (0.25-5 μM). The maximum final fluorescence intensity is observed at substoichiometric ratios of DPH to amylin. No significant increase in fluorescence was observed during the lag phase of amyloid formation, and the implications for the structure of amylin prefibril oligomers are discussed. h-Amylin contains three aromatic residues. A triple aromatic to leucine mutant forms amyloid, and DPH binds to the resulting fibrils, indicating that interactions with aromatic side chains are not required for DPH-amylin amyloid interactions. DPH may be especially useful for studies of mutant amylins and other polypeptides in which changes in charged residues might complicate interpretation of thioflavin-T fluorescence.

A colorimetric and fluorescence turn-on probe for the detection of ascorbic acid in living cells and beverages

A colorimetric and fluorescence turn-on dual-signal assay was developed for the determination of ascorbic acid (AA). Because the ultraviolet absorption of the oxidized 3,3′,5,5′-tetramethylbenzidine (oxTMB) overlapped with the fluorescence emission of glutathione stabilized Au nanoclusters (AuNCs), the fluorescence of AuNCs can be quenched by oxTMB. When AA was added, the blue oxTMB was reduced to colorless TMB, and the fluorescence of AuNCs was restored simultaneously. The decrease in absorbance and increase in fluorescence signal depended on the concentration of AA. In the determination range of 0.5 to 200 μM, the detection limits (LOD) for AA were as low as 0.15 µM and 0.22 µM for fluorometric and colorimetric, respectively. The established probe was used successfully for AA detection in living cells and beverages.

A comparison of cell death mechanisms of antioxidants, butylated hydroxyanisole and butylated hydroxytoluene

Butylated hydroxyanisole (BHA) and the chemically similar butylated hydroxytoluene (BHT) are widely used as antioxidants. Toxicity of BHA and BHT has been reported under in vitro and in vivo experimental conditions. However, the mechanism of BHA-induced toxic effects in cells is unclear. In this study, the cytotoxic effects of BHA and differences in cell death mechanism for BHA and BHT were investigated in rat thymocytes by flow cytometric analysis using a fluorescent probe. We observed a significant increase in propidium iodide fluorescence in the population of cells treated with 100 μM and 300 μM BHA (dead cells). Thymocytes treated with 100 µM BHA showed increased intracellular Ca2+ and Zn2+ levels and depolarized cell membranes. BHA (30–100 µM) decreased non-protein thiol content of cells, indicating decreased glutathione content. Co-stimulation with 100 µM BHA and 300 µM H2O2 acted synergistically to increase cell lethality. Moreover, BHA significantly increased caspase-3 activity and the number of annexin-V-positive cells in a concentration-dependent manner, indicating apoptosis. However, BHT reduced caspase-3 activity and increased the number of annexin-V-negative dead cells, indicating non-apoptotic cell death. Our results reveal the toxicity of BHA could be attributed to increased levels of intracellular Ca2+ and Zn2+, resulting in an increased vulnerability of rat thymocytes to oxidative stress. In addition, we demonstrate that whereas BHA induced apoptosis, BHT induced non-apoptotic cell death in rat thymocytes. Therefore, these results may support the safety of BHA, but also demonstrate the importance of performing toxicity evaluation at the cellular level besides the tissue level.

Applying foliar magnesium enhances wheat growth in acidic soil by stimulating exudation of malate and citrate

Aluminium toxicity in acidic soils adversely affects wheat growth. Foliar-applied magnesium (Mg) contributes to mitigating soil acidity stress in wheat, but the mechanisms are unknown. This study explored the mechanism of foliar-Mg mediated enhancement of wheat growth in acidic soil. Two contrasting near-isogenic wheat (Triticum aestivum L.) genotypes differing in Al resistance (Al-sensitive ES8 and Al-resistant ET8) were grown to the vegetative stage (Zadoks 24) in a reconstituted acidic soil (pH0.1 M CaCl2 4.0) profile with three rates of foliar Mg (0, 200 and 500 mg Mg L− 1 using MgSO4.7H2O). Magnesium was applied to the foliage twice [14 and 28 days after sowing (DAS)], and plant growth and root exuded carboxylates were measured up to 42 DAS. Applying 200 mg Mg L− 1 to the foliage increased organic acid anion exudation from wheat roots by ~ 2-fold compared to 0 foliar Mg treated plants. The Al-resistant wheat genotype exuded 1.3-fold more malate and citrate from roots than the Al-sensitive genotype in the absence of foliar Mg. Malate exudation was delayed relative to citrate following foliar Mg application. The foliar-applied Mg increased shoot and root dry weight (by ~ 38 %), total root length (by ~ 33 %) and intrinsic water-use efficiency (by ~ 80 %) compared to plants treated with no Mg. Applying foliar Mg decreased soil acidity stress (as shown by a significant increase in chlorophyll fluorescence Fv/Fm ratio) in wheat compared to 0 foliar Mg. Increased malate and citrate exudation is the underlying mechanism of enhanced wheat growth following foliar Mg application under acidic soil conditions.

Inducing dry eye disease using a custom engineered desiccation system: Impact on the ocular surface including keratin-14-positive limbal epithelial stem cells

PurposeDry eye disease (DED) is characterized by loss of tear film stability that becomes self-sustaining in a vicious cycle of pathophysiological events. Currently, desiccation stress (DS) is the dominant procedure for inducing DED in mice, however its’ effect on limbal epithelial stem cells (LESCs) has been overlooked. This study aimed to establish a DS model via the use of a novel hardware to investigate the impact on the ocular surface including LESCs. MethodsA mouse transporter unit was customized to generate a dehumidified environment. C57BL/6J mice were exposed to mild DS and injected with scopolamine hydrobromide (SH) or remained untreated (UT) under standard vivarium conditions for 10 consecutive days (n = 28/group). Clinical assessments included phenol red tear-thread test, fluorescein staining and optical coherence tomography assessments. Histopathological and immunofluorescence was used to evaluate tissue architecture, goblet cell (GC) status, lacrimal gland (LG) inflammation and epithelial phenotype on the ocular surface. Whole flat-mounted corneas were immunostained for keratin-14 (K14), then imaged by confocal microscopy and analyzed computationally to investigate the effect of DS on LESCs. ResultsCustom modifications made to the animal transporter unit resulted in dehumidified cage relative humidity (RH) of 43.5 ± 4.79% compared to the vivarium 53.9 ± 1.8% (p = 0.0243). Under these conditions, aqueous tear production in mice was suppressed whilst corneal permeability and corneal irregularity significantly increased. H&E staining indicated stressed corneal basal epithelial cells and increased desquamation. DS-exposed mice had reduced GC density (41.0 ± 5.10 GC/mm vs 46.9 ± 3.88 GC/mm, p = 0.0482) and LGs from these mice exhibited elevated CD4+ cell infiltration compared to controls. DS elicited K14+ epithelial cell displacement, as indicated by increased fluorescence signal at a distance of 50–100 μm radially inwards from the limbus [0.63 ± 0.053% (DS) vs 0.54 ± 0.060% (UT), p = 0.0317]. ConclusionsApplication of mild DS using customized hardware and SH injections generated features of DED in mice. Following DS, ocular surface epithelial cell health decreased and LESCs appeared stressed. This suggested that potential downstream effects of DS on corneal homeostasis are present, a phenomenon that is currently under-investigated. The method used to induce DED in this study enables the development of a chronic model which more closely resembles disease seen in the clinic.

Improved detection of reactive oxygen species by DCFH-DA: New insight into self-amplification of fluorescence signal by light irradiation

Abstract Dichlorodihydrofluorescein diacetate (DCFH-DA) has been widely used in detection of reactive oxygen species (ROS). Unfortunately, the detection process is easily affected by light irradiation during measurement, which cannot be overlooked especially for samples with a small amount of ROS. In the study, we revealed several factors that influence the ROS detection process and developed a new ROS detection method by DCFH-DA with higher stability and sensitivity. After light irradiation, the fluorescence signal of DCFH-DA solution increased significantly with pH value higher than 7.4. The increase of fluorescence signal was attributed to a self-oxidization process of DCFH-DA, which transferred dichlorodihydrofluorescein (DCFH) to 2′,7′-dichlorofluorescein (DCF) with the presence of DCF. At the beginning stage of light irradiation, i.e., when the amount of DCF was negligible compared to DCFH (DCFH-DA), the ratio of fluorescence density between samples keeps unchanged after light irradiation. On this basis, we developed a stable and sensitive ROS detection method via light irradiation. The method was successfully applied to detect ROS in HeLa cells which was induced by hydrogen peroxide (H2O2) and plastic nanoparticles. A new insight into the proportional increase of fluorescence signal by light irradiation was revealed.

Blue light promotes bactericidal action of plasma-activated water against Staphylococcus aureus on stainless steel surfaces

The effects of blue light on the promotion of the bactericidal action of plasma-activated water (PAW) against Staphylococcus aureus by inducing the photolysis of staphyloxanthin (STX) were examined. A light-emitting diode (466 nm) and atmospheric-pressure plasma were applied. The degradation of STX pigments extracted through pure methanol was facilitated by increased dosage of blue light (30, 60, 90, and 150 J/cm2). After combined treatment with blue light (150 J/cm2) and PAW (10 min), the surviving population of S. aureus in liquid decreased by 2.70 log CFU/mL, which was about 40 times greater than that of single PAW treatment. Blue light (150 J/cm2) treatment induced leakage of intracellular substances, indicating potential damage to the cell membrane, which was also confirmed as an increase in the SYTOX™ green fluorescence signal. The synergistic bactericidal effect of blue light and PAW was also observed for S. aureus attached to the surface of stainless steel.

Interventional NIR Fluorescence Imaging of Cancer: Review on Next Generation of Dye-Loaded Protein-Based Nanoparticles for Real-Time Feedback During Cancer Surgery

The use of fluorescence imaging technique for visualization, resection and treatment of cancerous tissue, attained plenty of interest once the promise of whole body and deep tissue near-infrared (NIR) imaging emerged. Why is NIR so desired? Contrast agents with optical properties in the NIR spectral range offer an upgrade for the diagnosis and treatment of cancer, by dint of the deep tissue penetration of light in the NIR region of the electromagnetic spectrum, also known as the optical window in biological tissue. Thus, the development of a new generation of NIR emitting and absorbing contrast agents able to overcome the shortcomings of the basic free dye administration is absolutely essential. Several examples of nanoparticles (NPs) have been successfully implemented as carriers for NIR dye molecules to the tumour site owing to their prolonged blood circulation time and enhanced accumulation within the tumour, as well as their increased fluorescence signal relative to free fluorophore emission and active targeting of cancerous cells. Due to their versatile structure, good biocompatibility and capability to efficiently load dyes and bioconjugate with diverse cancer-targeting ligands, the research area of developing protein-based NPs encapsulated or conjugated with NIR dyes is highly promising but still in its infancy. The current review aims to provide an up-to-date overview on the biocompatibility, specific targeting and versatility offered by protein-based NPs loaded with different classes of NIR dyes as next-generation fluorescent agents. Moreover, this study brings to light the newest and most relevant advances involving the state-of-the-art NIR fluorescent agents for the real-time interventional NIR fluorescence imaging of cancer in clinical trials.

Fast and cost-effective screening for SARS-CoV-2 variants in a routine diagnostic setting

ObjectiveThe aim is to recommend a fast and cost-effective screening procedure for UK/SA SARS-CoV-2 variants in a routing diagnostic setting. MethodsA rapid procedure using qPCR is described to provide clinicians with information about the two currently most prevalent variants (B1.1.7 and B1.351) that harbour receptor binding domain mutation N501Y. The N501Y specific assay only delivers an amplification signal if the Y501 variant is present. Results436 samples initially screened positive for SARS-CoV-2 were randomly selected. Only one of these samples showed a fluorescence signal increase indicative for the Y501 variant. The remaining 435 samples had a melting peak at 54 °C indicating the N501 wildtype. SignificanceThe screening of a broad population base can still be performed with the established test system. In case of a positive test for SARS-CoV-2 and corresponding clinical and anamnestic indications, a second qPCR for the mutation N501Y can follow and deliver the result to public health authorities and to the treating physician within a few hours.

Multifunctional nanostructured drug delivery carriers for cancer therapy: Multimodal imaging and ultrasound-induced drug release

Development of multimodal systems for therapy and diagnosis of neoplastic diseases is an unmet need in oncology. The possibility of simultaneous diagnostics, monitoring, and therapy of various diseases allows expanding the applicability of modern systems for drug delivery. We have developed hybrid particles based on biocompatible polymers containing magnetic nanoparticles (MNPs), photoacoustic (MNPs), fluorescent (Cy5 or Cy7 dyes), and therapeutic components (doxorubicin). To achieve high loading efficiency of MNP and Dox to nanostructured carriers, we utilized a novel freezing-induced loading technique. To reduce the systemic toxicity of antitumor drugs and increase their therapeutic efficacy, we can use targeted delivery followed by the remote control of drug release using high intensity-focused ultrasound (HIFU). Loading of MNPs allowed performing magnetic targeting of the carriers and enhanced optoacoustic signal after controlled destruction of the shell and release of therapeutics as well as MRI imaging. The raster scanning optoacoustic mesoscopy (PA, RSOM), MRI, and fluorescent tomography (FT) confirmed the ultrasound-induced release of doxorubicin from capsules: in vitro (in tubes and pieces of meat) and in vivo (after delivery to the liver). Disruption of capsules results in a significant increase of doxorubicin and Cy7 fluorescence initially quenched by magnetite nanoparticles that can be used for real-time monitoring of drug release in vivo. In addition, we explicitly studied cytotoxicity, intracellular localization, and biodistribution of these particles. Elaborated drug delivery carriers have a good perspective for simultaneous imaging and focal therapy of different cancer types, including liver cancer.

N,N-Dimethyl-4,4′-azodianiline functionalized magnetic nanoparticles for enhanced sensitivity of nucleic acid amplification tests

A facile molecular diagnostic method was devised for highly efficient detection of gram-negative bacterial pathogens with low abundance in urological samples, which contain various inhibitory factors of nucleic acid amplification. We developed a magnetic nanoparticle with N,N-Dimethyl-4,4′-azodianiline for bacterial pathogen sample preparation combining nucleic acid amplification test for bacterial pathogen detection and identification. We employed N,N-Dimethyl-4,4′-azodianiline functionalized nanoparticles to enable a dual strategy integrated within a single tube: (i) bacteria enrichment via the hydrophobic effect and (ii) DNA extraction and isolation via electrostatic interaction under pH control. It provided more yield and higher-quality DNA, resulting in a significant increase in fluorescent signal of quantitative PCR (qPCR). We evaluated our proposed method using gram-negative bacteria including Salmonella enterica serover Typhimurium (S. Typhimurium), Escherichia coli (E. coli), Klebsiella pneumonia (K. pneumonia) and Brucella ovis (B. ovis), found in human urine samples. Our proposed method resulted in a detection limit of five colony-forming units (CFU)/10 mL for S. Typhimurium and 10 CFU/10 mL for E. coli, and resulted in the detection of B. ovis and K. pneumonia. This novel method provides simple and sensitive detection of gram-negative bacteria species and can be used for diagnosis of urinary tract infections.

Impact of germination on phenolic composition, antioxidant properties, antinutritional factors, mineral content and Maillard reaction products of malted quinoa flour

The study aimed at improving and comparing the nutritional profile of black (BQ) and white quinoa (WQ) through malting at different germination periods (24, 48, 72 and 96 h), followed by drying at 50 °C, decluming, grinding and sieving to obtain malt flour. The changes in protein, reducing sugar, Maillard reaction products (MRPs), minerals, free and bound polyphenols and antioxidant activity were noted. Malting caused significant increase in polyphenolic content, antioxidant capacity and fluorescence of advanced MRP (FAST) index. The highest increment was noted in malts germinated for 48 and 72 h. BQ malts were marked by higher bound hydroxycinnamic acids, flavan-3-ols, magnesium, potassium and antioxidant activity, while, WQ malts had lower saponin, phytic acid but higher protein, iron, calcium, FAST index. WQ exhibited highest increment (27.23%) in antioxidant activity even though it had lower polyphenols than BQ after malting. Major loss in polyphenols and proteins occurred in malt germinated for 96 h.

Development of a dimer-based screening system for dimerization inhibitor of HIV-1 protease

An in vitro dimer-based screening system (DBSS) for selecting new HIV-1 protease dimerization inhibitor candidates from natural compounds had been established. This system utilizes a fusion between HIV-1 protease and dimer binding domain of AraC protein (proteaseHIV1-AraCDBD) where fluorescence signal will be emitted in the presence of HIV-1 protease inhibitor. However, this screening system had not been evaluated. Therefore, this study was aimed to evaluate it in recombinant Escherichia coli culture. The expression of proteaseHIV1-AraCDBD fusion gene was observed for 18 hours. Its crude lysate isolation was done once every 3 hours and analyzed using SDS PAGE. To test the DBSS, darunavir was used as positive control, and Nigella sativa extract (JH3) was used as the test compound. The results of SDS PAGE analysis on crude lysates presented a ~24.2 kDa band, which was the predicted size of the proteaseHIV1-AraCDBD fusion protein based on its amino acid sequence. The growth curve and protein expression profiles revealed that the 15 hours was the optimum culture age to be used in the screening system. Darunavir testing in DBSS showed an increase in fluorescence signal compared to the negative control. The same increase in fluorescence signal was also obtained from the JH3 compound test. In conclusion, DBSS could be used as an assay to screen for new HIV-1 protease inhibitors, and the JH3 compound demonstrated the ability to inhibit HIV-1 protease dimerization.

Positively charged Ag@Au core-shell nanoparticles as highly stable and enhanced fluorescence quenching platform for one-step nuclease activity detection

S1 nuclease plays an important role in DNA transcription, recombination and repair. Here, we synthesized a positively charged Ag@Au core-shell nanoparticles ((+)Ag@Au CSNPs) and used as highly stable and enhanced fluorescence quenching platform for one-step S1 nuclease activity detection. In the sensor, fluorophore labeled DNA was used as signal probe and its fluorescence signal could be effectively quenched based on the electrostatic interaction of it with (+)Ag@Au CSNPs. In the presence of S1 nuclease, DNA probe was hydrolyzed into DNA fragments and cannot interact with (+)Ag@Au CSNPs, inducing an obvious fluorescence signal increase that was proportional with S1 nuclease concentration in the range of 2.5 × 10−4–3.0 × 10−2 U/mL. The enhanced quenching efficiency of (+)Ag@Au CSNPs paved the way for sensitive S1 detection with a detection limit of 6.0 × 10−5 U/mL. Moreover, the high stability of such nanocomposite can achieve the one-step detection of S1 in complex solution containing salt, protein or other metal ions. In addition, the method also exhibited high selectivity for nuclease detection, making our sensing platform has a potential application value in bioanalysis and clinical diagnosis.

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide.

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)- and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/or the endosomal escape.